Administrative Appointments

  • Investigator, Howard Hughes Medical Institute (2005 - Present)

Current Research and Scholarly Interests

My laboratory studies the structural and functional basis of receptor/ligand interactions in systems which are relevant to human health and disease. Our investigations are aimed at understanding the molecular recognition properties governing the interactions of receptors with their ligands, and the subsequent molecular events which couple ligand recognition to receptor activation. Many of the systems we are studying in the laboratory are related to the interaction of the host with the environment. The structural studies are complemented by functional approaches using molecular biology and protein engineering to dissect the structural information, design new or altered proteins with modified specificities and activities, and ultimately contribute to the development of proteins or molecules with therapeutic potential. Molecules currently under study include receptors of the immune system involved in autoimmune disorders (T cell receptors, co-receptors, MHC, cytokines), proteins involved in host-pathogen interactions and molecular mimicry (CMV and Toxoplasma surface antigens), proteins of nervous system (peptide hormone receptors, neural guidance proteins), and membrane proteins (chemokine receptors). An emerging focus of our research is to develop. using combinatorial biology approaches, novel ligands for receptors, which may have altered activities, that may serve as therapeutic starting points.

Clinical Trials

  • Adaptive Immune Responses and Repertoire in Influenza Vaccination and Infection (SLVP031) Not Recruiting

    The purpose of this study is provide a better understanding of the adaptive immune response to the licensed flu vaccines. The investigators hope the information learned from this study will help identify and describe important factors of influenza immunity especially of or specific proteins associated with the T-cell immune response.

    Stanford is currently not accepting patients for this trial.

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2022-23 Courses

Stanford Advisees

All Publications

  • Design of cell-type-specific hyperstable IL-4 mimetics via modular de novo scaffolds. Nature chemical biology Yang, H., Ulge, U. Y., Quijano-Rubio, A., Bernstein, Z. J., Maestas, D. R., Chun, J., Wang, W., Lin, J., Jude, K. M., Singh, S., Orcutt-Jahns, B. T., Li, P., Mou, J., Chung, L., Kuo, Y., Ali, Y. H., Meyer, A. S., Grayson, W. L., Heller, N. M., Garcia, K. C., Leonard, W. J., Silva, D., Elisseeff, J. H., Baker, D., Spangler, J. B. 2023


    The interleukin-4 (IL-4) cytokine plays a critical role in modulating immune homeostasis. Although there is great interest in harnessing this cytokine as a therapeutic in natural or engineered formats, the clinical potential of native IL-4 is limited by its instability and pleiotropic actions. Here, we design IL-4 cytokine mimetics (denoted Neo-4) based on a de novo engineered IL-2 mimetic scaffold and demonstrate that these cytokines can recapitulate physiological functions of IL-4 in cellular and animal models. In contrast with natural IL-4, Neo-4 is hyperstable and signals exclusively through the type I IL-4 receptor complex, providing previously inaccessible insights into differential IL-4 signaling through type I versus type II receptors. Because of their hyperstability, our computationally designed mimetics can directly incorporate into sophisticated biomaterials that require heat processing, such as three-dimensional-printed scaffolds. Neo-4 should be broadly useful for interrogating IL-4 biology, and the design workflow will inform targeted cytokine therapeutic development.

    View details for DOI 10.1038/s41589-023-01313-6

    View details for PubMedID 37024727

  • Receptor Elimination by E3 Ubiquitin Ligase Recruitment (REULR): A Targeted Protein Degradation Toolbox. ACS synthetic biology Siepe, D. H., Picton, L. K., Garcia, K. C. 2023


    In recent years, targeted protein degradation (TPD) of plasma membrane proteins by hijacking the ubiquitin proteasome system (UPS) or the lysosomal pathway has emerged as a novel therapeutic avenue in drug development to address and inhibit canonically difficult targets. While TPD strategies have been successful in targeting cell surface receptors, these approaches are limited by the availability of suitable binders to generate heterobifunctional molecules. Here, we present the development of a nanobody (VHH)-based degradation toolbox termed REULR (Receptor Elimination by E3 Ubiquitin Ligase Recruitment). We generated human and mouse cross-reactive nanobodies against five transmembrane PA-TM-RING-type E3 ubiquitin ligases (RNF128, RNF130, RNF167, RNF43, and ZNRF3), covering a broad range and selectivity of tissue expression, with which we characterized the expression in human and mouse cell lines and immune cells (PBMCs). We demonstrate that heterobifunctional REULR molecules can enforce transmembrane E3 ligase interactions with a variety of disease-relevant target receptors (EGFR, EPOR, and PD-1) by induced proximity, resulting in effective membrane clearance of the target receptor at varying levels. In addition, we designed E3 ligase self-degrading molecules, "fratricide" REULRs (RNF128, RNF130, RENF167, RNF43, and ZNRF3), that allow downregulation of one or several E3 ligases from the cell surface and consequently modulate receptor signaling strength. REULR molecules represent a VHH-based modular and versatile "mix and match" targeting strategy for the facile modulation of cell surface proteins by induced proximity to transmembrane PA-TM-RING E3 ligases.

    View details for DOI 10.1021/acssynbio.2c00587

    View details for PubMedID 37011906

  • Structural insights into the mechanism of leptin receptor activation. Nature communications Saxton, R. A., Caveney, N. A., Moya-Garzon, M. D., Householder, K. D., Rodriguez, G. E., Burdsall, K. A., Long, J. Z., Garcia, K. C. 2023; 14 (1): 1797


    Leptin is an adipocyte-derived protein hormone that promotes satiety and energy homeostasis by activating the leptin receptor (LepR)-STAT3 signaling axis in a subset of hypothalamic neurons. Leptin signaling is dysregulated in obesity, however, where appetite remains elevated despite high levels of circulating leptin. To gain insight into the mechanism of leptin receptor activation, here we determine the structure of a stabilized leptin-bound LepR signaling complex using single particle cryo-EM. The structure reveals an asymmetric architecture in which a single leptin induces LepR dimerization via two distinct receptor-binding sites. Analysis of the leptin-LepR binding interfaces reveals the molecular basis for human obesity-associated mutations. Structure-based design of leptin variants that destabilize the asymmetric LepR dimer yield both partial and biased agonists that partially suppress STAT3 activation in the presence of wild-type leptin and decouple activation of STAT3 from LepR negative regulators. Together, these results reveal the structural basis for LepR activation and provide insights into the differential plasticity of signaling pathways downstream of LepR.

    View details for DOI 10.1038/s41467-023-37169-6

    View details for PubMedID 37002197

    View details for PubMedCentralID 4859313

  • Structure of the Wnt-Frizzled-LRP6 initiation complex reveals the basis for coreceptor discrimination. Proceedings of the National Academy of Sciences of the United States of America Tsutsumi, N., Hwang, S., Waghray, D., Hansen, S., Jude, K. M., Wang, N., Miao, Y., Glassman, C. R., Caveney, N. A., Janda, C. Y., Hannoush, R. N., Garcia, K. C. 2023; 120 (11): e2218238120


    Wnt morphogens are critical for embryonic development and tissue regeneration. Canonical Wnts form ternary receptor complexes composed of tissue-specific Frizzled (Fzd) receptors together with the shared LRP5/6 coreceptors to initiate β-catenin signaling. The cryo-EM structure of a ternary initiation complex of an affinity-matured XWnt8-Frizzled8-LRP6 complex elucidates the basis of coreceptor discrimination by canonical Wnts by means of their N termini and linker domains that engage the LRP6 E1E2 domain funnels. Chimeric Wnts bearing modular linker "grafts" were able to transfer LRP6 domain specificity between different Wnts and enable non-canonical Wnt5a to signal through the canonical pathway. Synthetic peptides comprising the linker domain serve as Wnt-specific antagonists. The structure of the ternary complex provides a topological blueprint for the orientation and proximity of Frizzled and LRP6 within the Wnt cell surface signalosome.

    View details for DOI 10.1073/pnas.2218238120

    View details for PubMedID 36893265

  • Structural basis of Janus kinase trans-activation. Cell reports Caveney, N. A., Saxton, R. A., Waghray, D., Glassman, C. R., Tsutsumi, N., Hubbard, S. R., Garcia, K. C. 2023; 42 (3): 112201


    Janus kinases (JAKs) mediate signal transduction downstream of cytokine receptors. Cytokine-dependent dimerization is conveyed across the cell membrane to drive JAK dimerization, trans-phosphorylation, and activation. Activated JAKs in turn phosphorylate receptor intracellular domains (ICDs), resulting in the recruitment, phosphorylation, and activation of signal transducer and activator of transcription (STAT)-family transcription factors. The structural arrangement of a JAK1 dimer complex with IFNlambdaR1 ICD was recently elucidated while bound by stabilizing nanobodies. While this revealed insights into the dimerization-dependent activation of JAKs and the role of oncogenic mutations in this process, the tyrosine kinase (TK) domains were separated by a distance not compatible with the trans-phosphorylation events between the TK domains. Here, we report the cryoelectron microscopy structure of a mouse JAK1 complex in a putative trans-activation state and expand these insights to other physiologically relevant JAK complexes, providing mechanistic insight into the crucial trans-activation step of JAK signaling and allosteric mechanisms of JAK inhibition.

    View details for DOI 10.1016/j.celrep.2023.112201

    View details for PubMedID 36867534

  • Facile repurposing of peptide-MHC-restricted antibodies for cancer immunotherapy. Nature biotechnology Yang, X., Nishimiya, D., Lochte, S., Jude, K. M., Borowska, M., Savvides, C. S., Dougan, M., Su, L., Zhao, X., Piehler, J., Garcia, K. C. 2023


    Monoclonal antibodies (Abs) that recognize major histocompatability complex (MHC)-presented tumor antigens in a manner similar to Tcell receptors (TCRs) have great potential as cancer immunotherapeutics. However, isolation of 'TCR-mimic' (TCRm) Abs is laborious because Abs have not evolved the structurally nuanced peptide-MHC restriction of alphabeta-TCRs. Here, we present a strategy for rapid isolation of highly peptide-specific and 'MHC-restricted' Abs by re-engineering preselected Abs that engage peptide-MHC in a manner structurally similar to that of conventional alphabeta-TCRs. We created structure-based libraries focused on the peptide-interacting residues of TCRm Ab complementarity-determining region (CDR) loops, and rapidly generated MHC-restricted Abs to both mouse and human tumor antigens that specifically killed target cells when formatted as IgG, bispecific T cell engager (BiTE) and chimeric antigen receptor-T (CAR-T). Crystallographic analysis of one selected pMHC-restricted Ab revealed highly peptide-specific recognition, validating the engineering strategy. This approach can yield tumor antigen-specific antibodies in several weeks, potentially enabling rapid clinical translation.

    View details for DOI 10.1038/s41587-022-01567-w

    View details for PubMedID 36593402

  • Prevention of acute GVHD disease using an orthogonal IL-2/IL-2Rbeta system to selectively expand regulatory T-cells in vivo. Blood Lopes Ramos, T., Bolivar-Wagers, S., Jin, S., Thangavelu, G., Simonetta, F., Lin, P., Hirai, T., Saha, A., Koehn, B. H., L Su, L., Picton, L. K., Baker, J., Lohmeyer, J. K., Riddle, M. J., Eide, C., Tolar, J., Panoskaltsis-Mortari, A., Wagner, J. E., Garcia, K. C., Negrin, R. S., Blazar, B. R. 2022


    Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative option for patients with hematological disorders and bone marrow failure syndromes. Graft-versus-Host Disease (GVHD) remains a leading cause of morbidity post-transplant. Regulatory T cell (Treg) therapies are efficacious in ameliorating GVHD but limited by variable suppressive capacities and the need for a high therapeutic dose. Here, we sought to expand Treg in vivo by expressing an orthogonal IL-2Rbeta receptor (oIL2Rbeta) that would selectively interact with orthogonal IL-2 (oIL2) cytokine and not wildtype IL2. To test whether the orthogonal system would preferentially drive donor Treg expansion, we used a murine MHC-disparate GVHD model of lethally irradiated BALB/c mice given T-cell depleted bone marrow from C57BL/6 (B6) mice alone, or together with B6Foxp3+GFP+ Treg or oIL2Rbeta transduced Treg at low cell numbers that typically do not control GVHD with WT Treg. On day 2, B6 Tcons were injected to induce GVHD. Recipients were treated with PBS or oIL2 daily for 14 days, then 3 times weekly for an additional 14 days. Mice treated with oIL2Rbeta Treg and oIL2 compared to PBS had enhanced GVHD survival, in vivo selective expansion of Tregs, and greater suppression of Tcon expansion in secondary lymphoid organs, and intestines. Importantly, oIL2Rbeta Treg maintained graft-versus-tumor (GVT) responses in two distinct tumor models (A20, MLL-AF9). These data demonstrate a novel approach to enhance the efficacy of Treg cell therapy in allo-HSCT using an oIL2/oIL2Rbeta system that allows for selective in vivo expansion of Treg leading to GVHD protection and GVT maintenance.

    View details for DOI 10.1182/blood.2022018440

    View details for PubMedID 36564052

  • Synthetic cytokine circuits that drive T cells into immune-excluded tumors. Science (New York, N.Y.) Allen, G. M., Frankel, N. W., Reddy, N. R., Bhargava, H. K., Yoshida, M. A., Stark, S. R., Purl, M., Lee, J., Yee, J. L., Yu, W., Li, A. W., Garcia, K. C., El-Samad, H., Roybal, K. T., Spitzer, M. H., Lim, W. A. 2022; 378 (6625): eaba1624


    Chimeric antigen receptor (CAR) T cells are ineffective against solid tumors with immunosuppressive microenvironments. To overcome suppression, we engineered circuits in which tumor-specific synNotch receptors locally induce production of the cytokine IL-2. These circuits potently enhance CAR T cell infiltration and clearance of immune-excluded tumors, without systemic toxicity. The most effective IL-2 induction circuit acts in an autocrine and T cell receptor (TCR)- or CAR-independent manner, bypassing suppression mechanisms including consumption of IL-2 or inhibition of TCR signaling. These engineered cells establish a foothold in the target tumors, with synthetic Notch-induced IL-2 production enabling initiation of CAR-mediated T cell expansion and cell killing. Thus, it is possible to reconstitute synthetic T cell circuits that activate the outputs ultimately required for an antitumor response, but in a manner that evades key points of tumor suppression.

    View details for DOI 10.1126/science.aba1624

    View details for PubMedID 36520915

  • Autoimmunity-associated T cell receptors recognize HLA-B*27-bound peptides. Nature Yang, X., Garner, L. I., Zvyagin, I. V., Paley, M. A., Komech, E. A., Jude, K. M., Zhao, X., Fernandes, R. A., Hassman, L. M., Paley, G. L., Savvides, C. S., Brackenridge, S., Quastel, M. N., Chudakov, D. M., Bowness, P., Yokoyama, W. M., McMichael, A. J., Gillespie, G. M., Garcia, K. C. 2022


    Human leucocyte antigen B*27 (HLA-B*27) is strongly associated with inflammatory diseases of the spine and pelvis (for example, ankylosing spondylitis (AS)) and the eye (that is, acute anterior uveitis (AAU))1. How HLA-B*27 facilitates disease remains unknown, but one possible mechanism could involve presentation of pathogenic peptides to CD8+ T cells. Here we isolated orphan T cell receptors (TCRs) expressing a disease-associated public β-chain variable region-complementary-determining region 3β (BV9-CDR3β) motif2-4 from blood and synovial fluid T cells from individuals with AS and from the eye in individuals with AAU. These TCRs showed consistent α-chain variable region (AV21) chain pairing and were clonally expanded in the joint and eye. We used HLA-B*27:05 yeast display peptide libraries to identify shared self-peptides and microbial peptides that activated the AS- and AAU-derived TCRs. Structural analysis revealed that TCR cross-reactivity for peptide-MHC was rooted in a shared binding motif present in both self-antigens and microbial antigens that engages the BV9-CDR3β TCRs. These findings support the hypothesis that microbial antigens and self-antigens could play a pathogenic role in HLA-B*27-associated disease.

    View details for DOI 10.1038/s41586-022-05501-7

    View details for PubMedID 36477533

  • Activation of Wnt/beta-catenin signaling by Zeb1 in endothelial progenitors induces vascular quiescence entry. Cell reports Yu, Q. C., Geng, A., Preusch, C. B., Chen, Y., Peng, G., Xu, Y., Jia, Y., Miao, Y., Xue, H., Gao, D., Bao, L., Pan, W., Chen, J., Garcia, K. C., Cheung, T. H., Zeng, Y. A. 2022; 41 (8): 111694


    The establishment of a functional vasculature requires endothelial cells to enter quiescence during the completion of development, otherwise pathological overgrowth occurs. How such a transition is regulated remains unclear. Here, we uncover a role of Zeb1 in defining vascular quiescence entry. During quiescence acquisition, Zeb1 increases along with the progressive decline of endothelial progenitors' activities, with Zeb1 loss resulting in endothelial overgrowth and vascular deformities. RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin sequencing (ATAC-seq) analyses reveal that Zeb1 represses Wif1, thereby activating Wnt/beta-catenin signaling. Knockdown of Wif1 rescues the overgrowth induced by Zeb1 deletion. Importantly, local administration of surrogate Wnt molecules in the retina ameliorates the overgrowth defects of Zeb1 mutants. These findings show a mechanism by which Zeb1 induces quiescence of endothelial progenitors during the establishing of vascular homeostasis, providing molecular insight into the inherited neovascular pathologies associated with human ZEB1 mutations, suggesting pharmacological activation of Wnt/beta-catenin signaling as a potential therapeutical approach.

    View details for DOI 10.1016/j.celrep.2022.111694

    View details for PubMedID 36417861

  • Publisher Correction: Potentiating adoptive cell therapy using synthetic IL-9 receptors. Nature Kalbasi, A., Siurala, M., Su, L. L., Tariveranmoshabad, M., Picton, L. K., Ravikumar, P., Li, P., Lin, J., Escuin-Ordinas, H., Da, T., Kremer, S. V., Sun, A. L., Castelli, S., Agarwal, S., Scholler, J., Song, D., Rommel, P. C., Radaelli, E., Young, R. M., Leonard, W. J., Ribas, A., June, C. H., Garcia, K. C. 2022

    View details for DOI 10.1038/s41586-022-05548-6

    View details for PubMedID 36380037

  • Epitope Editing in Hematopoietic Cells Enables CD45-Directed Immune Therapy Wellhausen, N., Rennels, A. K., Lesch, S., Agarwal, S., Charria, B., Choi, G., Young, R. M., Garcia, C., June, C. H., Gill, S. AMER SOC HEMATOLOGY. 2022
  • Membrane phosphoinositides regulate GPCR-beta-arrestin complex assembly and dynamics. Cell Janetzko, J., Kise, R., Barsi-Rhyne, B., Siepe, D. H., Heydenreich, F. M., Kawakami, K., Masureel, M., Maeda, S., Garcia, K. C., von Zastrow, M., Inoue, A., Kobilka, B. K. 2022


    Binding of arrestin to phosphorylated G protein-coupled receptors (GPCRs) is crucial for modulating signaling. Once internalized, some GPCRs remain complexed with beta-arrestins, while others interact only transiently; this difference affects GPCR signaling and recycling. Cell-based and invitro biophysical assays reveal the role of membrane phosphoinositides (PIPs) in beta-arrestin recruitment and GPCR-beta-arrestin complexdynamics. We find that GPCRs broadly stratify into two groups, one that requires PIP binding for beta-arrestin recruitment and one that does not. Plasma membrane PIPs potentiate an active conformation of beta-arrestin and stabilize GPCR-beta-arrestin complexes by promoting a fully engaged state of the complex. As allosteric modulators of GPCR-beta-arrestin complex dynamics, membrane PIPs allow for additional conformational diversity beyond that imposed by GPCR phosphorylation alone. For GPCRs that require membrane PIP binding for beta-arrestin recruitment, this provides a mechanism for beta-arrestin release upon translocation of the GPCR to endosomes, allowing for its rapid recycling.

    View details for DOI 10.1016/j.cell.2022.10.018

    View details for PubMedID 36368322

  • Biofunctional Nanodot Arrays in Living Cells Uncover Synergistic Co-Condensation of Wnt Signalodroplets. Small (Weinheim an der Bergstrasse, Germany) Philippi, M., Richter, C. P., Kappen, M., Watrinet, I., Miao, Y., Runge, M., Jorde, L., Korneev, S., Holtmannspotter, M., Kurre, R., Holthuis, J. C., Garcia, K. C., Pluckthun, A., Steinhart, M., Piehler, J., You, C. 2022: e2203723


    Qualitative and quantitative analysis of transient signaling platforms in the plasma membrane has remained a key experimental challenge. Here, biofunctional nanodot arrays (bNDAs) are developed to spatially control dimerization and clustering of cell surface receptors at the nanoscale. High-contrast bNDAs with spot diameters of 300nm are obtained by capillary nanostamping of bovine serum albumin bioconjugates, which are subsequently biofunctionalized by reaction with tandem anti-green fluorescence protein (GFP) clampfusions. Spatially controlled assembly of active Wnt signalosomes is achieved at the nanoscale in the plasma membrane of live cells by capturing the co-receptor Lrp6 into bNDAs via an extracellular GFP tag. Strikingly, co-recruitment is observed of co-receptor Frizzled-8 as well as the cytosolic scaffold proteins Axin-1 and Disheveled-2 into Lrp6 nanodots in the absence of ligand. Density variation and the high dynamics of effector proteins uncover highly cooperative liquid-liquid phase separation (LLPS)-driven assembly of Wnt "signalodroplets" at the plasma membrane, pinpointing the synergistic effects ofLLPSfor Wnt signaling amplification. These insights highlight the potential of bNDAs for systematically interrogating nanoscale signaling platforms and condensation at the plasma membrane of live cells.

    View details for DOI 10.1002/smll.202203723

    View details for PubMedID 36266931

  • Localized ablative immunotherapy drives de novo CD8+ T-cell responses to poorly immunogenic tumors. Journal for immunotherapy of cancer Hoover, A. R., Kaabinejadian, S., Krawic, J. R., Sun, X., Naqash, A. R., Yin, Q., Yang, X., Christopher Garcia, K., Davis, M. M., Hildebrand, W. H., Chen, W. R. 2022; 10 (10)


    BACKGROUND: Localized ablative immunotherapies hold great promise in stimulating antitumor immunity to treat metastatic and poorly immunogenic tumors. Tumor ablation is well known to release tumor antigens and danger-associated molecular patterns to stimulate T-cell immunity, but its immune stimulating effect is limited, particularly against metastatic tumors.METHODS: In this study, we combined photothermal therapy with a potent immune stimulant, N-dihydrogalactochitosan, to create a local ablative immunotherapy which we refer to as laser immunotherapy (LIT). Mice bearing B16-F10 tumors were treated with LIT when the tumors reached 0.5 cm3 and were monitored for survival, T-cell activation, and the ability to resist tumor rechallenge.RESULTS: We found that LIT stimulated a stronger and more consistent antitumor T-cell response to the immunologically 'cold' B16-F10 melanoma tumors and conferred a long-term antitumor memory on tumor rechallenge. Furthermore, we discovered that LIT generated de novo CD8+ T-cell responses that strongly correlated with animal survival and tumor rejection.CONCLUSION: In summary, our findings demonstrate that LIT enhances the activation of T cells and drives de novo antitumor T-cell responses. The data presented herein suggests that localized ablative immunotherapies have great potential to synergize with immune checkpoint therapies to enhance its efficacy, resulting in improved antitumor immunity.

    View details for DOI 10.1136/jitc-2022-004973

    View details for PubMedID 36253002

  • Identification of orphan ligand-receptor relationships using a cell-based CRISPRa enrichment screening platform. eLife Siepe, D. H., Henneberg, L. T., Wilson, S. C., Hess, G. T., Bassik, M. C., Zinn, K., Garcia, K. C. 2022; 11


    Secreted proteins, which include cytokines, hormones and growth factors, are extracellular ligands that control key signaling pathways mediating cell-cell communication within and between tissues and organs. Many drugs target secreted ligands and their cell-surface receptors. Still, there are hundreds of secreted human proteins that either have no identified receptors ('orphans') and are likely to act through cell surface receptors that have not yet been characterized. Discovery of secreted ligand-receptor interactions by high-throughput screening has been problematic, because the most commonly used high-throughput methods for protein-protein interaction (PPI) screening do not work well for extracellular interactions. Cell-based screening is a promising technology for definition of new ligand-receptor interactions, because multimerized ligands can enrich for cells expressing low affinity cell-surface receptors, and such methods do not require purification of receptor extracellular domains. Here, we present a proteo-genomic cell-based CRISPR activation (CRISPRa) enrichment screening platform employing customized pooled cell surface receptor sgRNA libraries in combination with a magnetic bead selection-based enrichment workflow for rapid, parallel ligand-receptor deorphanization. We curated 80 potentially high value orphan secreted proteins and ultimately screened 20 secreted ligands against two cell sgRNA libraries with targeted expression of all single-pass (TM1) or multi-pass (TM2+) receptors by CRISPRa. We identified previously unknown interactions in 12 of these screens, and validated several of them using surface plasmon resonance and/or cell binding. The newly deorphanized ligands include three receptor tyrosine phosphatase (RPTP) ligands and a chemokine like protein that binds to killer cell inhibitory receptors (KIR's). These new interactions provide a resource for future investigations of interactions between the human secreted and membrane proteomes.

    View details for DOI 10.7554/eLife.81398

    View details for PubMedID 36178190

  • Induced CD45 Proximity Potentiates Natural Killer Cell Receptor Antagonism. ACS synthetic biology Ren, J., Jo, Y., Picton, L. K., Su, L. L., Raulet, D. H., Garcia, K. C. 2022


    Natural killer (NK) cells are a major subset of innate immune cells that are essential for host defense against pathogens and cancer. Two main classes of inhibitory NK receptors (NKR), KIR and CD94/NKG2A, play a key role in suppressing NK activity upon engagement with tumor cells or virus-infected cells, limiting their antitumor and antiviral activities. Here, we find that single-chain NKR antagonists linked to a VHH that binds the cell surface phosphatase CD45 potentiate NK and T activities to a greater extent than NKR blocking antibodies alone in vitro. We also uncovered crosstalk between NKG2A and Ly49 that collectively inhibit NK cell activation, such that CD45-NKG2A and CD45-Ly49 bispecific molecules show synergistic effects in their ability to enhance NK cell activation. The basis of the activity enhancement by CD45 ligation may reflect greater antagonism of inhibitory signaling from engagement of MHC I on target cells, combined with other mechanisms, including avidity effects, tonic signaling, antagonism of weak inhibition from engagement of MHC I on non-target cells, and possible CD45 segregation within the NK cell-target cell synapse. These results uncover a strategy for enhancing the activity of NK and T cells that may improve cancer immunotherapies.

    View details for DOI 10.1021/acssynbio.2c00337

    View details for PubMedID 36169352

  • Emerging principles of cytokine pharmacology and therapeutics. Nature reviews. Drug discovery Saxton, R. A., Glassman, C. R., Garcia, K. C. 2022


    Cytokines are secreted signalling proteins that play essential roles in the initiation, maintenance and resolution of immune responses. Although the unique ability of cytokines to control immune function has garnered clinical interest in the context of cancer, autoimmunity and infectious disease, the use of cytokine-based therapeutics has been limited. This is due, in part, to the ability of cytokines to act on many cell types and impact diverse biological functions, resulting in dose-limiting toxicity or lack of efficacy. Recent studies combining structural biology, protein engineering and receptor pharmacology have unlocked new insights into the mechanisms of cytokine receptor activation, demonstrating that many aspects of cytokine function are highly tunable. Here, we discuss the pharmacological principles underlying these efforts to overcome cytokine pleiotropy and enhance the therapeutic potential of this important class of signalling molecules.

    View details for DOI 10.1038/s41573-022-00557-6

    View details for PubMedID 36131080

  • A bead-based method for high-throughput mapping of the sequence- and force-dependence of T cell activation. Nature methods Feng, Y., Zhao, X., White, A. K., Garcia, K. C., Fordyce, P. M. 2022


    Adaptive immunity relies on T lymphocytes that use alphabeta T cell receptors (TCRs) to discriminate among peptides presented by major histocompatibility complex molecules (pMHCs). Identifying pMHCs capable of inducing robust T cell responses will not only enable a deeper understanding of the mechanisms governing immune responses but could also have broad applications in diagnosis and treatment. T cell recognition of sparse antigenic pMHCs in vivo relies on biomechanical forces. However, in vitro screening methods test potential pMHCs without force and often at high (nonphysiological) pMHC densities and thus fail to predict potent agonists in vivo. Here, we present a technology termed BATTLES (biomechanically assisted T cell triggering for large-scale exogenous-pMHC screening) that uses biomechanical force to initiate T cell triggering for peptides and cells in parallel. BATTLES displays candidate pMHCs on spectrally encoded beads composed of a thermo-responsive polymer capable of applying shear loads to T cells, facilitating exploration of the force- and sequence-dependent landscape of T cell responses. BATTLES can be used to explore basic T cell mechanobiology and T cell-based immunotherapies.

    View details for DOI 10.1038/s41592-022-01592-2

    View details for PubMedID 36064771

  • IL-2 receptor engineering enhances regulatory T cell function suppressed by calcineurin inhibitor. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons Hirai, T., Lin, P., Ramos, T. L., Simonetta, F., Su, L. L., Picton, L. K., Baker, J., Lohmeyer, J. K., Garcia, K. C., Negrin, R. S. 2022


    Clinical trials utilizing regulatory T cell (Treg) therapy in organ transplantation have shown promising results, however, the choice of a standard immunosuppressive regimen is still controversial. Calcineurin inhibitors (CNIs) are one of the most common immunosuppressants for organ transplantation, although they may negatively affect Tregs by inhibiting IL-2 production by conventional T cells. As a strategy to replace IL-2 signaling selectively in Tregs, we have introduced an engineered orthogonal IL-2 (ortho IL-2) cytokine/cytokine receptor (R) pair that specifically binds with each other but does not bind with their wild type counterparts. Murine Tregs were isolated from recipients and retrovirally transduced with ortho IL-2Rbeta during ex vivo expansion. Transduced Tregs (ortho Tregs) were transferred into recipient mice in a mixed hematopoietic chimerism model with tacrolimus administration. Ortho IL-2 treatment significantly increased the ortho IL-2Rbeta(+) Treg population in the presence of tacrolimus without stimulating other T cell subsets. All the mice treated with tacrolimus plus ortho IL-2 achieved heart allograft tolerance, even after tacrolimus cessation, whereas those receiving tacrolimus treatment alone did not. These data demonstrate that Treg therapy can be adopted into a CNI-based regimen by utilizing cytokine receptor engineering.

    View details for DOI 10.1111/ajt.17181

    View details for PubMedID 36031344

  • Engineered IL13 variants direct specificity of IL13Rα2-targeted CAR T cell therapy. Proceedings of the National Academy of Sciences of the United States of America Stern, L. A., Gholamin, S., Moraga, I., Yang, X., Saravanakumar, S., Cohen, J. R., Starr, R., Aguilar, B., Salvary, V., Hibbard, J. C., Kalbasi, A., Shepphird, J. K., O'Hearn, J., Garcia, K. C., Brown, C. E. 2022; 119 (33): e2112006119


    IL13Rα2 is an attractive target due to its overexpression in a variety of cancers and rare expression in healthy tissue, motivating expansion of interleukin 13 (IL13)-based chimeric antigen receptor (CAR) T cell therapy from glioblastoma into systemic malignancies. IL13Rα1, the other binding partner of IL13, is ubiquitously expressed in healthy tissue, raising concerns about the therapeutic window of systemic administration. IL13 mutants with diminished binding affinity to IL13Rα1 were previously generated by structure-guided protein engineering. In this study, two such variants, termed C4 and D7, are characterized for their ability to mediate IL13Rα2-specific response as binding domains for CAR T cells. Despite IL13Rα1 and IL13Rα2 sharing similar binding interfaces on IL13, mutations to IL13 that decrease binding affinity for IL13Rα1 did not drastically change binding affinity for IL13Rα2. Micromolar affinity to IL13Rα1 was sufficient to pacify IL13-mutein CAR T cells in the presence of IL13Rα1-overexpressing cells in vitro. Interestingly, effector activity of D7 CAR T cells, but not C4 CAR T cells, was demonstrated when cocultured with IL13Rα1/IL4Rα-coexpressing cancer cells. While low-affinity interactions with IL13Rα1 did not result in observable toxicities in mice, in vivo biodistribution studies demonstrated that C4 and D7 CAR T cells were better able to traffic away from IL13Rα1+ lung tissue than were wild-type (WT) CAR T cells. These results demonstrate the utility of structure-guided engineering of ligand-based binding domains with appropriate selectivity while validating IL13-mutein CARs with improved selectivity for application to systemic IL13Rα2-expressing malignancies.

    View details for DOI 10.1073/pnas.2112006119

    View details for PubMedID 35939683

  • Organizing Structural Principles of the Interleukin-17 Ligand-Receptor Axis. Nature Wilson, S. C., Caveney, N. A., Yen, M., Pollmann, C., Xiang, X., Jude, K. M., Hafer, M., Tsutsumi, N., Piehler, J., Garcia, K. C. 2022


    The interleukin-17 (IL-17) family of cytokines and receptors play central roles in host defense against infection, and development of inflammatory diseases1. The compositions and structures of functional IL-17 family ligand-receptor signaling assemblies remain unclear. Interleukin-17E (IL-17E or IL-25) is a key regulator of Th2 immune responses and driver of inflammatory diseases such as allergic asthma and requires both IL-17 receptor A (IL-17RA) and IL-17RB to elicit functional responses2. Here, we studied IL-25-IL-17RB binary and IL-25-IL-17RB-IL-17RA ternary complexes using a combination of cryo-electron microscopy (cryo-EM), single-molecule imaging, and cell-based signaling approaches. The IL-25-IL-17RB-IL-17RA ternary signaling assembly is a c2-symmetric complex in which the IL-25-IL-17RB homodimer is flanked by two "wing-like" IL-17RA co-receptors through a "tip-to-tip" geometry that is the key receptor-receptor interaction required for initiation of signal transduction. IL-25 interacts solely with IL-17RB to allosterically promote the formation of the IL-17RB-IL-17RA tip-to-tip interface. The resulting large separation between the receptors at the membrane-proximal level may reflect proximity constraints by the intracellular domains for signaling. Cryo-EM structures of IL-17A-IL-17RA and IL-17A-IL-17RA-IL-17RC complexes reveal that this tip-to-tip architecture is a key organizing principle of the IL-17 receptor family, Furthermore, these studies reveal dual actions for IL-17RA sharing amongst IL-17 cytokine complexes, by either directly engaging IL-17 cytokines, or alternatively functioning as a co-receptor.

    View details for DOI 10.1038/s41586-022-05116-y

    View details for PubMedID 35863378

  • Protein-Binding Proteins Designed from Target Structural Information DeBouver, N., Cao, L., Coventry, B., Bera, A., Yang, W., Bernard, S., Stewart, L., Wilson, I., Reohola-Baker, H., Schiessinger, J., Lee, S., Savvides, S., Garcia, K. INT UNION CRYSTALLOGRAPHY. 2022: A281
  • Human chimeric orthogonal IL9 receptor signaling promotes stemness and polyfunctionality for adoptive T cell therapy of cancer Tariveranmoshabad, M., Su, L. L., Sun, A. L., Picton, L. K., Ribas, A., Garcia, K., Kalbasi, A. AMER ASSOC CANCER RESEARCH. 2022
  • Potentiating adoptive cell therapy using synthetic IL-9 receptors. Nature Kalbasi, A., Siurala, M., Su, L. L., Tariveranmoshabad, M., Picton, L. K., Ravikumar, P., Li, P., Lin, J., Escuin-Ordinas, H., Da, T., Kremer, S. V., Sun, A. L., Castelli, S., Agarwal, S., Scholler, J., Song, D., Rommel, P. C., Radaelli, E., Young, R. M., Leonard, W. J., Ribas, A., June, C. H., Garcia, K. C. 2022


    Synthetic receptor signalling has the potential to endow adoptively transferred T cells with new functions that overcome major barriers in the treatment of solid tumours, including the need for conditioning chemotherapy1,2. Here we designed chimeric receptors that have an orthogonal IL-2 receptor extracellular domain (ECD) fused with the intracellular domain (ICD) of receptors for common gamma-chain (gammac) cytokines IL-4, IL-7, IL-9 and IL-21 such that the orthogonal IL-2 cytokine elicits the corresponding gammac cytokine signal. Of these, T cells that signal through the chimeric orthogonal IL-2Rbeta-ECD-IL-9R-ICD (o9R) are distinguished by the concomitant activation of STAT1, STAT3 and STAT5 and assume characteristics of stem cell memory and effector T cells. Compared to o2R T cells,o9R T cells have superior anti-tumour efficacy in two recalcitrant syngeneic mouse solid tumour models of melanoma and pancreatic cancer and are effective even in the absence of conditioning lymphodepletion. Therefore, by repurposing IL-9R signalling using a chimeric orthogonal cytokine receptor, T cells gain new functions, and this results in improved anti-tumour activity for hard-to-treat solid tumours.

    View details for DOI 10.1038/s41586-022-04801-2

    View details for PubMedID 35676488

  • Viral G Protein-Coupled Receptors Encoded by beta- and gamma-Herpesviruses. Annual review of virology Rosenkilde, M. M., Tsutsumi, N., Knerr, J. M., Kildedal, D. F., Garcia, K. C. 2022


    Herpesviruses are ancient large DNA viruses that have exploited gene capture as part of their strategy to escape immune surveillance, promote virus spreading, or reprogram host cells to benefit their survival. Most acquired genes are transmembrane proteins and cytokines, such as viral G protein-coupled receptors (vGPCRs), chemokines, and chemokine-binding proteins. This review focuses on the vGPCRs encoded by the human beta- and gamma-herpesviruses. These include receptors from human cytomegalovirus, which encodes four vGPCRs: US27, US28, UL33, and UL78; human herpesvirus 6 and 7 with two receptors: U12 and U51; Epstein-Barr virus with one: BILF1; and Kaposi's sarcoma-associated herpesvirus with one: open reading frame 74. We discuss ligand binding, signaling, and structures of the vGPCRs in light of robust differences from endogenous receptors. Finally, we briefly discuss the therapeutic targeting of vGPCRs as future treatment of acute and chronic herpesvirus infections. Expected final online publication date for the Annual Review of Virology, Volume 9 is September 2022. Please see for revised estimates.

    View details for DOI 10.1146/annurev-virology-100220-113942

    View details for PubMedID 35671566

  • Engineered IL-2 Plus Calcineurin Inhibitor Synergistically Expands Regulatory T Cells Transduced with Engineered IL-2 Receptor and Facilitates Establishment of Transplantation Tolerance. Hirai, T., Lin, P., Ramos, T. L., Garcia, K., Negrin, R. S. WILEY. 2022: 499
  • Synergy of a STING agonist and an IL-2 superkine in cancer immunotherapy against MHC I-deficient and MHC I+ tumors. Proceedings of the National Academy of Sciences of the United States of America Wolf, N. K., Blaj, C., Picton, L. K., Snyder, G., Zhang, L., Nicolai, C. J., Ndubaku, C. O., McWhirter, S. M., Garcia, K. C., Raulet, D. H. 2022; 119 (22): e2200568119


    SignificanceImmunotherapy provides long-term remissions in numerous types of cancer but is ineffective in most tumors with poor immune cell infiltrates or lacking T cell epitopes or MHC I molecules. Agents that activate the STING protein showed promise in several MHC I+ and MHC-deficient tumor models in mice, where they induced powerful antitumor CD8+ T cell and natural killer (NK) cell responses, respectively. They were less effective in numerous other tumor models and yielded mixed results in the clinic in human patients. This report demonstrates strong synergy between a STING agonist and an IL-2 superkine in effectively curing difficult-to-treat MHC-deficient and MHC-positive tumor models in mice by mobilizing T cells and NK cells. This combination therapy shows considerable promise for clinical application.

    View details for DOI 10.1073/pnas.2200568119

    View details for PubMedID 35588144

  • Structure of the IL-27 quaternary receptor signaling complex. eLife Caveney, N. A., Glassman, C. R., Jude, K. M., Tsutsumi, N., Garcia, K. C. 2022; 11


    Interleukin 27 (IL-27) is a heterodimeric cytokine that functions to constrain T cell-mediated inflammation and plays an important role in immune homeostasis. Binding of IL-27 to cell surface receptors IL-27Ralpha and gp130 results in activation of receptor-associated Janus Kinases and nuclear translocation of Signal Transducer and Activator of Transcription 1 (STAT1) and STAT3 transcription factors. Despite the emerging therapeutic importance of this cytokine axis in cancer and autoimmunity, a molecular blueprint of the IL-27 receptor signaling complex, and its relation to other gp130/IL-12 family cytokines, is currently unclear. We used cryogenic-electron microscopy to determine the quaternary structure of IL-27, composed of p28 and Ebi3 subunits, bound to receptors, IL-27Ralpha and gp130. The resulting 3.47 A resolution structure revealed a three-site assembly mechanism nucleated by the central p28 subunit of the cytokine. The overall topology and molecular details of this binding are reminiscent of IL-6 but distinct from related heterodimeric cytokines IL-12 and IL-23. These results indicate distinct receptor assembly mechanisms used by heterodimeric cytokines with important consequences for targeted agonism and antagonism of IL-27 signaling.

    View details for DOI 10.7554/eLife.78463

    View details for PubMedID 35579417

  • Membrane Phosphoinositides Stabilize GPCR-arrestin Complexes and Provide Temporal Control of Complex Assembly and Dynamics. FASEB journal : official publication of the Federation of American Societies for Experimental Biology Janetzko, J., Kise, R., Barsi-Rhyne, B., Siepe, D. H., Heydenreich, F. M., Masureel, M., Kawakami, K., Garcia, K. C., von Zastrow, M., Inoue, A., Kobilka, B. K. 2022; 36 Suppl 1


    Binding of arrestin to phosphorylated G protein-coupled receptors (GPCRs) is crucial for gating signaling. Once internalized some GPCRs remain stably associated with arrestin, while others interact transiently; this difference affects signaling and recycling behaviors of these GPCRs. Using cell-based and in vitro biophysical assays we examined the role of membrane phosphoinositides (PIPs) in arrestin recruitment and GPCR-arrestin complex dynamics. We find that GPCRs broadly stratify into two groups, one which requires PIP-binding for arrestin recruitment and one that does not. Plasma membrane PIPs potentiate an active conformation of arrestin and stabilize GPCR-arrestin complexes by promoting a core-engaged state of the complex. As allosteric modulators of GPCR-arrestin complex dynamics, membrane PIPs allow for additional conformational diversity beyond that imposed by GPCR phosphorylation alone. The dependance on membrane PIPs provides a mechanism for arrestin release from transiently associated GPCRs, allowing their rapid recycling, while explaining how stably associated GPCRs are able to engage G proteins at endosomes.

    View details for DOI 10.1096/fasebj.2022.36.S1.R6320

    View details for PubMedID 35553909

  • Tuning T cell receptor sensitivity through catch bond engineering. Science (New York, N.Y.) Zhao, X., Kolawole, E. M., Chan, W., Feng, Y., Yang, X., Gee, M. H., Jude, K. M., Sibener, L. V., Fordyce, P. M., Germain, R. N., Evavold, B. D., Garcia, K. C. 2022; 376 (6589): eabl5282


    Adoptive cell therapy using engineered T cell receptors (TCRs) is a promising approach for targeting cancer antigens, but tumor-reactive TCRs are often weakly responsive to their target ligands, peptide-major histocompatibility complexes (pMHCs). Affinity-matured TCRs can enhance the efficacy of TCR-T cell therapy but can also cross-react with off-target antigens, resulting in organ immunopathology. We developed an alternative strategy to isolate TCR mutants that exhibited high activation signals coupled with low-affinity pMHC binding through the acquisition of catch bonds. Engineered analogs of a tumor antigen MAGE-A3-specific TCR maintained physiological affinities while exhibiting enhanced target killing potency and undetectable cross-reactivity, compared with a high-affinity clinically tested TCR that exhibited lethal cross-reactivity with a cardiac antigen. Catch bond engineering is a biophysically based strategy to tune high-sensitivity TCRs for T cell therapy with reduced potential for adverse cross-reactivity.

    View details for DOI 10.1126/science.abl5282

    View details for PubMedID 35389803

  • Design of protein binding proteins from target structure alone. Nature Cao, L., Coventry, B., Goreshnik, I., Huang, B., Park, J. S., Jude, K. M., Markovic, I., Kadam, R. U., Verschueren, K. H., Verstraete, K., Walsh, S. T., Bennett, N., Phal, A., Yang, A., Kozodoy, L., DeWitt, M., Picton, L., Miller, L., Strauch, E., DeBouver, N. D., Pires, A., Bera, A. K., Halabiya, S., Hammerson, B., Yang, W., Bernard, S., Stewart, L., Wilson, I. A., Ruohola-Baker, H., Schlessinger, J., Lee, S., Savvides, S. N., Garcia, K. C., Baker, D. 2022


    The design of proteins that bind to a specific site on the surface of a target protein using no information other than the three-dimensional structure of the target remains an outstanding challenge1-5. We describe a general solution to this problem which starts with a broad exploration of the very large space of possible binding modes to a selected region of a protein surface, and then intensifies the search in the vicinity of the most promising binding modes. We demonstrate its very broad applicability by de novo design of binding proteins to 12 diverse protein targets with very different shapes and surface properties. Biophysical characterization shows that the binders, which are all smaller than 65 amino acids, are hyperstable and following experimental optimization bind their targets with nanomolar to picomolar affinities. We succeeded in solving crystal structures of five of the binder-target complexes, and all five are very close to the corresponding computational design models. Experimental data on nearly half a million computational designs and hundreds of thousands of point mutants provide detailed feedback on the strengths and limitations of the method and of our current understanding of protein-protein interactions, and should guide improvement of both. Our approach now enables targeted design of binders to sites of interest on a wide variety of proteins for therapeutic and diagnostic applications.

    View details for DOI 10.1038/s41586-022-04654-9

    View details for PubMedID 35332283

  • Facile discovery of surrogate cytokine agonists. Cell Yen, M., Ren, J., Liu, Q., Glassman, C. R., Sheahan, T. P., Picton, L. K., Moreira, F. R., Rustagi, A., Jude, K. M., Zhao, X., Blish, C. A., Baric, R. S., Su, L. L., Garcia, K. C. 2022


    Cytokines are powerful immune modulators that initiate signaling through receptor dimerization, but natural cytokines have structural limitations as therapeutics. We present a strategy to discover cytokine surrogate agonists by using modular ligands that exploit induced proximity and receptor dimer geometry as pharmacological metrics amenable to high-throughput screening. Using VHH and scFv to human interleukin-2/15, type-I interferon, and interleukin-10 receptors, we generated combinatorial matrices of single-chain bispecific ligands that exhibited diverse spectrums of functional activities, including potent inhibition of SARS-CoV-2 by surrogate interferons. Crystal structures of IL-2R:VHH complexes revealed that variation in receptor dimer geometries resulted in functionally diverse signaling outputs. This modular platform enabled engineering of surrogate ligands that compelled assembly of an IL-2R/IL-10R heterodimer, which does not naturally exist, that signaled through pSTAT5 on T and natural killer (NK) cells. This "cytokine med-chem" approach, rooted in principles of induced proximity, is generalizable for discovery of diversified agonists for many ligand-receptor systems.

    View details for DOI 10.1016/j.cell.2022.02.025

    View details for PubMedID 35325595

  • Structure of a Janus kinase cytokine receptor complex reveals the basis for dimeric activation. Science (New York, N.Y.) Glassman, C. R., Tsutsumi, N., Saxton, R. A., Lupardus, P. J., Jude, K. M., Garcia, K. C. 2022: eabn8933


    Cytokines signal through cell surface receptor dimers to initiate activation of intracellular Janus Kinases (JAKs). We report the 3.6-A resolution cryo-EM structure of full-length JAK1 complexed with a cytokine receptor intracellular Box1/Box2 domain, captured as an activated homodimer bearing the ValPhe (VF) mutation prevalent in myeloproliferative neoplasms. The seven domains of JAK1 form an extended structural unit whose dimerization is mediated by close-packed pseudokinase (PK) domains. The oncogenic VF mutation lies within the core of the JAK1 PK dimer interface, enhancing packing complementarity to facilitate ligand-independent activation. The C-terminal tyrosine kinase domains are poised to phosphorylate the receptor STAT-recruiting motifs projecting from the overhanging FERM-SH2 domains. Mapping of constitutively active JAK mutants supports a two-step allosteric activation mechanism and reveals new opportunities for selective therapeutic targeting of oncogenic JAK signaling.

    View details for DOI 10.1126/science.abn8933

    View details for PubMedID 35271300

  • Mesenchymal-epithelial crosstalk shapes intestinal regionalisation via Wnt and Shh signalling. Nature communications Maimets, M., Pedersen, M. T., Guiu, J., Dreier, J., Thodberg, M., Antoku, Y., Schweiger, P. J., Rib, L., Bressan, R. B., Miao, Y., Garcia, K. C., Sandelin, A., Serup, P., Jensen, K. B. 2022; 13 (1): 715


    Organs are anatomically compartmentalised to cater for specialised functions. In the small intestine (SI), regionalisation enables sequential processing of food and nutrient absorption. While several studies indicate the critical importance of non-epithelial cells during development and homeostasis, the extent to which these cells contribute to regionalisation during morphogenesis remains unexplored. Here, we identify a mesenchymal-epithelial crosstalk that shapes the developing SI during late morphogenesis. We find that subepithelial mesenchymal cells are characterised by gradients of factors supporting Wnt signalling and stimulate epithelial growth in vitro. Such a gradient impacts epithelial gene expression and regional villus formation along the anterior-posterior axis of the SI. Notably, we further provide evidence that Wnt signalling directly regulates epithelial expression of Sonic Hedgehog (SHH), which, in turn, acts on mesenchymal cells to drive villi formation. Taken together our results uncover a mechanistic link between Wnt and Hedgehog signalling across different cellular compartments that is central for anterior-posterior regionalisation and correct formation of the SI.

    View details for DOI 10.1038/s41467-022-28369-7

    View details for PubMedID 35132078

  • Atypical structural snapshots of human cytomegalovirus GPCR interactions with host G proteins. Science advances Tsutsumi, N., Maeda, S., Qu, Q., Vogele, M., Jude, K. M., Suomivuori, C., Panova, O., Waghray, D., Kato, H. E., Velasco, A., Dror, R. O., Skiniotis, G., Kobilka, B. K., Garcia, K. C. 1800; 8 (3): eabl5442


    Human cytomegalovirus (HCMV) encodes G protein-coupled receptors (GPCRs) US28 and US27, which facilitate viral pathogenesis through engagement of host G proteins. Here we report cryo-electron microscopy structures of US28 and US27 forming nonproductive and productive complexes with Gi and Gq, respectively, exhibiting unusual features with functional implications. The "orphan" GPCR US27 lacks a ligand-binding pocket and has captured a guanosine diphosphate-bound inactive Gi through a tenuous interaction. The docking modes of CX3CL1-US28 and US27 to Gi favor localization to endosome-like curved membranes, where US28 and US27 can function as nonproductive Gi sinks to attenuate host chemokine-dependent Gi signaling. The CX3CL1-US28-Gq/11 complex likely represents a trapped intermediate during productive signaling, providing a view of a transition state in GPCR-G protein coupling for signaling. Our collective results shed new insight into unique G protein-mediated HCMV GPCR structural mechanisms, compared to mammalian GPCR counterparts, for subversion of host immunity.

    View details for DOI 10.1126/sciadv.abl5442

    View details for PubMedID 35061538

  • Clonally Expanded B Cells in Multiple Sclerosis Bind EBV EBNA1 and GlialCAM. Nature Lanz, T. V., Brewer, R. C., Ho, P. P., Moon, J. S., Jude, K. M., Fernandez, D., Fernandes, R. A., Gomez, A. M., Nadj, G. S., Bartley, C. M., Schubert, R. D., Hawes, I. A., Vazquez, S. E., Iyer, M., Zuchero, J. B., Teegen, B., Dunn, J. E., Lock, C. B., Kipp, L. B., Cotham, V. C., Ueberheide, B. M., Aftab, B. T., Anderson, M. S., DeRisi, J. L., Wilson, M. R., Bashford-Rogers, R. J., Platten, M., Garcia, K. C., Steinman, L., Robinson, W. H. 2022


    Multiple sclerosis (MS) is a heterogenous autoimmune disease in which autoreactive lymphocytes attack the myelin sheath of the central nervous system (CNS). B lymphocytes in the cerebrospinal fluid (CSF) of MS patients contribute to inflammation and secrete oligoclonal immunoglobulins1,2. Epstein-Barr virus (EBV) infection has been linked to MS epidemiologically, but its pathological role remains unclear3. Here we demonstrate high-affinity molecular mimicry between the EBV transcription factor EBNA1 and the CNS protein GlialCAM, and provide structural and in-vivo functional evidence for its relevance. A cross-reactive CSF-derived antibody was initially identified by single-cell sequencing of the paired-chain B cell repertoire of MS blood and CSF, followed by protein microarray-based testing of recombinantly expressed CSF-derived antibodies against MS-associated viruses. Sequence analysis, affinity measurements, and the crystal structure of the EBNA1-peptide epitope in complex with the autoreactive Fab fragment allowed for tracking the development of the naïve EBNA1-restricted antibody to a mature EBNA1/GlialCAM cross-reactive antibody. Molecular mimicry is facilitated by a post-translational modification of GlialCAM. EBNA1 immunization exacerbates the mouse model of MS and anti-EBNA1/GlialCAM antibodies are prevalent in MS patients. Our results provide a mechanistic link for the association between MS and EBV, and could guide the development of novel MS therapies.

    View details for DOI 10.1038/s41586-022-04432-7

    View details for PubMedID 35073561

  • Interleukin-2 superkines by computational design. Proceedings of the National Academy of Sciences of the United States of America Ren, J., Chu, A. E., Jude, K. M., Picton, L. K., Kare, A. J., Su, L., Montano Romero, A., Huang, P. S., Garcia, K. C. 2022; 119 (12): e2117401119


    SignificanceWhile computational engineering of therapeutic proteins is a desirable goal, in practice the optimization of protein-protein interactions requires substantial experimental intervention. We present here a computational approach that focuses on stabilizing core protein structures rather than engineering the protein-protein interface. Using this approach, we designed thermostabilized interleukin-2 (IL-2) variants that bind tightly to their receptor without experimental optimization, mimicking the properties of the yeast-display engineered IL-2 variant "super-2." Our results suggest that structure-guided stabilization may be a general method for in silico affinity maturation of protein-protein interactions.

    View details for DOI 10.1073/pnas.2117401119

    View details for PubMedID 35294290

  • A human orthogonal IL-2 and IL-2Rbeta system enhances CAR T cell expansion and antitumor activity in a murine model of leukemia. Science translational medicine Zhang, Q., Hresko, M. E., Picton, L. K., Su, L., Hollander, M. J., Nunez-Cruz, S., Zhang, Z., Assenmacher, C., Sockolosky, J. T., Garcia, K. C., Milone, M. C. 1800; 13 (625): eabg6986


    [Figure: see text].

    View details for DOI 10.1126/scitranslmed.abg6986

    View details for PubMedID 34936380

  • T cells targeted to TdT kill leukemic lymphoblasts while sparing normal lymphocytes. Nature biotechnology Ali, M., Giannakopoulou, E., Li, Y., Lehander, M., Virding Culleton, S., Yang, W., Knetter, C., Odabasi, M. C., Bollineni, R. C., Yang, X., Foldvari, Z., Boschen, M., Taraldsrud, E., Stronen, E., Toebes, M., Hillen, A., Mazzi, S., de Ru, A. H., Janssen, G. M., Kolstad, A., Tjonnfjord, G. E., Lie, B. A., Griffioen, M., Lehmann, S., Osnes, L. T., Buechner, J., Garcia, K. C., Schumacher, T. N., van Veelen, P. A., Leisegang, M., Jacobsen, S. E., Woll, P., Olweus, J. 2021


    Unlike chimeric antigen receptors, T-cell receptors (TCRs) can recognize intracellular targets presented on human leukocyte antigen (HLA) molecules. Here we demonstrate that Tcells expressing TCRs specific for peptides from the intracellular lymphoid-specific enzyme terminal deoxynucleotidyl transferase (TdT), presented in the context of HLA-A*02:01, specifically eliminate primary acute lymphoblastic leukemia (ALL) cells of T- and B-cell origin in vitro and in three mouse models of disseminated B-ALL. By contrast, the treatment spares normal peripheral T- and B-cell repertoires and normal myeloid cells in vitro, and in vivo in humanized mice. TdT is an attractive cancer target as it is highly and homogeneously expressed in 80-94% of B- and T-ALLs, but only transiently expressed during normal lymphoid differentiation, limiting on-target toxicity of TdT-specific Tcells. TCR-modified Tcells targeting TdT may be a promising immunotherapy for B-ALL and T-ALL that preserves normal lymphocytes.

    View details for DOI 10.1038/s41587-021-01089-x

    View details for PubMedID 34873326

  • Prevention of Acute Graft-Versus-Host Disease Using an Engineered Mouse Orthogonal IL-2/IL-2R ss Regulatory T Cells Ramos, T., Wagers, S. B., Lin, P., Hirai, T., Su, L., Picton, L., Baker, J., Lohmeyer, J., Mavers, M., Garcia, C., Blazar, B. R., Negrin, R. S. AMER SOC HEMATOLOGY. 2021
  • RTN4/NoGo-receptor binding to BAI adhesion-GPCRs regulates neuronal development. Cell Wang, J., Miao, Y., Wicklein, R., Sun, Z., Wang, J., Jude, K. M., Fernandes, R. A., Merrill, S. A., Wernig, M., Garcia, K. C., Sudhof, T. C. 2021


    RTN4-binding proteins were widely studied as "NoGo" receptors, but their physiological interactors and roles remain elusive. Similarly, BAI adhesion-GPCRs were associated with numerous activities, but their ligands and functions remain unclear. Using unbiased approaches, we observed an unexpected convergence: RTN4 receptors are high-affinity ligands for BAI adhesion-GPCRs. A single thrombospondin type 1-repeat (TSR) domain of BAIs binds to the leucine-rich repeat domain of all three RTN4-receptor isoforms with nanomolar affinity. In the 1.65A crystal structure of the BAI1/RTN4-receptor complex, C-mannosylation of tryptophan and O-fucosylation of threonine in the BAI TSR-domains creates a RTN4-receptor/BAI interface shaped by unusual glycoconjugates that enables high-affinity interactions. In human neurons, RTN4 receptors regulate dendritic arborization, axonal elongation, and synapse formation by differential binding to glial versus neuronal BAIs, thereby controlling neural network activity. Thus, BAI binding to RTN4/NoGo receptors represents a receptor-ligand axis that, enabled by rare post-translational modifications, controls development of synaptic circuits.

    View details for DOI 10.1016/j.cell.2021.10.016

    View details for PubMedID 34758294

  • POWERFUL SYNERGISTIC EFFECTS OF A STING AGONIST AND THE IL-2 SUPERKINE, H9, IN ELICITING NK AND T CELL RESPONSES AGAINST MHC I- AND MHC I plus TUMORS Wolf, N., Blaj, C., Picton, L., Snyder, G., Zhang, L., Nicolai, C., Ndubaku, C., Gauthier, K., McWhirter, S., Garcia, K., Raulet, D. BMJ PUBLISHING GROUP. 2021: A614
  • A Conversation with Dr. K. Christopher Garcia. Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research Garcia, K. C. 2021; 41 (10): 355-359

    View details for DOI 10.1089/jir.2021.29029.exp

    View details for PubMedID 34672799

  • Super-enhancer-based identification of a BATF3/IL-2R-module reveals vulnerabilities in anaplastic large cell lymphoma. Nature communications Liang, H., Costanza, M., Prutsch, N., Zimmerman, M. W., Gurnhofer, E., Montes-Mojarro, I. A., Abraham, B. J., Prokoph, N., Stoiber, S., Tangermann, S., Lobello, C., Oppelt, J., Anagnostopoulos, I., Hielscher, T., Pervez, S., Klapper, W., Zammarchi, F., Silva, D., Garcia, K. C., Baker, D., Janz, M., Schleussner, N., Fend, F., Pospisilova, S., Janikova, A., Wallwitz, J., Stoiber, D., Simonitsch-Klupp, I., Cerroni, L., Pileri, S., de Leval, L., Sibon, D., Fataccioli, V., Gaulard, P., Assaf, C., Knorr, F., Damm-Welk, C., Woessmann, W., Turner, S. D., Look, A. T., Mathas, S., Kenner, L., Merkel, O. 2021; 12 (1): 5577


    Anaplastic large cell lymphoma (ALCL), an aggressive CD30-positive T-cell lymphoma, comprises systemic anaplastic lymphoma kinase (ALK)-positive, and ALK-negative, primary cutaneous and breast implant-associated ALCL. Prognosis of some ALCL subgroups is still unsatisfactory, and already in second line effective treatment options are lacking. To identify genes defining ALCL cell state and dependencies, we here characterize super-enhancer regions by genome-wide H3K27ac ChIP-seq. In addition to known ALCL key regulators, the AP-1-member BATF3 and IL-2 receptor (IL2R)-components are among the top hits. Specific and high-level IL2R expression in ALCL correlates with BATF3 expression. Confirming a regulatory link, IL-2R-expression decreases following BATF3 knockout, and BATF3 is recruited to IL2R regulatory regions. Functionally, IL-2, IL-15 and Neo-2/15, a hyper-stable IL-2/IL-15 mimic, accelerate ALCL growth and activate STAT1, STAT5 and ERK1/2. In line, strong IL-2Ralpha-expression in ALCL patients is linked to more aggressive clinical presentation. Finally, an IL-2Ralpha-targeting antibody-drug conjugate efficiently kills ALCL cells in vitro and in vivo. Our results highlight the importance of the BATF3/IL-2R-module for ALCL biology and identify IL-2Ralpha-targeting as a promising treatment strategy for ALCL.

    View details for DOI 10.1038/s41467-021-25379-9

    View details for PubMedID 34552066

  • An engineered IL-2 partial agonist promotes CD8+ T cell stemness. Nature Mo, F., Yu, Z., Li, P., Oh, J., Spolski, R., Zhao, L., Glassman, C. R., Yamamoto, T. N., Chen, Y., Golebiowski, F. M., Hermans, D., Majri-Morrison, S., Picton, L. K., Liao, W., Ren, M., Zhuang, X., Mitra, S., Lin, J., Gattinoni, L., Powell, J. D., Restifo, N. P., Garcia, K. C., Leonard, W. J. 2021


    Adoptive transfer of antigen-specific T cells represents a major advance in cancer immunotherapy, with robust clinical outcomes in some patients1. Both the number of transferred T cells and their differentiation state are critical determinants of effective responses2,3. T cells can be expanded with T cell receptor (TCR)-mediated stimulation and interleukin-2, but this can lead to differentiation into effector T cells4,5 and lower therapeutic efficacy6, whereas maintenance of a more stem-cell-like state before adoptive transfer is beneficial7. Here we show that H9T, an engineered interleukin-2 partial agonist, promotes the expansion of CD8+T cells without driving terminal differentiation. H9T led to altered STAT5 signalling and mediated distinctive downstream transcriptional, epigenetic and metabolic programs. In addition, H9T treatmentsustained the expression of T cell transcription factor1 (TCF-1) and promoted mitochondrial fitness, thereby facilitating the maintenance of a stem-cell-like state. Moreover, TCR-transgenic and chimeric antigen receptor-modified CD8+ T cells that were expanded with H9T showed robust anti-tumour activity in vivo in mousemodels of melanoma and acute lymphoblastic leukaemia. Thus, engineering cytokine variants with distinctive properties is a promising strategyfor creating new molecules with translational potential.

    View details for DOI 10.1038/s41586-021-03861-0

    View details for PubMedID 34526724

  • Accurate prediction of protein structures and interactions using a three-track neural network. Science (New York, N.Y.) Baek, M., DiMaio, F., Anishchenko, I., Dauparas, J., Ovchinnikov, S., Lee, G. R., Wang, J., Cong, Q., Kinch, L. N., Schaeffer, R. D., Millan, C., Park, H., Adams, C., Glassman, C. R., DeGiovanni, A., Pereira, J. H., Rodrigues, A. V., van Dijk, A. A., Ebrecht, A. C., Opperman, D. J., Sagmeister, T., Buhlheller, C., Pavkov-Keller, T., Rathinaswamy, M. K., Dalwadi, U., Yip, C. K., Burke, J. E., Garcia, K. C., Grishin, N. V., Adams, P. D., Read, R. J., Baker, D. 2021


    DeepMind presented remarkably accurate predictions at the recent CASP14 protein structure prediction assessment conference. We explored network architectures incorporating related ideas and obtained the best performance with a three-track network in which information at the 1D sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The three-track network produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables the rapid solution of challenging X-ray crystallography and cryo-EM structure modeling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate protein-protein complex models from sequence information alone, short circuiting traditional approaches which require modeling of individual subunits followed by docking. We make the method available to the scientific community to speed biological research.

    View details for DOI 10.1126/science.abj8754

    View details for PubMedID 34282049

  • Orthogonal IL-9 receptor signaling reprograms T cells to obviate conditioning chemotherapy before adoptive cell therapy. Kalbasi, A., Tariveranmoshabad, M., Escuin-Ordenas, H., Kremer, S., Su, L. L., Picton, L., Parisi, A., Garcia, C., Ribas, A. AMER ASSOC CANCER RESEARCH. 2021
  • A role for the transcription factor STAT5 in antagonizing CD8+T cell exhaustion Beltra, J., Manne, S., Abdel-Hakeem, M. S., Nzingha, K., Zhang, Z., Huang, H., Kurachi, M., Muroyama, Y., Huang, Y., Su, L. L., Picton, L., Decaluwe, H., Huang, A. C., Berger, S. L., Garcia, C. K., Wherry, E. AMER ASSOC IMMUNOLOGISTS. 2021
  • Selective expansion of regulatory T cells using an orthogonal IL-2/IL-2 receptor system facilitates transplantation tolerance. The Journal of clinical investigation Hirai, T., Ramos, T. L., Lin, P., Simonetta, F., Su, L. L., Picton, L. K., Baker, J., Lin, J., Li, P., Seo, K., Lohmeyer, J. K., Wagers, S. B., Mavers, M., Leonard, W. J., Blazar, B. R., Garcia, K. C., Negrin, R. S. 2021; 131 (8)


    Adoptive transfer of Tregs has been shown to improve alloengraftment in animal models. However, it is technically challenging to expand Tregs ex vivo for the purpose of infusing large numbers of cells in the clinic. We demonstrate an innovative approach to engineering an orthogonal IL-2/IL-2 receptor (IL-2R) pair, the parts of which selectively interact with each other, transmitting native IL-2 signals, but do not interact with the natural IL-2 or IL-2R counterparts, thereby enabling selective stimulation of target cells in vivo. Here, we introduced this orthogonal IL-2R into Tregs. Upon adoptive transfer in a murine mixed hematopoietic chimerism model, orthogonal IL-2 injection significantly promoted orthogonal IL-2R+Foxp3GFP+CD4+ cell proliferation without increasing other T cell subsets and facilitated donor hematopoietic cell engraftment followed by acceptance of heart allografts. Our data indicate that selective target cell stimulation enabled by the engineered orthogonal cytokine receptor improves Treg potential for the induction of organ transplantation tolerance.

    View details for DOI 10.1172/JCI139991

    View details for PubMedID 33855972

  • The tissue protective functions of interleukin-22 can be decoupled from pro-inflammatory actions through structure-based design. Immunity Saxton, R. A., Henneberg, L. T., Calafiore, M., Su, L., Jude, K. M., Hanash, A. M., Garcia, K. C. 2021; 54 (4): 660


    Interleukin-22 (IL-22) acts on epithelial cells to promote tissue protection and regeneration, but can also elicit pro-inflammatory effects, contributing to disease pathology. Here, we engineered a high-affinity IL-22 super-agonist that enabled the structure determination of the IL-22-IL-22Ralpha-IL-10Rbeta ternary complex to a resolution of 2.6A. Using structure-based design, we systematically destabilized the IL-22-IL-10Rbeta binding interface to create partial agonist analogs that decoupled downstream STAT1 and STAT3 signaling. The extent of STAT bias elicited by a single ligand varied across tissues, ranging from full STAT3-biased agonism to STAT1/3 antagonism, correlating with IL-10Rbeta expression levels. Invivo, this tissue-selective signaling drove tissue protection in the pancreas and gastrointestinal tract without inducing local or systemic inflammation, thereby uncoupling these opposing effects of IL-22 signaling. Our findings provide insight into the mechanisms underlying the cytokine pleiotropy and illustrate how differential receptor expression levels and STAT response thresholds can be synthetically exploited to endow pleiotropic cytokines with enhanced functional specificity.

    View details for DOI 10.1016/j.immuni.2021.03.008

    View details for PubMedID 33852830

  • Tuning MPL signaling to influence hematopoietic stem cell differentiation and inhibit essential thrombocythemia progenitors. Proceedings of the National Academy of Sciences of the United States of America Cui, L., Moraga, I., Lerbs, T., Van Neste, C., Wilmes, S., Tsutsumi, N., Trotman-Grant, A. C., Gakovic, M., Andrews, S., Gotlib, J., Darmanis, S., Enge, M., Quake, S., Hitchcock, I. S., Piehler, J., Garcia, K. C., Wernig, G. 2021; 118 (2)


    Thrombopoietin (TPO) and the TPO-receptor (TPO-R, or c-MPL) are essential for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Agents that can modulate TPO-R signaling are highly desirable for both basic research and clinical utility. We developed a series of surrogate protein ligands for TPO-R, in the form of diabodies (DBs), that homodimerize TPO-R on the cell surface in geometries that are dictated by the DB receptor binding epitope, in effect "tuning" downstream signaling responses. These surrogate ligands exhibit diverse pharmacological properties, inducing graded signaling outputs, from full to partial TPO agonism, thus decoupling the dual functions of TPO/TPO-R. Using single-cell RNA sequencing and HSC self-renewal assays we find that partial agonistic diabodies preserved the stem-like properties of cultured HSCs, but also blocked oncogenic colony formation in essential thrombocythemia (ET) through inverse agonism. Our data suggest that dampening downstream TPO signaling is a powerful approach not only for HSC preservation in culture, but also for inhibiting oncogenic signaling through the TPO-R.

    View details for DOI 10.1073/pnas.2017849118

    View details for PubMedID 33384332

  • Global analysis of shared T cell specificities in human non-small cell lung cancer enables HLA inference and antigen discovery. Immunity Chiou, S. H., Tseng, D. n., Reuben, A. n., Mallajosyula, V. n., Molina, I. S., Conley, S. n., Wilhelmy, J. n., McSween, A. M., Yang, X. n., Nishimiya, D. n., Sinha, R. n., Nabet, B. Y., Wang, C. n., Shrager, J. B., Berry, M. F., Backhus, L. n., Lui, N. S., Wakelee, H. A., Neal, J. W., Padda, S. K., Berry, G. J., Delaidelli, A. n., Sorensen, P. H., Sotillo, E. n., Tran, P. n., Benson, J. A., Richards, R. n., Labanieh, L. n., Klysz, D. D., Louis, D. M., Feldman, S. A., Diehn, M. n., Weissman, I. L., Zhang, J. n., Wistuba, I. I., Futreal, P. A., Heymach, J. V., Garcia, K. C., Mackall, C. L., Davis, M. M. 2021; 54 (3): 586–602.e8


    To identify disease-relevant T cell receptors (TCRs) with shared antigen specificity, we analyzed 778,938 TCRβ chain sequences from 178 non-small cell lung cancer patients using the GLIPH2 (grouping of lymphocyte interactions with paratope hotspots 2) algorithm. We identified over 66,000 shared specificity groups, of which 435 were clonally expanded and enriched in tumors compared to adjacent lung. The antigenic epitopes of one such tumor-enriched specificity group were identified using a yeast peptide-HLA A∗02:01 display library. These included a peptide from the epithelial protein TMEM161A, which is overexpressed in tumors and cross-reactive epitopes from Epstein-Barr virus and E. coli. Our findings suggest that this cross-reactivity may underlie the presence of virus-specific T cells in tumor infiltrates and that pathogen cross-reactivity may be a feature of multiple cancers. The approach and analytical pipelines generated in this work, as well as the specificity groups defined here, present a resource for understanding the T cell response in cancer.

    View details for DOI 10.1016/j.immuni.2021.02.014

    View details for PubMedID 33691136

  • Calibration of cell-intrinsic interleukin-2 response thresholds guides design of a regulatory T cell biased agonist. eLife Glassman, C. R., Su, L. n., Majri-Morrison, S. S., Winkelmann, H. n., Mo, F. n., Li, P. n., Pérez-Cruz, M. n., Ho, P. P., Koliesnik, I. n., Nagy, N. n., Hnizdilova, T. n., Picton, L. K., Kovar, M. n., Bollyky, P. n., Steinman, L. n., Meyer, E. n., Piehler, J. n., Leonard, W. J., Garcia, K. C. 2021; 10


    Interleukin-2 is a pleiotropic cytokine that mediates both pro- and anti-inflammatory functions. Immune cells naturally differ in their sensitivity to IL-2 due to cell type and activation state-dependent expression of receptors and signaling pathway components. To probe differences in IL-2 signaling across cell types, we used structure-based design to create and profile a series of IL-2 variants with the capacity to titrate maximum signal strength in fine increments. One of these partial agonists, IL-2-REH, specifically expanded Foxp3+ regulatory T cells with reduced activity on CD8+ T cells due to cell type-intrinsic differences in IL-2 signaling. IL-2-REH elicited cell type-dependent differences in gene expression and provided mixed therapeutic results: showing benefit in the in vivo mouse dextran sulfate sodium (DSS) model of colitis, but no therapeutic efficacy in a transfer colitis model. Our findings show that cytokine partial agonists can be used to calibrate intrinsic differences in response thresholds across responding cell types to narrow pleiotropic actions, which may be generalizable to other cytokine and growth factor systems.

    View details for DOI 10.7554/eLife.65777

    View details for PubMedID 34003116

  • Selective targeting of ligand-dependent and -independent signaling by GPCR conformation-specific anti-US28 intrabodies. Nature communications De Groof, T. W., Bergkamp, N. D., Heukers, R., Giap, T., Bebelman, M. P., Goeij-de Haas, R., Piersma, S. R., Jimenez, C. R., Garcia, K. C., Ploegh, H. L., Siderius, M., Smit, M. J. 2021; 12 (1): 4357


    While various GPCRs, including US28, display constitutive, ligand-independent activity, it remains to be established whether ligand-dependent and -independent active conformations differ and can be selectively modulated. Previously, the agonist-bound conformation of US28 was stabilized and its structure was solved using the anti-US28 nanobody Nb7. Here we report the recognition of the constitutively active, apo-conformation of US28 by another nanobody VUN103. While the Nb7 intrabody selectively inhibits ligand-induced signaling, the VUN103 intrabody blocks constitutive signaling, indicating the existence of distinct US28 conformational states. By displacing Galphaq protein, VUN103 prevents US28 signaling and reduces tumor spheroids growth. Overall, nanobodies specific for distinct GPCR conformational states, i.e. apo- and agonist-bound, can selectively target and discern functional consequences of ligand-dependent versus independent signaling.

    View details for DOI 10.1038/s41467-021-24574-y

    View details for PubMedID 34272386

  • Structural basis for IL-12 and IL-23 receptor sharing reveals a gateway for shaping actions on T versus NK cells. Cell Glassman, C. R., Mathiharan, Y. K., Jude, K. M., Su, L. n., Panova, O. n., Lupardus, P. J., Spangler, J. B., Ely, L. K., Thomas, C. n., Skiniotis, G. n., Garcia, K. C. 2021; 184 (4): 983–99.e24


    Interleukin-12 (IL-12) and IL-23 are heterodimeric cytokines that are produced by antigen-presenting cells to regulate the activation and differentiation of lymphocytes, and they share IL-12Rβ1 as a receptor signaling subunit. We present a crystal structure of the quaternary IL-23 (IL-23p19/p40)/IL-23R/IL-12Rβ1 complex, together with cryoelectron microscopy (cryo-EM) maps of the complete IL-12 (IL-12p35/p40)/IL-12Rβ2/IL-12Rβ1 and IL-23 receptor (IL-23R) complexes, which reveal "non-canonical" topologies where IL-12Rβ1 directly engages the common p40 subunit. We targeted the shared IL-12Rβ1/p40 interface to design a panel of IL-12 partial agonists that preserved interferon gamma (IFNγ) induction by CD8+ T cells but impaired cytokine production from natural killer (NK) cells in vitro. These cell-biased properties were recapitulated in vivo, where IL-12 partial agonists elicited anti-tumor immunity to MC-38 murine adenocarcinoma absent the NK-cell-mediated toxicity seen with wild-type IL-12. Thus, the structural mechanism of receptor sharing used by IL-12 family cytokines provides a protein interface blueprint for tuning this cytokine axis for therapeutics.

    View details for DOI 10.1016/j.cell.2021.01.018

    View details for PubMedID 33606986

  • Cryo-EM structure of the IL-10 receptor complex provides a blueprint for ligand engineering. The FEBS journal Saxton, R. A., Garcia, K. C. 2021


    Interleukin-10 (IL-10) is an immunomodulatory cytokine that plays important roles in terminating inflammatory responses and preventing tissue damage resulting from autoimmunity. Although these anti-inflammatory actions have led to considerable clinical interest, efforts to exploit IL-10 therapeutically have been hindered by the highly pleiotropic nature of IL-10 and its ability to elicit pro-inflammatory effects in vivo. In this structural snapshot, we review the recent cryo-EM structure of the IL-10 receptor signaling complex, highlighting its unique structural features, insights into the mechanism of receptor sharing by the IL-10 cytokine family, and the implications for manipulating IL-10 signaling therapeutically.

    View details for DOI 10.1111/febs.16207

    View details for PubMedID 34543517

  • Structural basis for the constitutive activity and immunomodulatory properties of the Epstein-Barr virus-encoded G protein-coupled receptor BILF1. Immunity Tsutsumi, N., Qu, Q., Mavri, M., Baggesen, M. S., Maeda, S., Waghray, D., Berg, C., Kobilka, B. K., Rosenkilde, M. M., Skiniotis, G., Garcia, K. C. 2021


    Epstein-Barr virus (EBV) encodes a G protein-coupled receptor (GPCR) termed BILF1 that is essential for EBV-mediated immunosuppression and oncogenesis. BILF1 couples with inhibitory G protein (Gi), the major intracellular signaling effector for human chemokine receptors, and exhibits constitutive signaling activity; the ligand(s) for BILF1 are unknown. We studied the origins of BILF1's constitutive activity through structure determination of BILF1 bound to the inhibitory G protein (Gi) heterotrimer. The 3.2-Å resolution cryo-electron microscopy structure revealed an extracellular loop within BILF1 that blocked the typical chemokine binding site, suggesting ligand-autonomous receptor activation. Rather, amino acid substitutions within BILF1 transmembrane regions at hallmark ligand-activated class A GPCR "microswitches" stabilized a constitutively active BILF1 conformation for Gi coupling in a ligand-independent fashion. Thus, the constitutive activity of BILF1 promotes immunosuppression and virulence independent of ligand availability, with implications for the function of GPCRs encoded by related viruses and for therapeutic targeting of EBV.

    View details for DOI 10.1016/j.immuni.2021.06.001

    View details for PubMedID 34216564

  • Structure-based decoupling of the pro- and anti-inflammatory functions of interleukin-10. Science (New York, N.Y.) Saxton, R. A., Tsutsumi, N., Su, L. L., Abhiraman, G. C., Mohan, K., Henneberg, L. T., Aduri, N. G., Gati, C., Garcia, K. C. 2021; 371 (6535)


    Interleukin-10 (IL-10) is an immunoregulatory cytokine with both anti-inflammatory and immunostimulatory properties and is frequently dysregulated in disease. We used a structure-based approach to deconvolute IL-10 pleiotropy by determining the structure of the IL-10 receptor (IL-10R) complex by cryo-electron microscopy at a resolution of 3.5 angstroms. The hexameric structure shows how IL-10 and IL-10Ralpha form a composite surface to engage the shared signaling receptor IL-10Rbeta, enabling the design of partial agonists. IL-10 variants with a range of IL-10Rbeta binding strengths uncovered substantial differences in response thresholds across immune cell populations, providing a means of manipulating IL-10 cell type selectivity. Some variants displayed myeloid-biased activity by suppressing macrophage activation without stimulating inflammatory CD8+ T cells, thereby uncoupling the major opposing functions of IL-10. These results provide a mechanistic blueprint for tuning the pleiotropic actions of IL-10.

    View details for DOI 10.1126/science.abc8433

    View details for PubMedID 33737461

  • Tuning MPL signaling to influence hematopoietic stem cell differentiation and inhibit essential thrombocythemia progenitors Proceedings of the National Academy of Sciences Wernig, G. 2021; 118 (2) (Jan 2021)

    View details for DOI 10.1073/pnas.2017849118

  • Progenitor identification and SARS-CoV-2 infection in human distal lung organoids. Nature Salahudeen, A. A., Choi, S. S., Rustagi, A., Zhu, J., van Unen, V., de la O, S. M., Flynn, R. A., Margalef-Catala, M., Santos, A. J., Ju, J., Batish, A., Usui, T., Zheng, G. X., Edwards, C. E., Wagar, L. E., Luca, V., Anchang, B., Nagendran, M., Nguyen, K., Hart, D. J., Terry, J. M., Belgrader, P., Ziraldo, S. B., Mikkelsen, T. S., Harbury, P. B., Glenn, J. S., Garcia, K. C., Davis, M. M., Baric, R. S., Sabatti, C., Amieva, M. R., Blish, C. A., Desai, T. J., Kuo, C. J. 2020


    The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Three-dimensional in vitro human distal lung culture systems would strongly facilitate investigation of pathologies including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. We generated long-term feeder-free, chemically defined culture of distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids exhibited AT1 transdifferentiation potential while basal cell organoids developed lumens lined by differentiated club and ciliated cells. Single cell analysis of basal organoid KRT5+ cells revealed a distinct ITGA6+ITGB4+ mitotic population whose proliferation further segregated to a TNFRSF12Ahi subfraction comprising ~10% of KRT5+ basal cells, residing in clusters within terminal bronchioles and exhibiting enriched clonogenic organoid growth activity. Distal lung organoids were created with apical-out polarity to display ACE2 on the exposed external surface, facilitating SARS-CoV-2 infection of AT2 and basal cultures and identifying club cells as a novel target population. This long-term, feeder-free organoid culture of human distal lung, coupled with single cell analysis, identifies unsuspected basal cell functional heterogeneity and establishes a facile in vitro organoid model for human distal lung infections including COVID-19-associated pneumonia.

    View details for DOI 10.1038/s41586-020-3014-1

    View details for PubMedID 33238290

  • A Human IgSF Cell-Surface Interactome Reveals a Complex Network of Protein-Protein Interactions. Cell Wojtowicz, W. M., Vielmetter, J., Fernandes, R. A., Siepe, D. H., Eastman, C. L., Chisholm, G. B., Cox, S., Klock, H., Anderson, P. W., Rue, S. M., Miller, J. J., Glaser, S. M., Bragstad, M. L., Vance, J., Lam, A. W., Lesley, S. A., Zinn, K., Garcia, K. C. 2020; 182 (4): 1027


    Cell-surface protein-protein interactions (PPIs) mediate cell-cell communication, recognition, and responses. We executed an interactome screen of 564 human cell-surface and secreted proteins, most of which are immunoglobulin superfamily (IgSF) proteins, using a high-throughput, automated ELISA-based screening platform employing a pooled-protein strategy to test all 318,096 PPI combinations. Screen results, augmented by phylogenetic homology analysis, revealed 380 previously unreported PPIs. We validated a subset using surface plasmon resonance and cell binding assays. Observed PPIs reveal a large and complex network of interactions both within and across biological systems. We identified new PPIs for receptors with well-characterized ligands and binding partners for "orphan" receptors. New PPIs include proteins expressed on multiple cell types and involved in diverse processes including immune and nervous system development and function, differentiation/proliferation, metabolism, vascularization, and reproduction. These PPIs provide a resource for further biological investigation into their functional relevance and may offer new therapeutic drug targets.

    View details for DOI 10.1016/j.cell.2020.07.025

    View details for PubMedID 32822567

  • Discovery of surrogate agonists for visceral fat Treg cells that modulate metabolic indices in vivo. eLife Fernandes, R. A., Li, C., Wang, G., Yang, X., Savvides, C. S., Glassman, C. R., Dong, S., Luxenberg, E., Sibener, L. V., Birnbaum, M. E., Benoist, C., Mathis, D., Garcia, K. C. 2020; 9


    T regulatory (Treg) cells play vital roles in modulating immunity and tissue homeostasis. Their actions depend on TCR recognition of peptide-MHC molecules; yet the degree of peptide specificity of Treg-cell function, and whether Treg ligands can be used to manipulate Treg cell biology are unknown. Here, we developed an Ab-peptide library that enabled unbiased screening of peptides recognized by a bona fide murine Treg cell clone isolated from the visceral adipose tissue (VAT), and identified surrogate agonist peptides, with differing affinities and signaling potencies. The VAT-Treg cells expanded in vivo by one of the surrogate agonists preserved the typical VAT-Treg transcriptional programs. Immunization with this surrogate, especially when coupled with blockade of TNFa signaling, expanded VAT-Treg cells, resulting in protection from inflammation and improved metabolic indices, including promotion of insulin sensitivity. These studies suggest that antigen-specific targeting of VAT-localized Treg cells could eventually be a strategy for improving metabolic disease.

    View details for DOI 10.7554/eLife.58463

    View details for PubMedID 32773038

  • Interrogating the recognition landscape of a conserved HIV-specific TCR reveals distinct bacterial peptide cross-reactivity. eLife Mendoza, J. L., Fischer, S., Gee, M. H., Lam, L. H., Brackenridge, S., Powrie, F. M., Birnbaum, M., McMichael, A. J., Garcia, K. C., Gillespie, G. M. 2020; 9


    T cell cross-reactivity ensures that diverse pathogen-derived epitopes encountered during a lifetime are recognized by the available TCR repertoire. A feature of cross-reactivity where previous exposure to one microbe can alter immunity to subsequent, non-related pathogens has been mainly explored for viruses. Yet cross-reactivity to additional microbes is important to consider, especially in HIV infection where gut-intestinal barrier dysfunction could facilitate T cell exposure to commensal/pathogenic microbes. Here we evaluated the cross-reactivity of a 'public', HIV-specific, CD8 T cell-derived TCR (AGA1 TCR) using MHC class I yeast display technology. Via screening of MHC-restricted libraries comprising ~2*108 sequence-diverse peptides, AGA1 TCR specificity was mapped to a central peptide di-motif. Using the top TCR-enriched library peptides to probe the non-redundant protein database, bacterial peptides that elicited functional responses by AGA1-expressing T cells were identified. The possibility that in context-specific settings, MHC class I proteins presenting microbial peptides influence virus-specific T cell populations in vivo is discussed.

    View details for DOI 10.7554/eLife.58128

    View details for PubMedID 32716298

  • Discovery of a novel shared tumor antigen in human lung cancer. Tseng, D., Chiou, S., Yang, X., Reuben, A., Wilhelmy, J., McSween, A., Conley, S., Sinha, R., Nabet, B., Wang, C., Shrager, J. B., Berry, M. F., Backhus, L., Lui, n., Wakelee, H. A., Neal, J. W., Zhang, J., Garcia, K., Mackall, C., Davis, M. AMER SOC CLINICAL ONCOLOGY. 2020
  • The Power Of Single Cell Technologies; From T Cell Receptor To Antigen(s) In Multiple Sclerosis Saligrama, N., Fernandes, R. A., Pai, J., Yao, W., Louis, D. M., Oksenberg, J., Kipp, L., Dunn, J. E., Satpathy, A., Garcia, K. C., Davis, M. M. SAGE PUBLICATIONS LTD. 2020: 5–6
  • An engineered IL-2 partial agonist promoted CD8+T cell stemness and anti-tumor efficacy Mo, F., Yu, Z., Li, P., Oh, J., Spolski, R., Zhao, L., Glassman, C. R., Yamamoto, T. N., Chen, Y., Golebiowski, F. M., Hermans, D., Marjri, S. S., Picton, L., Liao, W., Ren, M., Zhuang, X., Mitra, S., Lin, J., Gattinoni, L., Powell, J. D., Restifo, N. P., Garcia, K., Leonard, W. J. AMER ASSOC IMMUNOLOGISTS. 2020
  • Combination therapy to enhance NK cell anti-tumor responses Wolf, N. K., Nicolai, C., Dang, S., Synder, G., Blaj, C., McWhirter, S., Picton, L., Garcia, K., Raulet, D. H. AMER ASSOC IMMUNOLOGISTS. 2020
  • Orthogonal IL-2 Cytokine-Receptor Pair Capable of Selective Expansion of Regulatory T Cells Facilitates Alloengraftment Hirai, T., Simonetta, F., Su, L. L., Picton, L., Baker, J., Lin, J., Li, P., Leonard, W. J., Garcia, K. C., Negrin, R. S. WILEY. 2020: 331
  • Interleukin-2 druggability is modulated by global conformational transitions controlled by a helical capping switch. Proceedings of the National Academy of Sciences of the United States of America De Paula, V. S., Jude, K. M., Nerli, S., Glassman, C. R., Garcia, K. C., Sgourakis, N. G. 2020


    Interleukin-2 (IL-2) is a small alpha-helical cytokine that regulates immune cell homeostasis through its recruitment to a high-affinity heterotrimeric receptor complex (IL-2Ralpha/IL-2Rbeta/gammac). IL-2 has been shown to have therapeutic efficacy for immune diseases by preferentially expanding distinct T cell compartments, and several regulatory T cell (Treg)-biasing anti-IL-2 antibodies have been developed for combination therapies. The conformational plasticity of IL-2 plays an important role in its biological actions by modulating the strength of receptor and drug interactions. Through an NMR analysis of milliseconds-timescale dynamics of free mouse IL-2 (mIL-2), we identify a global transition to a sparse conformation which is regulated by an alpha-helical capping "switch" at the loop between the A and B helices (AB loop). Binding to either an anti-mouse IL-2 monoclonal antibody (mAb) or a small molecule inhibitor near the loop induces a measurable response at the core of the structure, while locking the switch to a single conformation through a designed point mutation leads to a global quenching of core dynamics accompanied by a pronounced effect in mAb binding. By elucidating key details of the long-range allosteric communication between the receptor binding surfaces and the core of the IL-2 structure, our results offer a direct blueprint for designing precision therapeutics targeting a continuum of conformational states.

    View details for DOI 10.1073/pnas.2000419117

    View details for PubMedID 32184322

  • Towards the identification of novel tumor antigens in human lung cancer. Chiou, S., Tseng, D., Wang, C., Reuben, A., Yang, X., Wilhelmy, J., McSween, A., Zhang, J., Shrager, J., Garcia, K., Davis, M. AMER ASSOC CANCER RESEARCH. 2020: 44–45
  • Engineered IL-2 Cytokine-Cytokine Receptor Complex Enables Selective Expansion of Regulatory T Cells and Facilitates Establishment of Organ Transplantation Tolerance Hirai, T., Simonetta, F., Su, L. L., Picton, L., Baker, J., Seo, K., Lohmeyer, J., Mavers, M., Blazar, B. R., Garcia, C., Negrin, R. S. ELSEVIER SCIENCE INC. 2020: S59–S60
  • Interleukin-2 Druggability is Modulated by Global Conformational Transitions that are Controlled by a Helical Capping Switch De Paula, V., Jude, K. M., Nerli, S., Glassman, C. R., Garcia, K., Sgourakis, N. CELL PRESS. 2020: 51A
  • Immune receptor inhibition through enforced phosphatase recruitment. Nature Fernandes, R. A., Su, L. n., Nishiga, Y. n., Ren, J. n., Bhuiyan, A. M., Cheng, N. n., Kuo, C. J., Picton, L. K., Ohtsuki, S. n., Majzner, R. G., Rietberg, S. P., Mackall, C. L., Yin, Q. n., Ali, L. R., Yang, X. n., Savvides, C. S., Sage, J. n., Dougan, M. n., Garcia, K. C. 2020


    Antibodies that antagonize extracellular receptor-ligand interactions are used as therapeutic agents for many diseases to inhibit signalling by cell-surface receptors1. However, this approach does not directly prevent intracellular signalling, such as through tonic or sustained signalling after ligand engagement. Here we present an alternative approach for attenuating cell-surface receptor signalling, termed receptor inhibition by phosphatase recruitment (RIPR). This approach compels cis-ligation of cell-surface receptors containing ITAM, ITIM or ITSM tyrosine phosphorylation motifs to the promiscuous cell-surface phosphatase CD452,3, which results in the direct intracellular dephosphorylation of tyrosine residues on the receptor target. As an example, we found that tonic signalling by the programmed cell death-1 receptor (PD-1) results in residual suppression of T cell activation, but is not inhibited by ligand-antagonist antibodies. We engineered a PD-1 molecule, which we denote RIPR-PD1, that induces cross-linking of PD-1 to CD45 and inhibits both tonic and ligand-activated signalling. RIPR-PD1 demonstrated enhanced inhibition of checkpoint blockade compared with ligand blocking by anti-PD1 antibodies, and increased therapeutic efficacy over anti-PD1 in mouse tumour models. We also show that the RIPR strategy extends to other immune-receptor targets that contain activating or inhibitory ITIM, ITSM or ITAM motifs; for example, inhibition of the macrophage SIRPα 'don't eat me' signal with a SIRPα-CD45 RIPR molecule potentiates antibody-dependent cellular phagocytosis beyond that of SIRPα blockade alone. RIPR represents a general strategy for direct attenuation of signalling by kinase-activated cell-surface receptors.

    View details for DOI 10.1038/s41586-020-2851-2

    View details for PubMedID 33087934

  • Structure of human Frizzled5 by fiducial-assisted cryo-EM supports a heterodimeric mechanism of canonical Wnt signaling. eLife Tsutsumi, N. n., Mukherjee, S. n., Waghray, D. n., Janda, C. Y., Jude, K. M., Miao, Y. n., Burg, J. S., Aduri, N. G., Kossiakoff, A. A., Gati, C. n., Garcia, K. C. 2020; 9


    Frizzleds (Fzd) are the primary receptors for Wnt morphogens, which are essential regulators of stem cell biology, yet the structural basis of Wnt signaling through Fzd remains poorly understood. Here we report the structure of an unliganded human Fzd5 determined by single-particle cryo-EM at 3.7 Å resolution, with the aid of an antibody chaperone acting as a fiducial marker. We also analyzed the topology of low-resolution XWnt8/Fzd5 complex particles, which revealed extreme flexibility between the Wnt/Fzd-CRD and the Fzd-TM regions. Analysis of Wnt/β-catenin signaling in response to Wnt3a versus a 'surrogate agonist' that cross-links Fzd to LRP6, revealed identical structure-activity relationships. Thus, canonical Wnt/β-catenin signaling appears to be principally reliant on ligand-induced Fzd/LRP6 heterodimerization, versus the allosteric mechanisms seen in structurally analogous class A G protein-coupled receptors, and Smoothened. These findings deepen our mechanistic understanding of Wnt signal transduction, and have implications for harnessing Wnt agonism in regenerative medicine.

    View details for DOI 10.7554/eLife.58464

    View details for PubMedID 32762848

  • Next-Generation Surrogate Wnts Support Organoid Growth and Deconvolute Frizzled Pleiotropy In Vivo. Cell stem cell Miao, Y. n., Ha, A. n., de Lau, W. n., Yuki, K. n., Santos, A. J., You, C. n., Geurts, M. H., Puschhof, J. n., Pleguezuelos-Manzano, C. n., Peng, W. C., Senlice, R. n., Piani, C. n., Buikema, J. W., Gbenedio, O. M., Vallon, M. n., Yuan, J. n., de Haan, S. n., Hemrika, W. n., Rösch, K. n., Dang, L. T., Baker, D. n., Ott, M. n., Depeille, P. n., Wu, S. M., Drost, J. n., Nusse, R. n., Roose, J. P., Piehler, J. n., Boj, S. F., Janda, C. Y., Clevers, H. n., Kuo, C. J., Garcia, K. C. 2020


    Modulation of Wnt signaling has untapped potential in regenerative medicine due to its essential functions in stem cell homeostasis. However, Wnt lipidation and Wnt-Frizzled (Fzd) cross-reactivity have hindered translational Wnt applications. Here, we designed and engineered water-soluble, Fzd subtype-specific "next-generation surrogate" (NGS) Wnts that hetero-dimerize Fzd and Lrp6. NGS Wnt supports long-term expansion of multiple different types of organoids, including kidney, colon, hepatocyte, ovarian, and breast. NGS Wnts are superior to Wnt3a conditioned media in organoid expansion and single-cell organoid outgrowth. Administration of Fzd subtype-specific NGS Wnt in vivo reveals that adult intestinal crypt proliferation can be promoted by agonism of Fzd5 and/or Fzd8 receptors, while a broad spectrum of Fzd receptors can induce liver zonation. Thus, NGS Wnts offer a unified organoid expansion protocol and a laboratory "tool kit" for dissecting the functions of Fzd subtypes in stem cell biology.

    View details for DOI 10.1016/j.stem.2020.07.020

    View details for PubMedID 32818433

  • Mutational signature in colorectal cancer caused by genotoxic pks+ E. coli. Nature Pleguezuelos-Manzano, C. n., Puschhof, J. n., Huber, A. R., van Hoeck, A. n., Wood, H. M., Nomburg, J. n., Gurjao, C. n., Manders, F. n., Dalmasso, G. n., Stege, P. B., Paganelli, F. L., Geurts, M. H., Beumer, J. n., Mizutani, T. n., Miao, Y. n., van der Linden, R. n., van Elst, S. n., Garcia, K. C., Top, J. n., Willems, R. J., Giannakis, M. n., Bonnet, R. n., Quirke, P. n., Meyerson, M. n., Cuppen, E. n., van Boxtel, R. n., Clevers, H. n. 2020


    Various species of the intestinal microbiota have been associated with the development of colorectal cancer (CRC)1,2, but it has not been demonstrated that bacteria have a direct role in the occurrence of oncogenic mutations. Escherichia coli can carry the pathogenicity island pks, which encodes a set of enzymes that synthesize colibactin3. This compound is believed to alkylate DNA on adenine residues4,5 and induces double-strand breaks in cultured cells3. Here we expose human intestinal organoids to genotoxic pks+ E. coli by repeated luminal injection over five months. Whole-genome sequencing of clonal organoids before and after this exposure revealed a distinct mutational signature that was absent from organoids injected with isogenic pks-mutant bacteria. The same mutational signature was detected in a subset of 5,876 human cancer genomes from two independent cohorts, predominantly in CRC. Our study describes a distinct mutational signature in CRC and implies that the underlying mutational process results directly from past exposure to bacteria carrying the colibactin-producing pks pathogenicity island.

    View details for DOI 10.1038/s41586-020-2080-8

    View details for PubMedID 32106218

  • Wnt Activation and Reduced Cell-Cell Contact Synergistically Induce Massive Expansion of Functional Human iPSC-Derived Cardiomyocytes. Cell stem cell Buikema, J. W., Lee, S. n., Goodyer, W. R., Maas, R. G., Chirikian, O. n., Li, G. n., Miao, Y. n., Paige, S. L., Lee, D. n., Wu, H. n., Paik, D. T., Rhee, S. n., Tian, L. n., Galdos, F. X., Puluca, N. n., Beyersdorf, B. n., Hu, J. n., Beck, A. n., Venkamatran, S. n., Swami, S. n., Wijnker, P. n., Schuldt, M. n., Dorsch, L. M., van Mil, A. n., Red-Horse, K. n., Wu, J. Y., Geisen, C. n., Hesse, M. n., Serpooshan, V. n., Jovinge, S. n., Fleischmann, B. K., Doevendans, P. A., van der Velden, J. n., Garcia, K. C., Wu, J. C., Sluijter, J. P., Wu, S. M. 2020; 27 (1): 50–63.e5


    Modulating signaling pathways including Wnt and Hippo can induce cardiomyocyte proliferation in vivo. Applying these signaling modulators to human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro can expand CMs modestly (<5-fold). Here, we demonstrate massive expansion of hiPSC-CMs in vitro (i.e., 100- to 250-fold) by glycogen synthase kinase-3β (GSK-3β) inhibition using CHIR99021 and concurrent removal of cell-cell contact. We show that GSK-3β inhibition suppresses CM maturation, while contact removal prevents CMs from cell cycle exit. Remarkably, contact removal enabled 10 to 25 times greater expansion beyond GSK-3β inhibition alone. Mechanistically, persistent CM proliferation required both LEF/TCF activity and AKT phosphorylation but was independent from yes-associated protein (YAP) signaling. Engineered heart tissues from expanded hiPSC-CMs showed comparable contractility to those from unexpanded hiPSC-CMs, demonstrating uncompromised cellular functionality after expansion. In summary, we uncovered a molecular interplay that enables massive hiPSC-CM expansion for large-scale drug screening and tissue engineering applications.

    View details for DOI 10.1016/j.stem.2020.06.001

    View details for PubMedID 32619518

  • Surrogate R-spondins for tissue-specific potentiation of Wnt Signaling. PloS one Luca, V. C., Miao, Y. n., Li, X. n., Hollander, M. J., Kuo, C. J., Garcia, K. C. 2020; 15 (1): e0226928


    Secreted R-spondin1-4 proteins (RSPO1-4) orchestrate stem cell renewal and tissue homeostasis by potentiating Wnt/β-catenin signaling. RSPOs induce the turnover of negative Wnt regulators RNF43 and ZNRF3 through a process that requires RSPO interactions with Leucine-rich repeat-containing G-protein coupled receptors (LGRs), or through an LGR-independent mechanism that is enhanced by RSPO binding to heparin sulfate proteoglycans (HSPGs). Here, we describe the engineering of 'surrogate RSPOs' that function independently of LGRs to potentiate Wnt signaling on cell types expressing a target surface marker. These bispecific proteins were generated by fusing an RNF43- or ZNRF3-specific single chain antibody variable fragment (scFv) to the immune cytokine IL-2. Surrogate RSPOs mimic the function of natural RSPOs by crosslinking the extracellular domain (ECD) of RNF43 or ZNRF3 to the ECD of the IL-2 receptor CD25, which sequesters the complex and results in highly selective amplification of Wnt signaling on CD25+ cells. Furthermore, surrogate RSPOs were able substitute for wild type RSPO in a colon organoid growth assay when intestinal stem cells were transduced to express CD25. Our results provide proof-of-concept for a technology that may be adapted for use on a broad range of cell- or tissue-types and will open new avenues for the development of Wnt-based therapeutics for regenerative medicine.

    View details for DOI 10.1371/journal.pone.0226928

    View details for PubMedID 31914456

  • Structure and selectivity engineering of the M1 muscarinic receptor toxin complex. Science (New York, N.Y.) Maeda, S. n., Xu, J. n., N Kadji, F. M., Clark, M. J., Zhao, J. n., Tsutsumi, N. n., Aoki, J. n., Sunahara, R. K., Inoue, A. n., Garcia, K. C., Kobilka, B. K. 2020; 369 (6500): 161–67


    Muscarinic toxins (MTs) are natural toxins produced by mamba snakes that primarily bind to muscarinic acetylcholine receptors (MAChRs) and modulate their function. Despite their similar primary and tertiary structures, MTs show distinct binding selectivity toward different MAChRs. The molecular details of how MTs distinguish MAChRs are not well understood. Here, we present the crystal structure of M1AChR in complex with MT7, a subtype-selective anti-M1AChR snake venom toxin. The structure reveals the molecular basis of the extreme subtype specificity of MT7 for M1AChR and the mechanism by which it regulates receptor function. Through in vitro engineering of MT7 finger regions that was guided by the structure, we have converted the selectivity from M1AChR toward M2AChR, suggesting that the three-finger fold is a promising scaffold for developing G protein-coupled receptor modulators.

    View details for DOI 10.1126/science.aax2517

    View details for PubMedID 32646996

  • Mechanism of homodimeric cytokine receptor activation and dysregulation by oncogenic mutations. Science (New York, N.Y.) Wilmes, S. n., Hafer, M. n., Vuorio, J. n., Tucker, J. A., Winkelmann, H. n., Löchte, S. n., Stanly, T. A., Pulgar Prieto, K. D., Poojari, C. n., Sharma, V. n., Richter, C. P., Kurre, R. n., Hubbard, S. R., Garcia, K. C., Moraga, I. n., Vattulainen, I. n., Hitchcock, I. S., Piehler, J. n. 2020; 367 (6478): 643–52


    Homodimeric class I cytokine receptors are assumed to exist as preformed dimers that are activated by ligand-induced conformational changes. We quantified the dimerization of three prototypic class I cytokine receptors in the plasma membrane of living cells by single-molecule fluorescence microscopy. Spatial and spatiotemporal correlation of individual receptor subunits showed ligand-induced dimerization and revealed that the associated Janus kinase 2 (JAK2) dimerizes through its pseudokinase domain. Oncogenic receptor and hyperactive JAK2 mutants promoted ligand-independent dimerization, highlighting the formation of receptor dimers as the switch responsible for signal activation. Atomistic modeling and molecular dynamics simulations based on a detailed energetic analysis of the interactions involved in dimerization yielded a mechanistic blueprint for homodimeric class I cytokine receptor activation and its dysregulation by individual mutations.

    View details for DOI 10.1126/science.aaw3242

    View details for PubMedID 32029621

  • The combination of a STING agonist with cytokines results in robust anti-tumor effects in autochthonous tumor models Blaj, C., Li, Y., Chen, A., Descien, A., Francica, B., McWhirter, S., Picton, L., Garcia, K., Raulet, D. BMC. 2019
  • In vivo molecular imaging for immunotherapy using ultra-bright near-infrared-IIb rare-earth nanoparticles. Nature biotechnology Zhong, Y., Ma, Z., Wang, F., Wang, X., Yang, Y., Liu, Y., Zhao, X., Li, J., Du, H., Zhang, M., Cui, Q., Zhu, S., Sun, Q., Wan, H., Tian, Y., Liu, Q., Wang, W., Garcia, K. C., Dai, H. 2019


    The near-infrared-IIb (NIR-IIb) (1,500-1,700nm) window is ideal for deep-tissue optical imaging in mammals, but lacks bright and biocompatible probes. Here, we developed biocompatible cubic-phase (alpha-phase) erbium-based rare-earth nanoparticles (ErNPs) exhibiting bright downconversion luminescence at ~1,600nm for dynamic imaging of cancer immunotherapy in mice. We used ErNPs functionalized with cross-linked hydrophilic polymer layers attached to anti-PD-L1 (programmed cell death-1 ligand-1) antibody for molecular imaging of PD-L1 in a mouse model of colon cancer and achieved tumor-to-normal tissue signal ratios of ~40. The long luminescence lifetime of ErNPs (~4.6ms) enabled simultaneous imaging of ErNPs and lead sulfide quantum dots emitting in the same ~1,600nm window. In vivo NIR-IIb molecular imaging of PD-L1 and CD8 revealed cytotoxic T lymphocytes in the tumor microenvironment in response to immunotherapy, and altered CD8 signals in tumor and spleen due to immune activation. The cross-linked functionalization layer facilitated 90% ErNP excretion within 2weeks without detectable toxicity in mice.

    View details for DOI 10.1038/s41587-019-0262-4

    View details for PubMedID 31570897

  • Dual Arms of Adaptive Immunity: Division of Labor and Collaboration between B and T Cells. Cell Garcia, K. C. 2019


    This year's Lasker Basic Medical Research Award honors Max Cooper and Jacques Miller for discoveries that revealed the organizing principles of adaptive immunity. Their collective contributions have had broad clinical impact in the treatment of immune disease.

    View details for DOI 10.1016/j.cell.2019.08.022

    View details for PubMedID 31519306

  • A strategy for the selection of monovalent antibodies that span protein dimer interfaces. The Journal of biological chemistry Spangler, J. B., Moraga, I., Jude, K. M., Savvides, C. S., Garcia, K. C. 2019


    Ligand-induced dimerization is the predominant mechanism through which secreted proteins activate cell surface receptors to transmit essential biological signals. Cytokines are a large class of soluble proteins that dimerize transmembrane receptors into precise signaling topologies, but there is a need for alternative, engineerable ligand scaffolds that specifically recognize and stabilize these protein interactions. Recombinant antibodies can potentially serve as robust and versatile platforms for cytokine complex stabilization, and their specificity allows for tunable modulation of dimerization equilibrium. Here, we devised an evolutionary strategy to isolate monovalent antibody agonists that bridge together two different receptor subunits in a cytokine-receptor complex, precisely as the receptors are disposed in their natural signaling orientations. To do this, we screened a naive antibody library against a stabilized ligand/receptor ternary complex that acted as a 'molecular cast' of the natural receptor dimer conformation. Our selections elicited 'stapler' single-chain variable fragments (scFvs) of antibodies that specifically engage the interleukin-4 receptor heterodimer. The 3.1 A resolution crystal structure of one such stapler revealed that, as intended, this scFv recognizes a composite epitope between the two receptors as they are positioned in the complex. Extending our approach, we evolved a stapler scFv that specifically binds to and stabilizes the interface between the interleukin-2 cytokine and one of its receptor subunits, leading to a 15-fold enhancement in interaction affinity. This demonstration that scFvs can be selected to recognize epitopes that span protein interfaces presents new opportunities to engineer structurally defined antibodies for a broad range of research and therapeutic applications.

    View details for DOI 10.1074/jbc.RA119.009213

    View details for PubMedID 31387945

  • Spatiotemporal Dynamic of Assembly and Activation of Class II Cytokine Receptors Belton, J., Wilmes, S., Richter, C. P., Mendoza, J. L., Moraga, I., Garcia, K. C., Walter, M. R., Piehler, J. SPRINGER. 2019: S187
  • Receptor subtype discrimination using extensive shape complementary designed interfaces NATURE STRUCTURAL & MOLECULAR BIOLOGY Dang, L. T., Miao, Y., Ha, A., Yuki, K., Park, K., Janda, C. Y., Jude, K. M., Mohan, K., Ha, N., Vallon, M., Yuan, J., Vilches-Moure, J. G., Kuo, C. J., Garcia, K., Baker, D. 2019; 26 (6): 407-+
  • Topological control of cytokine receptor signaling induces differential effects in hematopoiesis. Science (New York, N.Y.) Mohan, K., Ueda, G., Kim, A. R., Jude, K. M., Fallas, J. A., Guo, Y., Hafer, M., Miao, Y., Saxton, R. A., Piehler, J., Sankaran, V. G., Baker, D., Garcia, K. C. 2019; 364 (6442)


    Although tunable signaling by G protein-coupled receptors can be exploited through medicinal chemistry, a comparable pharmacological approach has been lacking for the modulation of signaling through dimeric receptors, such as those for cytokines. We present a strategy to modulate cytokine receptor signaling output by use of a series of designed C2-symmetric cytokine mimetics, based on the designed ankyrin repeat protein (DARPin) scaffold, that can systematically control erythropoietin receptor (EpoR) dimerization orientation and distance between monomers. We sampled a range of EpoR geometries by varying intermonomer angle and distance, corroborated by several ligand-EpoR complex crystal structures. Across the range, we observed full, partial, and biased agonism as well as stage-selective effects on hematopoiesis. This surrogate ligand strategy opens access to pharmacological modulation of therapeutically important cytokine and growth factor receptor systems.

    View details for DOI 10.1126/science.aav7532

    View details for PubMedID 31123111

  • Receptor subtype discrimination using extensive shape complementary designed interfaces. Nature structural & molecular biology Dang, L. T., Miao, Y., Ha, A., Yuki, K., Park, K., Janda, C. Y., Jude, K. M., Mohan, K., Ha, N., Vallon, M., Yuan, J., Vilches-Moure, J. G., Kuo, C. J., Garcia, K. C., Baker, D. 2019


    To discriminate between closely related members of a protein family that differ at a limited number of spatially distant positions is a challenge for drug discovery. We describe a combined computational design and experimental selection approach for generating binders targeting functional sites with large, shape complementary interfaces to read out subtle sequence differences for subtype-specific antagonism. Repeat proteins are computationally docked against a functionally relevant region of the target protein surface that varies in the different subtypes, and the interface sequences are optimized for affinity and specificity first computationally and then experimentally. We used this approach to generate a series of human Frizzled (Fz) subtype-selective antagonists with extensive shape complementary interaction surfaces considerably larger than those of repeat proteins selected from random libraries. In vivo administration revealed that Wnt-dependent pericentral liver gene expression involves multiple Fz subtypes, while maintenance of the intestinal crypt stem cell compartment involves only a limited subset.

    View details for PubMedID 31086346

  • Structure of the IFN gamma receptor complex guides design of biased agonists NATURE Mendoza, J. L., Escalante, N. K., Jude, K. M., Bellon, J., Su, L., Horton, T. M., Tsutsumi, N., Berardinelli, S. J., Haltiwanger, R. S., Piehler, J., Engleman, E. G., Garcia, K. 2019; 567 (7746): 56-+
  • Structure of the IFNgamma receptor complex guides design of biased agonists. Nature Mendoza, J. L., Escalante, N. K., Jude, K. M., Sotolongo Bellon, J., Su, L., Horton, T. M., Tsutsumi, N., Berardinelli, S. J., Haltiwanger, R. S., Piehler, J., Engleman, E. G., Garcia, K. C. 2019


    The cytokine interferon-gamma (IFNgamma) is a central coordinator of innate and adaptive immunity, but its highly pleiotropic actions have diminished its prospects for use as an immunotherapeutic agent. Here, we took a structure-based approach to decoupling IFNgamma pleiotropy. We engineered an affinity-enhanced variant of the ligand-binding chain of the IFNgamma receptor IFNgammaR1, which enabled us to determine the crystal structure of the complete hexameric (2:2:2) IFNgamma-IFNgammaR1-IFNgammaR2 signalling complex at 3.25A resolution. The structure reveals the mechanism underlying deficits in IFNgamma responsiveness in mycobacterial disease syndrome resulting from a T168N mutation in IFNgammaR2, which impairs assembly of the full signalling complex. The topology of the hexameric complex offers a blueprint for engineering IFNgamma variants to tune IFNgamma receptor signalling output. Unexpectedly, we found that several partial IFNgamma agonists exhibited biased gene-expression profiles. These biased agonists retained the ability to induce upregulation of major histocompatibility complex class I antigen expression, but exhibited impaired induction of programmed death-ligand 1 expression in a wide range of human cancer cell lines, offering a route to decoupling immunostimulatory and immunosuppressive functions of IFNgamma for therapeutic applications.

    View details for PubMedID 30814731

  • De novo design of potent and selective mimics of IL-2 and IL-15 NATURE Silva, D., Yu, S., Ulge, U. Y., Spangler, J. B., Jude, K. M., Labao-Almeida, C., Ali, L. R., Quijano-Rubio, A., Ruterbusch, M., Leung, I., Biary, T., Crowley, S. J., Marcos, E., Walkey, C. D., Weitzner, B. D., Pardo-Avila, F., Castellanos, J., Carter, L., Stewart, L., Riddell, S. R., Pepper, M., Bernardes, G. L., Dougan, M., Garcia, K., Baker, D. 2019; 565 (7738): 186-+
  • Trans-endocytosis of intact IL-15Rα-IL-15 complex from presenting cells into NK cells favors signaling for proliferation. Proceedings of the National Academy of Sciences of the United States of America Anton, O. M., Peterson, M. E., Hollander, M. J., Dorward, D. W., Arora, G. n., Traba, J. n., Rajagopalan, S. n., Snapp, E. L., Garcia, K. C., Waldmann, T. A., Long, E. O. 2019


    Interleukin 15 (IL-15) is an essential cytokine for the survival and proliferation of natural killer (NK) cells. IL-15 activates signaling by the β and common γ (γc) chain heterodimer of the IL-2 receptor through trans-presentation by cells expressing IL-15 bound to the α chain of the IL-15 receptor (IL-15Rα). We show here that membrane-associated IL-15Rα-IL-15 complexes are transferred from presenting cells to NK cells through trans-endocytosis and contribute to the phosphorylation of ribosomal protein S6 and NK cell proliferation. NK cell interaction with soluble or surface-bound IL-15Rα-IL-15 complex resulted in Stat5 phosphorylation and NK cell survival at a concentration or density of the complex much lower than required to stimulate S6 phosphorylation. Despite this efficient response, Stat5 phosphorylation was reduced after inhibition of metalloprotease-induced IL-15Rα-IL-15 shedding from trans-presenting cells, whereas S6 phosphorylation was unaffected. Conversely, inhibition of trans-endocytosis by silencing of the small GTPase TC21 or expression of a dominant-negative TC21 reduced S6 phosphorylation but not Stat5 phosphorylation. Thus, trans-endocytosis of membrane-associated IL-15Rα-IL-15 provides a mode of regulating NK cells that is not afforded to IL-2 and is distinct from activation by soluble IL-15. These results may explain the strict IL-15 dependence of NK cells and illustrate how the cellular compartment in which receptor-ligand interaction occurs can influence functional outcome.

    View details for DOI 10.1073/pnas.1911678117

    View details for PubMedID 31871169

  • RasGRP1 is a potential biomarker to stratify anti-EGFR therapy response in colorectal cancer. JCI insight Gbenedio, O. M., Bonnans, C. n., Grun, D. n., Wang, C. Y., Hatch, A. J., Mahoney, M. R., Barras, D. n., Matli, M. n., Miao, Y. n., Garcia, K. C., Tejpar, S. n., Delorenzi, M. n., Venook, A. P., Nixon, A. B., Warren, R. S., Roose, J. P., Depeille, P. n. 2019; 5


    Colorectal cancer (CRC) is the third most frequent neoplastic disorder and is a main cause of tumor-related mortality as many patients progress to stage IV metastatic CRC. Standard care consists of combination chemotherapy (FOLFIRI or FOLFOX). Patients with WT KRAS typing are eligible to receive anti-EGFR therapy combined with chemotherapy. Unfortunately, predicting efficacy of CRC anti-EGFR therapy has remained challenging. Here we uncover that the EGFR-pathway component RasGRP1 acts as CRC tumor suppressor in the context of aberrant Wnt signaling. We find that RasGRP1 suppresses EGF-driven proliferation of colonic epithelial organoids. Having established that RasGRP1 dosage levels impacts biology, we focused on CRC patients next. Mining five different data platforms, we establish that RasGRP1 expression levels decrease with CRC progression and predict poor clinical outcome of patients. Lastly, deletion of one or two Rasgrp1 alleles makes CRC spheroids more susceptible to EGFR inhibition. Retrospective analysis of the CALGB80203 clinical trial shows that addition of anti-EGFR therapy to chemotherapy significantly improves outcome for CRC patients when tumors express low RasGRP1 suppressor levels. In sum, RasGRP1 is a unique biomarker positioned in the EGFR pathway and of potential relevance to anti-EGFR therapy for CRC patients.

    View details for DOI 10.1172/jci.insight.127552

    View details for PubMedID 31237864

  • Opposing T cell responses in experimental autoimmune encephalomyelitis. Nature Saligrama, N. n., Zhao, F. n., Sikora, M. J., Serratelli, W. S., Fernandes, R. A., Louis, D. M., Yao, W. n., Ji, X. n., Idoyaga, J. n., Mahajan, V. B., Steinmetz, L. M., Chien, Y. H., Hauser, S. L., Oksenberg, J. R., Garcia, K. C., Davis, M. M. 2019


    Experimental autoimmune encephalomyelitis is a model for multiple sclerosis. Here we show that induction generates successive waves of clonally expanded CD4+, CD8+ and γδ+ T cells in the blood and central nervous system, similar to gluten-challenge studies of patients with coeliac disease. We also find major expansions of CD8+ T cells in patients with multiple sclerosis. In autoimmune encephalomyelitis, we find that most expanded CD4+ T cells are specific for the inducing myelin peptide MOG35-55. By contrast, surrogate peptides derived from a yeast peptide major histocompatibility complex library of some of the clonally expanded CD8+ T cells inhibit disease by suppressing the proliferation of MOG-specific CD4+ T cells. These results suggest that the induction of autoreactive CD4+ T cells triggers an opposing mobilization of regulatory CD8+ T cells.

    View details for DOI 10.1038/s41586-019-1467-x

    View details for PubMedID 31391585

  • De novo design of potent and selective mimics of IL-2 and IL-15. Nature Silva, D., Yu, S., Ulge, U. Y., Spangler, J. B., Jude, K. M., Labao-Almeida, C., Ali, L. R., Quijano-Rubio, A., Ruterbusch, M., Leung, I., Biary, T., Crowley, S. J., Marcos, E., Walkey, C. D., Weitzner, B. D., Pardo-Avila, F., Castellanos, J., Carter, L., Stewart, L., Riddell, S. R., Pepper, M., Bernardes, G. J., Dougan, M., Garcia, K. C., Baker, D. 2019; 565 (7738): 186–91


    We describe a de novo computational approach for designing proteins that recapitulate the binding sites of natural cytokines, but are otherwise unrelated in topology or amino acid sequence. We use this strategy to design mimics of the central immune cytokine interleukin-2 (IL-2) that bind to the IL-2 receptor betagammac heterodimer (IL-2Rbetagammac) but have no binding site for IL-2Ralpha (also called CD25) or IL-15Ralpha (also known as CD215). The designs are hyper-stable, bind human and mouse IL-2Rbetagammac with higher affinity than the natural cytokines, and elicit downstream cell signalling independently of IL-2Ralpha and IL-15Ralpha. Crystal structures of the optimized design neoleukin-2/15 (Neo-2/15), both alone and in complex with IL-2Rbetagammac, are very similar to the designed model. Neo-2/15 has superior therapeutic activity to IL-2 in mouse models of melanoma and colon cancer, with reduced toxicity and undetectable immunogenicity. Our strategy for building hyper-stable de novo mimetics could be applied generally to signalling proteins, enabling the creation of superior therapeutic candidates.

    View details for PubMedID 30626941

  • Differential induction of interferon stimulated genes between type I and type III interferons is independent of interferon receptor abundance. PLoS pathogens Pervolaraki, K., Rastgou Talemi, S., Albrecht, D., Bormann, F., Bamford, C., Mendoza, J. L., Garcia, K. C., McLauchlan, J., Hofer, T., Stanifer, M. L., Boulant, S. 2018; 14 (11): e1007420


    It is currently believed that type I and III interferons (IFNs) have redundant functions. However, the preferential distribution of type III IFN receptor on epithelial cells suggests functional differences at epithelial surfaces. Here, using human intestinal epithelial cells we could show that although both type I and type III IFNs confer an antiviral state to the cells, they do so with distinct kinetics. Type I IFN signaling is characterized by an acute strong induction of interferon stimulated genes (ISGs) and confers fast antiviral protection. On the contrary, the slow acting type III IFN mediated antiviral protection is characterized by a weaker induction of ISGs in a delayed manner compared to type I IFN. Moreover, while transcript profiling revealed that both IFNs induced a similar set of ISGs, their temporal expression strictly depended on the IFNs, thereby leading to unique antiviral environments. Using a combination of data-driven mathematical modeling and experimental validation, we addressed the molecular reason for this differential kinetic of ISG expression. We could demonstrate that these kinetic differences are intrinsic to each signaling pathway and not due to different expression levels of the corresponding IFN receptors. We report that type III IFN is specifically tailored to act in specific cell types not only due to the restriction of its receptor but also by providing target cells with a distinct antiviral environment compared to type I IFN. We propose that this specific environment is key at surfaces that are often challenged with the extracellular environment.

    View details for PubMedID 30485383

  • A RECK-WNT7 Receptor-Ligand Interaction Enables Isoform-Specific Regulation of Wnt Bioavailability. Cell reports Vallon, M., Yuki, K., Nguyen, T. D., Chang, J., Yuan, J., Siepe, D., Miao, Y., Essler, M., Noda, M., Garcia, K. C., Kuo, C. J. 2018; 25 (2): 339


    WNT7A and WNT7B control CNS angiogenesis and blood-brain barrier formation by activating endothelial Wnt/beta-catenin signaling. The GPI-anchored protein RECK and adhesion G protein-coupled receptor GPR124 critically regulate WNT7-specific signaling in concert with FZD and LRP co-receptors. Here, we demonstrate that primarily the GPR124 ectodomain, but not its transmembrane and intracellular domains, mediates RECK/WNT7-induced canonical Wnt signaling. Moreover, RECK is the predominant binding partner of GPR124 in rat brain blood vessels in situ. WNT7A and WNT7B, but not WNT3A, directly bind to purified recombinant soluble RECK, full-length cell surface RECK, and the GPR124:RECK complex. Chemical cross-linking indicates that RECK and WNT7A associate with 1:1 stoichiometry, which stabilizes short-lived, active, monomeric, hydrophobic WNT7A. In contrast, free WNT7A rapidly converts into inactive, hydrophilic aggregates. Overall, RECK is a selective WNT7 receptor that mediates GPR124/FZD/LRP-dependent canonical Wnt/beta-catenin signaling by stabilizing active cell surface WNT7, suggesting isoform-specific regulation of Wnt bioavailability.

    View details for PubMedID 30304675

  • T cell receptor cross-reactivity expanded by dramatic peptide-MHC adaptability NATURE CHEMICAL BIOLOGY Riley, T. P., Hellman, L. M., Gee, M. H., Mendoza, J. L., Alonso, J. A., Foley, K. C., Nishimura, M. I., Vander Kooi, C. W., Garcia, K., Baker, B. M. 2018; 14 (10): 934-+


    T cell receptor cross-reactivity allows a fixed T cell repertoire to respond to a much larger universe of potential antigens. Recent work has emphasized the importance of peptide structural and chemical homology, as opposed to sequence similarity, in T cell receptor cross-reactivity. Surprisingly, though, T cell receptors can also cross-react between ligands with little physiochemical commonalities. Studying the clinically relevant receptor DMF5, we demonstrate that cross-recognition of such divergent antigens can occur through mechanisms that involve heretofore unanticipated rearrangements in the peptide and presenting MHC protein, including binding-induced peptide register shifts and extensions from MHC peptide binding grooves. Moreover, cross-reactivity can proceed even when such dramatic rearrangements do not translate into structural or chemical molecular mimicry. Beyond demonstrating new principles of T cell receptor cross-reactivity, our results have implications for efforts to predict and control T cell specificity and cross-reactivity and highlight challenges associated with predicting T cell reactivities.

    View details for PubMedID 30224695

  • A polymorphic residue that attenuates the antiviral potential of interferon lambda 4 in hominid lineages. PLoS pathogens Bamford, C. G., Aranday-Cortes, E., Filipe, I. C., Sukumar, S., Mair, D., Filipe, A. d., Mendoza, J. L., Garcia, K. C., Fan, S., Tishkoff, S. A., McLauchlan, J. 2018; 14 (10): e1007307


    As antimicrobial signalling molecules, type III or lambda interferons (IFNlambdas) are critical for defence against infection by diverse pathogens, including bacteria, fungi and viruses. Counter-intuitively, expression of one member of the family, IFNlambda4, is associated with decreased clearance of hepatitis C virus (HCV) in the human population; by contrast, a natural frameshift mutation that abrogates IFNlambda4 production improves HCV clearance. To further understand how genetic variation between and within species affects IFNlambda4 function, we screened a panel of all known extant coding variants of human IFNlambda4 for their antiviral potential and identify three that substantially affect activity: P70S, L79F and K154E. The most notable variant was K154E, which was found in African Congo rainforest 'Pygmy' hunter-gatherers. K154E greatly enhanced in vitro activity in a range of antiviral (HCV, Zika virus, influenza virus and encephalomyocarditis virus) and gene expression assays. Remarkably, E154 is the ancestral residue in mammalian IFNlambda4s and is extremely well conserved, yet K154 has been fixed throughout evolution of the hominid genus Homo, including Neanderthals. Compared to chimpanzee IFNlambda4, the human orthologue had reduced activity due to amino acid K154. Comparison of published gene expression data from humans and chimpanzees showed that this difference in activity between K154 and E154 in IFNlambda4 correlates with differences in antiviral gene expression in vivo during HCV infection. Mechanistically, our data show that the human-specific K154 negatively affects IFNlambda4 activity through a novel means by reducing its secretion and potency. We thus demonstrate that attenuated activity of IFNlambda4 is conserved among humans and postulate that differences in IFNlambda4 activity between species contribute to distinct host-specific responses to-and outcomes of-infection, such as HCV infection. The driver of reduced IFNlambda4 antiviral activity in humans remains unknown but likely arose between 6 million and 360,000 years ago in Africa.

    View details for PubMedID 30308076

  • Engineering a Single-Agent Cytokine/Antibody Fusion That Selectively Expands Regulatory T Cells for Autoimmune Disease Therapy. Journal of immunology (Baltimore, Md. : 1950) Spangler, J. B., Trotta, E., Tomala, J., Peck, A., Young, T. A., Savvides, C. S., Silveria, S., Votavova, P., Salafsky, J., Pande, V. S., Kovar, M., Bluestone, J. A., Garcia, K. C. 2018


    IL-2 has been used to treat diseases ranging from cancer to autoimmune disorders, but its concurrent immunostimulatory and immunosuppressive effects hinder efficacy. IL-2 orchestrates immune cell function through activation of a high-affinity heterotrimeric receptor (composed of IL-2Ralpha, IL-2Rbeta, and common gamma [gammac]). IL-2Ralpha, which is highly expressed on regulatory T (TReg) cells, regulates IL-2 sensitivity. Previous studies have shown that complexation of IL-2 with the JES6-1 Ab preferentially biases cytokine activity toward TReg cells through a unique mechanism whereby IL-2 is exchanged from the Ab to IL-2Ralpha. However, clinical adoption of a mixed Ab/cytokine complex regimen is limited by stoichiometry and stability concerns. In this study, through structure-guided design, we engineered a single agent fusion of the IL-2 cytokine and JES6-1 Ab that, despite being covalently linked, preserves IL-2 exchange, selectively stimulating TReg expansion and exhibiting superior disease control to the mixed IL-2/JES6-1 complex in a mouse colitis model. These studies provide an engineering blueprint for resolving a major barrier to the implementation of functionally similar IL-2/Ab complexes for treatment of human disease.

    View details for PubMedID 30104245

  • Isolation of a Structural Mechanism for Uncoupling T Cell Receptor Signaling from Peptide-MHC Binding. Cell Sibener, L. V., Fernandes, R. A., Kolawole, E. M., Carbone, C. B., Liu, F., McAffee, D., Birnbaum, M. E., Yang, X., Su, L. F., Yu, W., Dong, S., Gee, M. H., Jude, K. M., Davis, M. M., Groves, J. T., Goddard, W. A., Heath, J. R., Evavold, B. D., Vale, R. D., Garcia, K. C. 2018; 174 (3): 672


    TCR-signaling strength generally correlates with peptide-MHC binding affinity; however, exceptions exist. We find high-affinity, yet non-stimulatory, interactions occur with high frequency in the human Tcell repertoire. Here, we studied human TCRs that are refractory to activation by pMHC ligands despite robust binding. Analysis of 3D affinity, 2D dwell time, and crystal structures of stimulatory versus non-stimulatory TCR-pMHC interactions failed to account for their different signaling outcomes. Using yeast pMHC display, we identified peptide agonists of a formerly non-responsive TCR. Single-molecule force measurements demonstrated the emergence of catch bonds in the activating TCR-pMHC interactions, correlating with exclusion of CD45 from the TCR-APC contact site. Molecular dynamics simulations of TCR-pMHC disengagement distinguished agonist from non-agonist ligands based on the acquisition of catch bonds withinthe TCR-pMHC interface. The isolation of catch bonds as a parameter mediating the coupling of TCR binding and signaling has important implications for TCR and antigen engineering for immunotherapy.

    View details for PubMedID 30053426

  • Stress-testing the relationship between T cell receptor/peptide-MHC affinity and cross-reactivity using peptide velcro. Proceedings of the National Academy of Sciences of the United States of America Gee, M. H., Sibener, L. V., Birnbaum, M. E., Jude, K. M., Yang, X., Fernandes, R. A., Mendoza, J. L., Glassman, C. R., Garcia, K. C. 2018


    T cell receptors (TCRs) bind to peptide-major histocompatibility complex (pMHC) with low affinity (Kd muM), which is generally assumed to facilitate cross-reactive TCR "scanning" of ligands. To understand the relationship between TCR/pMHC affinity and cross-reactivity, we sought to engineer an additional weak interaction, termed "velcro," between the TCR and pMHC to probe the specificities of TCRs at relatively low and high affinities. This additional interaction was generated through an eight-amino acid peptide library covalently linked to the N terminus of the MHC-bound peptide. Velcro was selected through an affinity-based isolation and was subsequently shown to enhance the cognate TCR/pMHC affinity in a peptide-dependent manner by 10-fold. This was sufficient to convert a nonstimulatory ultra-low-affinity ligand into a stimulatory ligand. An X-ray crystallographic structure revealed how velcro interacts with the TCR. To probe TCR cross-reactivity, we screened TCRs against yeast-displayed pMHC libraries with and without velcro, and found that the peptide cross-reactivity profiles of low-affinity (Kd > 100 muM) and high-affinity (Kd muM) TCR/pMHC interactions are remarkably similar. The conservation of recognition of the TCR for pMHC across affinities reveals the nature of low-affinity ligands for which there are important biological functions and has implications for understanding the specificities of affinity-matured TCRs.

    View details for PubMedID 30021852

  • A human anti-IL-2 antibody that potentiates regulatory T cells by a structure-based mechanism NATURE MEDICINE Trotta, E., Bessette, P. H., Silveria, S. L., Ely, L. K., Jude, K. M., Le, D. T., Holst, C. R., Coyle, A., Potempa, M., Lanier, L. L., Garcia, K., Crellin, N. K., Rondon, I. J., Bluestone, J. A. 2018; 24 (7): 1005-+


    Interleukin-2 (IL-2) has been shown to suppress immune pathologies by preferentially expanding regulatory T cells (Tregs). However, this therapy has been limited by off-target complications due to pathogenic cell expansion. Recent efforts have been focused on developing a more selective IL-2. It is well documented that certain anti-mouse IL-2 antibodies induce conformational changes that result in selective targeting of Tregs. We report the generation of a fully human anti-IL-2 antibody, F5111.2, that stabilizes IL-2 in a conformation that results in the preferential STAT5 phosphorylation of Tregs in vitro and selective expansion of Tregs in vivo. When complexed with human IL-2, F5111.2 induced remission of type 1 diabetes in the NOD mouse model, reduced disease severity in a model of experimental autoimmune encephalomyelitis and protected mice against xenogeneic graft-versus-host disease. These results suggest that IL-2-F5111.2 may provide an immunotherapy to treat autoimmune diseases and graft-versus-host disease.

    View details for PubMedID 29942088

  • Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T cells NATURE Fraietta, J. A., Nobles, C. L., Sammons, M. A., Lundh, S., Carty, S. A., Reich, T. J., Cogdill, A. P., Morrissette, J. D., DeNizio, J. E., Reddy, S., Hwang, Y., Gohil, M., Kulikovskaya, I., Nazimuddin, F., Gupta, M., Chen, F., Everett, J. K., Alexander, K. A., Lin-Shiao, E., Gee, M. H., Liu, X., Young, R. M., Ambrose, D., Wang, Y., Xu, J., Jordan, M. S., Marcucci, K. T., Levine, B. L., Garcia, K., Zhao, Y., Kalos, M., Porter, D. L., Kohli, R. M., Lacey, S. F., Berger, S. L., Bushman, F. D., June, C. H., Melenhorst, J. 2018; 558 (7709): 307-+


    Cancer immunotherapy based on genetically redirecting T cells has been used successfully to treat B cell malignancies1-3. In this strategy, the T cell genome is modified by integration of viral vectors or transposons encoding chimaeric antigen receptors (CARs) that direct tumour cell killing. However, this approach is often limited by the extent of expansion and persistence of CAR T cells4,5. Here we report mechanistic insights from studies of a patient with chronic lymphocytic leukaemia treated with CAR T cells targeting the CD19 protein. Following infusion of CAR T cells, anti-tumour activity was evident in the peripheral blood, lymph nodes and bone marrow; this activity was accompanied by complete remission. Unexpectedly, at the peak of the response, 94% of CAR T cells originated from a single clone in which lentiviral vector-mediated insertion of the CAR transgene disrupted the methylcytosine dioxygenase TET2 gene. Further analysis revealed a hypomorphic mutation in this patient's second TET2 allele. TET2-disrupted CAR T cells exhibited an epigenetic profile consistent with altered T cell differentiation and, at the peak of expansion, displayed a central memory phenotype. Experimental knockdown of TET2 recapitulated the potency-enhancing effect of TET2 dysfunction in this patient's CAR T cells. These findings suggest that the progeny of a single CAR T cell induced leukaemia remission and that TET2 modification may be useful for improving immunotherapies.

    View details for PubMedID 29849141

  • Viral GPCR US28 can signal in response to chemokine agonists of nearly unlimited structural degeneracy ELIFE Miles, T. F., Spiess, K., Jude, K. M., Tsutsumi, N., Burg, J. S., Ingram, J. R., Waghray, D., Hjorto, G. M., Larsen, O., Ploegh, H. L., Rosenkilde, M. M., Garcia, K. 2018; 7


    Human cytomegalovirus has hijacked and evolved a human G-protein-coupled receptor into US28, which functions as a promiscuous chemokine 'sink' to facilitate evasion of host immune responses. To probe the molecular basis of US28's unique ligand cross-reactivity, we deep-sequenced CX3CL1 chemokine libraries selected on 'molecular casts' of the US28 active-state and find that US28 can engage thousands of distinct chemokine sequences, many of which elicit diverse signaling outcomes. The structure of a G-protein-biased CX3CL1-variant in complex with US28 revealed an entirely unique chemokine amino terminal peptide conformation and remodeled constellation of receptor-ligand interactions. Receptor signaling, however, is remarkably robust to mutational disruption of these interactions. Thus, US28 accommodates and functionally discriminates amongst highly degenerate chemokine sequences by sensing the steric bulk of the ligands, which distort both receptor extracellular loops and the walls of the ligand binding pocket to varying degrees, rather than requiring sequence-specific bonding chemistries for recognition and signaling.

    View details for PubMedID 29882741

  • Discovery of a novel human anti-IL-2 antibody that potentiates Regulatory T cells by a structure-based mechanism Trotta, E., Bessette, P. H., Silveria, S. L., Ely, L. K., Jude, K., Holst, C. R., Coyle, A., Garcia, C. K., Crellin, N. K., Rondon, I. J., Bluestone, J. A. AMER ASSOC IMMUNOLOGISTS. 2018
  • Selective targeting of engineered T cells using orthogonal IL-2 cytokine-receptor complexes SCIENCE Sockolosky, J. T., Trotta, E., Parisi, G., Picton, L., Su, L. L., Le, A. C., Chhabra, A., Silveria, S. L., George, B. M., King, I. C., Tiffany, M. R., Jude, K., Sibener, L. V., Baker, D., Shizuru, J. A., Ribas, A., Bluestone, J. A., Garcia, K. 2018; 359 (6379): 1037-+


    Interleukin-2 (IL-2) is a cytokine required for effector T cell expansion, survival, and function, especially for engineered T cells in adoptive cell immunotherapy, but its pleiotropy leads to simultaneous stimulation and suppression of immune responses as well as systemic toxicity, limiting its therapeutic use. We engineered IL-2 cytokine-receptor orthogonal (ortho) pairs that interact with one another, transmitting native IL-2 signals, but do not interact with their natural cytokine and receptor counterparts. Introduction of orthoIL-2Rβ into T cells enabled the selective cellular targeting of orthoIL-2 to engineered CD4+ and CD8+ T cells in vitro and in vivo, with limited off-target effects and negligible toxicity. OrthoIL-2 pairs were efficacious in a preclinical mouse cancer model of adoptive cell therapy and may therefore represent a synthetic approach to achieving selective potentiation of engineered cells.

    View details for PubMedID 29496879

    View details for PubMedCentralID PMC5947856

  • From T Cell Receptor to Antigen, Systems Approach to Discovering T Cell Antigen(s) in Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis Saligrama, N., Zhao, F., Fernandes, R. A., Serratelli, W. S., Louis, D. M., Chien, Y., Garcia, C. K., Oksenberg, J., Davis, M. M. SAGE PUBLICATIONS LTD. 2018: 5
  • From T Cell Receptor to Antigen, Systems Approach to Discovering T Cell Antigen(s) in Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis Saligrama, N., Zhao, F., Fernandes, R. A., Serratelli, W. S., Louis, D. M., Chien, Y., Garcia, C. K., Oksenberg, J., Davis, M. M. SAGE PUBLICATIONS LTD. 2018: 85
  • Antigen Identification for Orphan T Cell Receptors Expressed on Tumor-Infiltrating Lymphocytes CELL Gee, M. H., Han, A., Lofgren, S. M., Beausang, J. F., Mendoza, J. L., Birnbaum, M. E., Bethune, M. T., Fischer, S., Yang, X., Gomez-Eerland, R., Bingham, D. B., Sibener, L. V., Fernandes, R. A., Velasco, A., Baltimore, D., Schumacher, T. N., Khatri, P., Quake, S. R., Davis, M. M., Garcia, K. 2018; 172 (3): 549-+


    The immune system can mount T cell responses against tumors; however, the antigen specificities of tumor-infiltrating lymphocytes (TILs) are not well understood. We used yeast-display libraries of peptide-human leukocyte antigen (pHLA) to screen for antigens of "orphan" T cell receptors (TCRs) expressed on TILs from human colorectal adenocarcinoma. Four TIL-derived TCRs exhibited strong selection for peptides presented in a highly diverse pHLA-A∗02:01 library. Three of the TIL TCRs were specific for non-mutated self-antigens, two of which were present in separate patient tumors, and shared specificity for a non-mutated self-antigen derived from U2AF2. These results show that the exposed recognition surface of MHC-bound peptides accessible to the TCR contains sufficient structural information to enable the reconstruction of sequences of peptide targets for pathogenic TCRs of unknown specificity. This finding underscores the surprising specificity of TCRs for their cognate antigens and enables the facile indentification of tumor antigens through unbiased screening.

    View details for PubMedID 29275860

    View details for PubMedCentralID PMC5786495

  • Inhibition of Delta-induced Notch signaling using fucose analogs NATURE CHEMICAL BIOLOGY Schneider, M., Kumar, V., Nordstrom, L., Feng, L., Takeuchi, H., Hao, H., Luca, V. C., Garcia, K., Stanley, P., Wu, P., Haltiwanger, R. S. 2018; 14 (1): 65-+


    Notch is a cell-surface receptor that controls cell-fate decisions and is regulated by O-glycans attached to epidermal growth factor-like (EGF) repeats in its extracellular domain. Protein O-fucosyltransferase 1 (Pofut1) modifies EGF repeats with O-fucose and is essential for Notch signaling. Constitutive activation of Notch signaling has been associated with a variety of human malignancies. Therefore, tools that inhibit Notch activity are being developed as cancer therapeutics. To this end, we screened L-fucose analogs for their effects on Notch signaling. Two analogs, 6-alkynyl and 6-alkenyl fucose, were substrates of Pofut1 and were incorporated directly into Notch EGF repeats in cells. Both analogs were potent inhibitors of binding to and activation of Notch1 by Notch ligands Dll1 and Dll4, but not by Jag1. Mutagenesis and modeling studies suggest that incorporation of the analogs into EGF8 of Notch1 markedly reduces the ability of Delta ligands to bind and activate Notch1.

    View details for PubMedID 29176671

    View details for PubMedCentralID PMC5726916

  • Novel and shared neoantigen derived from histone 3 variant H3.3K27M mutation for glioma T cell therapy JOURNAL OF EXPERIMENTAL MEDICINE Chheda, Z. S., Kohanbash, G., Okada, K., Jahan, N., Sidney, J., Pecoraro, M., Yang, X., Carrera, D. A., Downey, K. M., Shrivastav, S., Liu, S., Lin, Y., Lagisetti, C., Chuntova, P., Watchmaker, P. B., Mueller, S., Pollack, I. F., Rajalingam, R., Carcaboso, A. M., Mann, M., Sette, A., Garcia, K., Hou, Y., Okada, H. 2018; 215 (1): 141–57


    The median overall survival for children with diffuse intrinsic pontine glioma (DIPG) is less than one year. The majority of diffuse midline gliomas, including more than 70% of DIPGs, harbor an amino acid substitution from lysine (K) to methionine (M) at position 27 of histone 3 variant 3 (H3.3). From a CD8+ T cell clone established by stimulation of HLA-A2+ CD8+ T cells with synthetic peptide encompassing the H3.3K27M mutation, complementary DNA for T cell receptor (TCR) α- and β-chains were cloned into a retroviral vector. TCR-transduced HLA-A2+ T cells efficiently killed HLA-A2+H3.3K27M+ glioma cells in an antigen- and HLA-specific manner. Adoptive transfer of TCR-transduced T cells significantly suppressed the progression of glioma xenografts in mice. Alanine-scanning assays suggested the absence of known human proteins sharing the key amino acid residues required for recognition by the TCR, suggesting that the TCR could be safely used in patients. These data provide us with a strong basis for developing T cell-based therapy targeting this shared neoepitope.

    View details for PubMedID 29203539

    View details for PubMedCentralID PMC5748856

  • Structure of an engineered IFN-lambda/IFN-lambda R1/IL-10R beta complex provides insight into the functional dichotomy of type III versus type I IFNs Mendoza, J., Schneider, W. M., Hoffman, H., Vercauteren, K., Jude, K. M., Xiong, A., Moraga, I., Horton, T. M., Glenn, J. S., de Jonga, Y. P., Garcia, K. ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD. 2017: 46
  • In vitro reconstitution of T cell receptor-mediated segregation of the CD45 phosphatase PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Carbone, C. B., Kern, N., Fernandes, R. A., Hui, E., Su, X., Garcia, K., Vale, R. D. 2017; 114 (44): E9338–E9345


    T cell signaling initiates upon the binding of peptide-loaded MHC (pMHC) on an antigen-presenting cell to the T cell receptor (TCR) on a T cell. TCR phosphorylation in response to pMHC binding is accompanied by segregation of the transmembrane phosphatase CD45 away from TCR-pMHC complexes. The kinetic segregation hypothesis proposes that CD45 exclusion shifts the local kinase-phosphatase balance to favor TCR phosphorylation. Spatial partitioning may arise from the size difference between the large CD45 extracellular domain and the smaller TCR-pMHC complex, although parsing potential contributions of extracellular protein size, actin activity, and lipid domains is difficult in living cells. Here, we reconstitute segregation of CD45 from bound receptor-ligand pairs using purified proteins on model membranes. Using a model receptor-ligand pair (FRB-FKBP), we first test physical and computational predictions for protein organization at membrane interfaces. We then show that the TCR-pMHC interaction causes partial exclusion of CD45. Comparing two developmentally regulated isoforms of CD45, the larger RABC variant is excluded more rapidly and efficiently (∼50%) than the smaller R0 isoform (∼20%), suggesting that CD45 isotypes could regulate signaling thresholds in different T cell subtypes. Similar to the sensitivity of T cell signaling, TCR-pMHC interactions with Kds of ≤15 µM were needed to exclude CD45. We further show that the coreceptor PD-1 with its ligand PD-L1, immunotherapy targets that inhibit T cell signaling, also exclude CD45. These results demonstrate that the binding energies of physiological receptor-ligand pairs on the T cell are sufficient to create spatial organization at membrane-membrane interfaces.

    View details for PubMedID 29042512

  • CD47 blockade enhances therapeutic activity of TCR mimic antibodies to ultra-low density cancer epitopes LEUKEMIA Mathias, M. D., Sockolosky, J. T., Chang, A. Y., Tan, K. S., Liu, C., Garcia, K. C., Scheinberg, D. A. 2017; 31 (10): 2254–57

    View details for PubMedID 28745331

    View details for PubMedCentralID PMC5628131

  • Ligand-induced type II interleukin-4 receptor dimers are sustained by rapid re-association within plasma membrane microcompartments. Nature communications Richter, D., Moraga, I., Winkelmann, H., Birkholz, O., Wilmes, S., Schulte, M., Kraich, M., Kenneweg, H., Beutel, O., Selenschik, P., Paterok, D., Gavutis, M., Schmidt, T., Garcia, K. C., Müller, T. D., Piehler, J. 2017; 8: 15976


    The spatiotemporal organization of cytokine receptors in the plasma membrane is still debated with models ranging from ligand-independent receptor pre-dimerization to ligand-induced receptor dimerization occurring only after receptor uptake into endosomes. Here, we explore the molecular and cellular determinants governing the assembly of the type II interleukin-4 receptor, taking advantage of various agonists binding the receptor subunits with different affinities and rate constants. Quantitative kinetic studies using artificial membranes confirm that receptor dimerization is governed by the two-dimensional ligand-receptor interactions and identify a critical role of the transmembrane domain in receptor dimerization. Single molecule localization microscopy at physiological cell surface expression levels, however, reveals efficient ligand-induced receptor dimerization by all ligands, largely independent of receptor binding affinities, in line with the similar STAT6 activation potencies observed for all IL-4 variants. Detailed spatiotemporal analyses suggest that kinetic trapping of receptor dimers in actin-dependent microcompartments sustains robust receptor dimerization and signalling.

    View details for DOI 10.1038/ncomms15976

    View details for PubMedID 28706306

    View details for PubMedCentralID PMC5519985

  • Intratumoural heterogeneity generated by Notch signalling promotes small-cell lung cancer NATURE Lim, J. S., Ibaseta, A., Fischer, M. M., Cancilla, B., O'Young, G., Cristea, S., Luca, V. C., Yang, D., Jahchan, N. S., Hamard, C., Antoine, M., Wislez, M., Kong, C., Cain, J., Liu, Y., Kapoun, A. M., Garcia, K. C., Hoey, T., Murriel, C. L., Sage, J. 2017; 545 (7654): 360-?


    The Notch signalling pathway mediates cell fate decisions and is tumour suppressive or oncogenic depending on the context. During lung development, Notch pathway activation inhibits the differentiation of precursor cells to a neuroendocrine fate. In small-cell lung cancer, an aggressive neuroendocrine lung cancer, loss-of-function mutations in NOTCH genes and the inhibitory effects of ectopic Notch activation indicate that Notch signalling is tumour suppressive. Here we show that Notch signalling can be both tumour suppressive and pro-tumorigenic in small-cell lung cancer. Endogenous activation of the Notch pathway results in a neuroendocrine to non-neuroendocrine fate switch in 10-50% of tumour cells in a mouse model of small-cell lung cancer and in human tumours. This switch is mediated in part by Rest (also known as Nrsf), a transcriptional repressor that inhibits neuroendocrine gene expression. Non-neuroendocrine Notch-active small-cell lung cancer cells are slow growing, consistent with a tumour-suppressive role for Notch, but these cells are also relatively chemoresistant and provide trophic support to neuroendocrine tumour cells, consistent with a pro-tumorigenic role. Importantly, Notch blockade in combination with chemotherapy suppresses tumour growth and delays relapse in pre-clinical models. Thus, small-cell lung cancer tumours generate their own microenvironment via activation of Notch signalling in a subset of tumour cells, and the presence of these cells may serve as a biomarker for the use of Notch pathway inhibitors in combination with chemotherapy in select patients with small-cell lung cancer.

    View details for DOI 10.1038/nature22323

    View details for PubMedID 28489825

  • The Intergenic Recombinant HLA-B*46:01 Has a Distinctive Peptidome that Includes KIR2DL3 Ligands. Cell reports Hilton, H. G., McMurtrey, C. P., Han, A. S., Djaoud, Z., Guethlein, L. A., Blokhuis, J. H., Pugh, J. L., Goyos, A., Horowitz, A., Buchli, R., Jackson, K. W., Bardet, W., Bushnell, D. A., Robinson, P. J., Mendoza, J. L., Birnbaum, M. E., Nielsen, M., Garcia, K. C., Hildebrand, W. H., Parham, P. 2017; 19 (7): 1394-1405


    HLA-B(∗)46:01 was formed by an intergenic mini-conversion, between HLA-B(∗)15:01 and HLA-C(∗)01:02, in Southeast Asia during the last 50,000 years, and it has since become the most common HLA-B allele in the region. A functional effect of the mini-conversion was introduction of the C1 epitope into HLA-B(∗)46:01, making it an exceptional HLA-B allotype that is recognized by the C1-specific natural killer (NK) cell receptor KIR2DL3. High-resolution mass spectrometry showed that HLA-B(∗)46:01 has a low-diversity peptidome that is distinct from those of its parents. A minority (21%) of HLA-B(∗)46:01 peptides, with common C-terminal characteristics, form ligands for KIR2DL3. The HLA-B(∗)46:01 peptidome is predicted to be enriched for peptide antigens derived from Mycobacterium leprae. Overall, the results indicate that the distinctive peptidome and functions of HLA-B(∗)46:01 provide carriers with resistance to leprosy, which drove its rapid rise in frequency in Southeast Asia.

    View details for DOI 10.1016/j.celrep.2017.04.059

    View details for PubMedID 28514659

  • Synthekines are surrogate cytokine and growth factor agonists that compel signaling through non-natural receptor dimers ELIFE Moraga, I., Spangler, J. B., Mendoza, J. L., Gakovic, M., Wehrman, T. S., Krutzik, P., Garcia, K. C. 2017; 6


    Cytokine and growth-factor ligands typically signal through homo- or hetero-dimeric cell surface receptors via Janus Kinase (JAK/TYK), or Receptor Tyrosine Kinase (RTK)-mediated trans-phosphorylation. However, the number of receptor dimer pairings occurring in nature is limited to those driven by natural ligands encoded within our genome. We have engineered synthethic cytokines (synthekines) that drive formation of cytokine receptor dimer pairings that are not formed by endogenous cytokines and that are not found in nature, and which activate distinct signaling programs. We show that a wide range of non-natural cytokine receptor hetero-dimers are competent to elicit a signaling output. We engineered synthekine ligands that assembled IL-2Rβ/IL-4Rα or IL-4Rα/IFNAR2 receptor heterodimers, that do not occur naturally, triggering signaling and functional responses distinct from those activated by the endogenous cytokines IL-2, IL-4, and IFN. Furthermore, hybrid synthekine ligands that dimerized a JAK/STAT cytokine receptor with a receptor tyrosine kinase (RTK) also elicited a signaling response. Synthekines represent a new family of synthetic ligands with pre-defined receptors, but 'orphan' functions, that enable the full combinatorial scope of dimeric signaling receptors encoded within the human genome to be exploited for basic research and drug discovery.

    View details for DOI 10.7554/eLife.22882

    View details for Web of Science ID 000401062800001

    View details for PubMedID 28498099

  • Non-equivalence of Wnt and R-spondin ligands during Lgr5(+) intestinal stem-cell self-renewal NATURE Yan, K. S., Janda, C. Y., Chang, J., Zheng, G. X., Larkin, K. A., Luca, V. C., Chia, L. A., Mah, A. T., Han, A., Terry, J. M., Ootani, A., Roelf, K., Lee, M., Yuan, J., Li, X., Bolen, C. R., Wilhelmy, J., Davies, P. S., Ueno, H., von Furstenberg, R. J., Belgrader, P., Ziraldo, S. B., Ordonez, H., Henning, S. J., Wong, M. H., Snyder, M. P., Weissman, I. L., Hsueh, A. J., Mikkelsen, T. S., Garcia, K. C., Kuo, C. J. 2017; 545 (7653): 238-?


    The canonical Wnt/β-catenin signalling pathway governs diverse developmental, homeostatic and pathological processes. Palmitoylated Wnt ligands engage cell-surface frizzled (FZD) receptors and LRP5 and LRP6 co-receptors, enabling β-catenin nuclear translocation and TCF/LEF-dependent gene transactivation. Mutations in Wnt downstream signalling components have revealed diverse functions thought to be carried out by Wnt ligands themselves. However, redundancy between the 19 mammalian Wnt proteins and 10 FZD receptors and Wnt hydrophobicity have made it difficult to attribute these functions directly to Wnt ligands. For example, individual mutations in Wnt ligands have not revealed homeostatic phenotypes in the intestinal epithelium-an archetypal canonical, Wnt pathway-dependent, rapidly self-renewing tissue, the regeneration of which is fueled by proliferative crypt Lgr5(+) intestinal stem cells (ISCs). R-spondin ligands (RSPO1-RSPO4) engage distinct LGR4-LGR6, RNF43 and ZNRF3 receptor classes, markedly potentiate canonical Wnt/β-catenin signalling, and induce intestinal organoid growth in vitro and Lgr5(+) ISCs in vivo. However, the interchangeability, functional cooperation and relative contributions of Wnt versus RSPO ligands to in vivo canonical Wnt signalling and ISC biology remain unknown. Here we identify the functional roles of Wnt and RSPO ligands in the intestinal crypt stem-cell niche. We show that the default fate of Lgr5(+) ISCs is to differentiate, unless both RSPO and Wnt ligands are present. However, gain-of-function studies using RSPO ligands and a new non-lipidated Wnt analogue reveal that these ligands have qualitatively distinct, non-interchangeable roles in ISCs. Wnt proteins are unable to induce Lgr5(+) ISC self-renewal, but instead confer a basal competency by maintaining RSPO receptor expression that enables RSPO ligands to actively drive and specify the extent of stem-cell expansion. This functionally non-equivalent yet cooperative interaction between Wnt and RSPO ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precise control of tissue regeneration.

    View details for DOI 10.1038/nature22313

    View details for Web of Science ID 000400963800037

  • Surrogate Wnt agonists that phenocopy canonical Wnt and beta-catenin signalling NATURE Janda, C. Y., Dang, L. T., You, C., Chang, J., de Lau, W., Zhong, Z. A., Yan, K. S., Marecic, O., Siepe, D., Li, X., Moody, J. D., Williams, B. O., Clevers, H., Piehler, J., Baker, D., Kuo, C. J., Garcia, K. C. 2017; 545 (7653): 234-?


    Wnt proteins modulate cell proliferation and differentiation and the self-renewal of stem cells by inducing β-catenin-dependent signalling through the Wnt receptor frizzled (FZD) and the co-receptors LRP5 and LRP6 to regulate cell fate decisions and the growth and repair of several tissues. The 19 mammalian Wnt proteins are cross-reactive with the 10 FZD receptors, and this has complicated the attribution of distinct biological functions to specific FZD and Wnt subtype interactions. Furthermore, Wnt proteins are modified post-translationally by palmitoylation, which is essential for their secretion, function and interaction with FZD receptors. As a result of their acylation, Wnt proteins are very hydrophobic and require detergents for purification, which presents major obstacles to the preparation and application of recombinant Wnt proteins. This hydrophobicity has hindered the determination of the molecular mechanisms of Wnt signalling activation and the functional importance of FZD subtypes, and the use of Wnt proteins as therapeutic agents. Here we develop surrogate Wnt agonists, water-soluble FZD-LRP5/LRP6 heterodimerizers, with FZD5/FZD8-specific and broadly FZD-reactive binding domains. Similar to WNT3A, these Wnt agonists elicit a characteristic β-catenin signalling response in a FZD-selective fashion, enhance the osteogenic lineage commitment of primary mouse and human mesenchymal stem cells, and support the growth of a broad range of primary human organoid cultures. In addition, the surrogates can be systemically expressed and exhibit Wnt activity in vivo in the mouse liver, regulating metabolic liver zonation and promoting hepatocyte proliferation, resulting in hepatomegaly. These surrogates demonstrate that canonical Wnt signalling can be activated by bi-specific ligands that induce receptor heterodimerization. Furthermore, these easily produced, non-lipidated Wnt surrogate agonists facilitate functional studies of Wnt signalling and the exploration of Wnt agonists for translational applications in regenerative medicine.

    View details for DOI 10.1038/nature22306

    View details for PubMedID 28467818

  • intestinal stem-cell self-renewal. Nature Yan, K. S., Janda, C. Y., Chang, J., Zheng, G. X., Larkin, K. A., Luca, V. C., Chia, L. A., Mah, A. T., Han, A., Terry, J. M., Ootani, A., Roelf, K., Lee, M., Yuan, J., Li, X., Bolen, C. R., Wilhelmy, J., Davies, P. S., Ueno, H., von Furstenberg, R. J., Belgrader, P., Ziraldo, S. B., Ordonez, H., Henning, S. J., Wong, M. H., Snyder, M. P., Weissman, I. L., Hsueh, A. J., Mikkelsen, T. S., Garcia, K. C., Kuo, C. J. 2017; 545 (7653): 238-242


    The canonical Wnt/β-catenin signalling pathway governs diverse developmental, homeostatic and pathological processes. Palmitoylated Wnt ligands engage cell-surface frizzled (FZD) receptors and LRP5 and LRP6 co-receptors, enabling β-catenin nuclear translocation and TCF/LEF-dependent gene transactivation. Mutations in Wnt downstream signalling components have revealed diverse functions thought to be carried out by Wnt ligands themselves. However, redundancy between the 19 mammalian Wnt proteins and 10 FZD receptors and Wnt hydrophobicity have made it difficult to attribute these functions directly to Wnt ligands. For example, individual mutations in Wnt ligands have not revealed homeostatic phenotypes in the intestinal epithelium-an archetypal canonical, Wnt pathway-dependent, rapidly self-renewing tissue, the regeneration of which is fueled by proliferative crypt Lgr5(+) intestinal stem cells (ISCs). R-spondin ligands (RSPO1-RSPO4) engage distinct LGR4-LGR6, RNF43 and ZNRF3 receptor classes, markedly potentiate canonical Wnt/β-catenin signalling, and induce intestinal organoid growth in vitro and Lgr5(+) ISCs in vivo. However, the interchangeability, functional cooperation and relative contributions of Wnt versus RSPO ligands to in vivo canonical Wnt signalling and ISC biology remain unknown. Here we identify the functional roles of Wnt and RSPO ligands in the intestinal crypt stem-cell niche. We show that the default fate of Lgr5(+) ISCs is to differentiate, unless both RSPO and Wnt ligands are present. However, gain-of-function studies using RSPO ligands and a new non-lipidated Wnt analogue reveal that these ligands have qualitatively distinct, non-interchangeable roles in ISCs. Wnt proteins are unable to induce Lgr5(+) ISC self-renewal, but instead confer a basal competency by maintaining RSPO receptor expression that enables RSPO ligands to actively drive and specify the extent of stem-cell expansion. This functionally non-equivalent yet cooperative interaction between Wnt and RSPO ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precise control of tissue regeneration.

    View details for DOI 10.1038/nature22313

    View details for PubMedID 28467820

  • The IFN-?-IFN-?R1-IL-10Rß Complex Reveals Structural Features Underlying Type III IFN Functional Plasticity. Immunity Mendoza, J. L., Schneider, W. M., Hoffmann, H., Vercauteren, K., Jude, K. M., Xiong, A., Moraga, I., Horton, T. M., Glenn, J. S., de Jong, Y. P., Rice, C. M., Garcia, K. C. 2017; 46 (3): 379-392


    Type III interferons (IFN-λs) signal through a heterodimeric receptor complex composed of the IFN-λR1 subunit, specific for IFN-λs, and interleukin-10Rβ (IL-10Rβ), which is shared by multiple cytokines in the IL-10 superfamily. Low affinity of IL-10Rβ for cytokines has impeded efforts aimed at crystallizing cytokine-receptor complexes. We used yeast surface display to engineer a higher-affinity IFN-λ variant, H11, which enabled crystallization of the ternary complex. The structure revealed that IL-10Rβ uses a network of tyrosine residues as hydrophobic anchor points to engage IL-10 family cytokines that present complementary hydrophobic binding patches, explaining its role as both a cross-reactive but cytokine-specific receptor. H11 elicited increased anti-proliferative and antiviral activities in vitro and in vivo. In contrast, engineered higher-affinity type I IFNs did not increase antiviral potency over wild-type type I IFNs. Our findings provide insight into cytokine recognition by the IL-10R family and highlight the plasticity of type III interferon signaling and its therapeutic potential.

    View details for DOI 10.1016/j.immuni.2017.02.017

    View details for PubMedID 28329704

  • Decoupling the Functional Pleiotropy of Stem Cell Factor by Tuning c-Kit Signaling CELL Ho, C. C., Chhabra, A., Starkl, P., Schnorr, P., Wilmes, S., Moraga, I., Kwon, H., Gaudenzio, N., Sibilano, R., Wehrman, T. S., Gakovic, M., Sockolosky, J. T., Tiffany, M. R., Ring, A. M., Piehler, J., Weissman, I. L., Galli, S. J., Shizuru, J. A., Garcia, K. C. 2017; 168 (6): 1041-?


    Most secreted growth factors and cytokines are functionally pleiotropic because their receptors are expressed on diverse cell types. While important for normal mammalian physiology, pleiotropy limits the efficacy of cytokines and growth factors as therapeutics. Stem cell factor (SCF) is a growth factor that acts through the c-Kit receptor tyrosine kinase to elicit hematopoietic progenitor expansion but can be toxic when administered in vivo because it concurrently activates mast cells. We engineered a mechanism-based SCF partial agonist that impaired c-Kit dimerization, truncating downstream signaling amplitude. This SCF variant elicited biased activation of hematopoietic progenitors over mast cells in vitro and in vivo. Mouse models of SCF-mediated anaphylaxis, radioprotection, and hematopoietic expansion revealed that this SCF partial agonist retained therapeutic efficacy while exhibiting virtually no anaphylactic off-target effects. The approach of biasing cell activation by tuning signaling thresholds and outputs has applications to many dimeric receptor-ligand systems.

    View details for DOI 10.1016/j.cell.2017.02.011

    View details for Web of Science ID 000396287900012

    View details for PubMedID 28283060

  • Functional Selectivity in Cytokine Signaling Revealed Through a Pathogenic EPO Mutation CELL Kim, A., Ulirsch, J. C., Wilmes, S., Unal, E., Moraga, I., Karakukcu, M., Yuan, D., Kazerounian, S., Abdulhay, N. J., King, D. S., Gupta, N., Gabriel, S. B., Lander, E. S., Patiroglu, T., Ozcan, A., Ozdemir, M., Garcia, K., Piehler, J., Gazda, H. T., Klein, D. E., Sankaran, V. G. 2017; 168 (6): 1053-+


    Cytokines are classically thought to stimulate downstream signaling pathways through monotonic activation of receptors. We describe a severe anemia resulting from a homozygous mutation (R150Q) in the cytokine erythropoietin (EPO). Surprisingly, the EPO R150Q mutant shows only a mild reduction in affinity for its receptor but has altered binding kinetics. The EPO mutant is less effective at stimulating erythroid cell proliferation and differentiation, even at maximally potent concentrations. While the EPO mutant can stimulate effectors such as STAT5 to a similar extent as the wild-type ligand, there is reduced JAK2-mediated phosphorylation of select downstream targets. This impairment in downstream signaling mechanistically arises from altered receptor dimerization dynamics due to extracellular binding changes. These results demonstrate how variation in a single cytokine can lead to biased downstream signaling and can thereby cause human disease. Moreover, we have defined a distinct treatable form of anemia through mutation identification and functional studies.

    View details for PubMedID 28283061

    View details for PubMedCentralID PMC5376096

  • Notch-Jagged complex structure implicates a catch bond in tuning ligand sensitivity. Science Luca, V. C., Kim, B. C., Ge, C., Kakuda, S., Wu, D., Roein-Peikar, M., Haltiwanger, R. S., Zhu, C., Ha, T., Garcia, K. C. 2017


    Notch receptor activation initiates cell fate decisions and is distinctive in its reliance on mechanical force and protein glycosylation. The 2.5-angstrom-resolution crystal structure of the extracellular interacting region of Notch1 complexed with an engineered, high-affinity variant of Jagged1 (Jag1) reveals a binding interface that extends ~120 angstroms along five consecutive domains of each protein. O-Linked fucose modifications on Notch1 epidermal growth factor-like (EGF) domains 8 and 12 engage the EGF3 and C2 domains of Jag1, respectively, and different Notch1 domains are favored in binding to Jag1 than those that bind to the Delta-like 4 ligand. Jag1 undergoes conformational changes upon Notch binding, exhibiting catch bond behavior that prolongs interactions in the range of forces required for Notch activation. This mechanism enables cellular forces to regulate binding, discriminate among Notch ligands, and potentiate Notch signaling.

    View details for DOI 10.1126/science.aaf9739

    View details for PubMedID 28254785

  • Deconstruction of the Beaten Path-Sidestep interaction network provides insights into neuromuscular system development. eLife Li, H. n., Watson, A. n., Olechwier, A. n., Anaya, M. n., Sorooshyari, S. K., Harnett, D. P., Lee, H. P., Vielmetter, J. n., Fares, M. A., Garcia, K. C., Özkan, E. n., Labrador, J. P., Zinn, K. n. 2017; 6


    An "interactome" screen of all Drosophila cell-surface and secreted proteins containing immunoglobulin superfamily (IgSF) domains discovered a network formed by paralogs of Beaten Path (Beat) and Sidestep (Side), a ligand-receptor pair that is central to motor axon guidance. Here we describe a new method for interactome screening, the Bio-Plex Interactome Assay (BPIA), which allows identification of many interactions in a single sample. Using the BPIA, we "deorphanized" four more members of the Beat-Side network. We confirmed interactions using surface plasmon resonance. The expression patterns of beat and side genes suggest that Beats are neuronal receptors for Sides expressed on peripheral tissues. side-VI is expressed in muscle fibers targeted by the ISNb nerve, as well as at growth cone choice points and synaptic targets for the ISN and TN nerves. beat-V genes, encoding Side-VI receptors, are expressed in ISNb and ISN motor neurons.

    View details for PubMedID 28829740

  • Localized CD47 blockade enhances immunotherapy for murine melanoma. Proceedings of the National Academy of Sciences of the United States of America Ingram, J. R., Blomberg, O. S., Sockolosky, J. T., Ali, L. n., Schmidt, F. I., Pishesha, N. n., Espinosa, C. n., Dougan, S. K., Garcia, K. C., Ploegh, H. L., Dougan, M. n. 2017; 114 (38): 10184–89


    CD47 is an antiphagocytic ligand broadly expressed on normal and malignant tissues that delivers an inhibitory signal through the receptor signal regulatory protein alpha (SIRPα). Inhibitors of the CD47-SIRPα interaction improve antitumor antibody responses by enhancing antibody-dependent cellular phagocytosis (ADCP) in xenograft models. Endogenous expression of CD47 on a variety of cell types, including erythrocytes, creates a formidable antigen sink that may limit the efficacy of CD47-targeting therapies. We generated a nanobody, A4, that blocks the CD47-SIRPα interaction. A4 synergizes with anti-PD-L1, but not anti-CTLA4, therapy in the syngeneic B16F10 melanoma model. Neither increased dosing nor half-life extension by fusion of A4 to IgG2a Fc (A4Fc) overcame the issue of an antigen sink or, in the case of A4Fc, systemic toxicity. Generation of a B16F10 cell line that secretes the A4 nanobody showed that an enhanced response to several immune therapies requires near-complete blockade of CD47 in the tumor microenvironment. Thus, strategies to localize CD47 blockade to tumors may be particularly valuable for immune therapy.

    View details for PubMedID 28874561

  • Receptor dimer stabilization by hierarchical plasma membrane microcompartments regulates cytokine signaling. Science advances You, C., Marquez-Lago, T. T., Richter, C. P., Wilmes, S., Moraga, I., Garcia, K. C., Leier, A., Piehler, J. 2016; 2 (12): e1600452


    The interaction dynamics of signaling complexes is emerging as a key determinant that regulates the specificity of cellular responses. We present a combined experimental and computational study that quantifies the consequences of plasma membrane microcompartmentalization for the dynamics of type I interferon receptor complexes. By using long-term dual-color quantum dot (QD) tracking, we found that the lifetime of individual ligand-induced receptor heterodimers depends on the integrity of the membrane skeleton (MSK), which also proved important for efficient downstream signaling. By pair correlation tracking and localization microscopy as well as by fast QD tracking, we identified a secondary confinement within ~300-nm-sized zones. A quantitative spatial stochastic diffusion-reaction model, entirely parameterized on the basis of experimental data, predicts that transient receptor confinement by the MSK meshwork allows for rapid reassociation of dissociated receptor dimers. Moreover, the experimentally observed apparent stabilization of receptor dimers in the plasma membrane was reproduced by simulations of a refined, hierarchical compartment model. Our simulations further revealed that the two-dimensional association rate constant is a key parameter for controlling the extent of MSK-mediated stabilization of protein complexes, thus ensuring the specificity of this effect. Together, experimental evidence and simulations support the hypothesis that passive receptor confinement by MSK-based microcompartmentalization promotes maintenance of signaling complexes in the plasma membrane.

    View details for DOI 10.1126/sciadv.1600452

    View details for PubMedID 27957535

    View details for PubMedCentralID PMC5135388

  • Salmonella Infection Enhances Erythropoietin Production by the Kidney and Liver, Which Correlates with Elevated Bacterial Burdens INFECTION AND IMMUNITY Li, L., Benoun, J. M., Weiskopf, K., Garcia, K. C., McSorley, S. J. 2016; 84 (10): 2833-2841


    Salmonella infection profoundly affects host erythroid development, but the mechanisms responsible for this effect remain poorly understood. We monitored the impact of Salmonella infection on erythroid development and found that systemic infection induced anemia, splenomegaly, elevated erythropoietin (EPO) levels, and extramedullary erythropoiesis in a process independent of Salmonella pathogenicity island 2 (SPI2) or flagellin. The circulating EPO level was also constitutively higher in mice lacking the expression of signal-regulatory protein α (SIRPα). The expression level of EPO mRNA was elevated in the kidney and liver but not increased in the spleens of infected mice despite the presence of extramedullary erythropoiesis in this tissue. In contrast to data from a previous report, mice lacking EPO receptor (EPOR) expression on nonerythroid cells (EPOR rescued) had bacterial loads similar to those of wild-type mice following Salmonella infection. Indeed, treatment to reduce splenic erythroblasts and mature red blood cells correlated with elevated bacterial burdens, implying that extramedullary erythropoiesis benefits the host. Together, these findings emphasize the profound effect of Salmonella infection on erythroid development and suggest that the modulation of erythroid development has both positive and negative consequences for host immunity.

    View details for DOI 10.1128/IAI.00337-16

    View details for Web of Science ID 000384077800011

    View details for PubMedID 27456828

    View details for PubMedCentralID PMC5038055

  • Genetic variation in MHC proteins is associated with T cell receptor expression biases. Nature genetics Sharon, E., Sibener, L. V., Battle, A., Fraser, H. B., Garcia, K. C., Pritchard, J. K. 2016; 48 (9): 995-1002


    In each individual, a highly diverse T cell receptor (TCR) repertoire interacts with peptides presented by major histocompatibility complex (MHC) molecules. Despite extensive research, it remains controversial whether germline-encoded TCR-MHC contacts promote TCR-MHC specificity and, if so, whether differences exist in TCR V gene compatibilities with different MHC alleles. We applied expression quantitative trait locus (eQTL) mapping to test for associations between genetic variation and TCR V gene usage in a large human cohort. We report strong trans associations between variation in the MHC locus and TCR V gene usage. Fine-mapping of the association signals identifies specific amino acids from MHC genes that bias V gene usage, many of which contact or are spatially proximal to the TCR or peptide in the TCR-peptide-MHC complex. Hence, these MHC variants, several of which are linked to autoimmune diseases, can directly affect TCR-MHC interaction. These results provide the first examples of trans-QTL effects mediated by protein-protein interactions and are consistent with intrinsic TCR-MHC specificity.

    View details for DOI 10.1038/ng.3625

    View details for PubMedID 27479906

    View details for PubMedCentralID PMC5010864

  • Durable antitumor responses to CD47 blockade require adaptive immune stimulation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sockolosky, J. T., Dougan, M., Ingram, J. R., Ho, C. C., Kauke, M. J., Almo, S. C., Ploegh, H. L., Garcia, K. C. 2016; 113 (19): E2646-E2654


    Therapeutic antitumor antibodies treat cancer by mobilizing both innate and adaptive immunity. CD47 is an antiphagocytic ligand exploited by tumor cells to blunt antibody effector functions by transmitting an inhibitory signal through its receptor signal regulatory protein alpha (SIRPα). Interference with the CD47-SIRPα interaction synergizes with tumor-specific monoclonal antibodies to eliminate human tumor xenografts by enhancing macrophage-mediated antibody-dependent cellular phagocytosis (ADCP), but synergy between CD47 blockade and ADCP has yet to be demonstrated in immunocompetent hosts. Here, we show that CD47 blockade alone or in combination with a tumor-specific antibody fails to generate antitumor immunity against syngeneic B16F10 tumors in mice. Durable tumor immunity required programmed death-ligand 1 (PD-L1) blockade in combination with an antitumor antibody, with incorporation of CD47 antagonism substantially improving response rates. Our results highlight an underappreciated contribution of the adaptive immune system to anti-CD47 adjuvant therapy and suggest that targeting both innate and adaptive immune checkpoints can potentiate the vaccinal effect of antitumor antibody therapy.

    View details for DOI 10.1073/pnas.1604268113

    View details for Web of Science ID 000375478800015

    View details for PubMedID 27091975

    View details for PubMedCentralID PMC4868409

  • Data publication with the structural biology data grid supports live analysis NATURE COMMUNICATIONS Meyer, P. A., Socias, S., Key, J., Ransey, E., Tjon, E. C., Buschiazzo, A., Lei, M., Botka, C., Withrow, J., Neau, D., Rajashankar, K., Anderson, K. S., Baxter, R. H., Blacklow, S. C., Boggon, T. J., Bonvin, A. M., Borek, D., Brett, T. J., Caflisch, A., Chang, C., Chazin, W. J., Corbett, K. D., Cosgrove, M. S., Crosson, S., dhe-Paganon, S., Di Cera, E., Drennan, C. L., Eck, M. J., Eichman, B. F., Fan, Q. R., Ferre-D'Amare, A. R., Fromme, J. C., Garcia, K. C., Gaudet, R., Gong, P., Harrison, S. C., Heldwein, E. E., Jia, Z., Keenan, R. J., Kruse, A. C., Kvansakul, M., McLellan, J. S., Modis, Y., Nam, Y., Otwinowski, Z., Pai, E. F., Barbosa Pereira, P. J., Petosa, C., Raman, S., Rapoport, T. A., Roll-Mecak, A., Rosen, M. K., Rudenko, G., Schlessinger, J., Schwartz, T. U., Shamoo, Y., Sondermann, H., Tao, Y. J., Tolia, N. H., Tsodikov, O. V., Westover, K. D., Wu, H., Foster, I., Fraser, J. S., Maia, F. R., Gonen, T., Kirchhausen, T., Diederichs, K., Crosas, M., Sliz, P. 2016; 7


    Access to experimental X-ray diffraction image data is fundamental for validation and reproduction of macromolecular models and indispensable for development of structural biology processing methods. Here, we established a diffraction data publication and dissemination system, Structural Biology Data Grid (SBDG;, to preserve primary experimental data sets that support scientific publications. Data sets are accessible to researchers through a community driven data grid, which facilitates global data access. Our analysis of a pilot collection of crystallographic data sets demonstrates that the information archived by SBDG is sufficient to reprocess data to statistics that meet or exceed the quality of the original published structures. SBDG has extended its services to the entire community and is used to develop support for other types of biomedical data sets. It is anticipated that access to the experimental data sets will enhance the paradigm shift in the community towards a much more dynamic body of continuously improving data analysis.

    View details for DOI 10.1038/ncomms10882

    View details for Web of Science ID 000371719200001

    View details for PubMedCentralID PMC4786681

  • Alpha and Beta Type 1 Interferon Signaling: Passage for Diverse Biologic Outcomes. Cell Ng, C. T., Mendoza, J. L., Garcia, K. C., Oldstone, M. B. 2016; 164 (3): 349-352


    Type I interferon (IFN-I) elicits a complex cascade of events in response to microbial infection. Here, we review recent developments illuminating the large number of IFN-I species and describing their unique biologic functions.

    View details for DOI 10.1016/j.cell.2015.12.027

    View details for PubMedID 26824652

    View details for PubMedCentralID PMC4733246

  • Structural interplay between germline interactions and adaptive recognition determines the bandwidth of TCR-peptide-MHC cross-reactivity NATURE IMMUNOLOGY Adams, J. J., Narayanan, S., Birnbaum, M. E., Sidhu, S. S., Blevins, S. J., Gee, M. H., Sibener, L. V., Baker, B. M., Kranz, D. M., Garcia, K. C. 2016; 17 (1): 87-?

    View details for DOI 10.1038/ni.3310

    View details for Web of Science ID 000366675800014

    View details for PubMedID 26523866

  • Data publication with the structural biology data grid supports live analysis. Nature communications Meyer, P. A., Socias, S., Key, J., Ransey, E., Tjon, E. C., Buschiazzo, A., Lei, M., Botka, C., Withrow, J., Neau, D., Rajashankar, K., Anderson, K. S., Baxter, R. H., Blacklow, S. C., Boggon, T. J., Bonvin, A. M., Borek, D., Brett, T. J., Caflisch, A., Chang, C., Chazin, W. J., Corbett, K. D., Cosgrove, M. S., Crosson, S., dhe-Paganon, S., Di Cera, E., Drennan, C. L., Eck, M. J., Eichman, B. F., Fan, Q. R., Ferré-D'Amaré, A. R., Christopher Fromme, J., Garcia, K. C., Gaudet, R., Gong, P., Harrison, S. C., Heldwein, E. E., Jia, Z., Keenan, R. J., Kruse, A. C., Kvansakul, M., McLellan, J. S., Modis, Y., Nam, Y., Otwinowski, Z., Pai, E. F., Pereira, P. J., Petosa, C., Raman, C. S., Rapoport, T. A., Roll-Mecak, A., Rosen, M. K., Rudenko, G., Schlessinger, J., Schwartz, T. U., Shamoo, Y., Sondermann, H., Tao, Y. J., Tolia, N. H., Tsodikov, O. V., Westover, K. D., Wu, H., Foster, I., Fraser, J. S., Maia, F. R., Gonen, T., Kirchhausen, T., Diederichs, K., Crosas, M., Sliz, P. 2016; 7: 10882-?


    Access to experimental X-ray diffraction image data is fundamental for validation and reproduction of macromolecular models and indispensable for development of structural biology processing methods. Here, we established a diffraction data publication and dissemination system, Structural Biology Data Grid (SBDG;, to preserve primary experimental data sets that support scientific publications. Data sets are accessible to researchers through a community driven data grid, which facilitates global data access. Our analysis of a pilot collection of crystallographic data sets demonstrates that the information archived by SBDG is sufficient to reprocess data to statistics that meet or exceed the quality of the original published structures. SBDG has extended its services to the entire community and is used to develop support for other types of biomedical data sets. It is anticipated that access to the experimental data sets will enhance the paradigm shift in the community towards a much more dynamic body of continuously improving data analysis.

    View details for DOI 10.1038/ncomms10882

    View details for PubMedID 26947396

  • Control of Synaptic Connectivity by a Network of Drosophila IgSF Cell Surface Proteins CELL Carrillo, R. A., Oezkan, E., Menon, K. P., Nagarkar-Jaiswal, S., Lee, P., Jeon, M., Birnbaum, M. E., Bellen, H. J., Garcia, K. C., Zinn, K. 2015; 163 (7): 1770-1782


    We have defined a network of interacting Drosophila cell surface proteins in which a 21-member IgSF subfamily, the Dprs, binds to a nine-member subfamily, the DIPs. The structural basis of the Dpr-DIP interaction code appears to be dictated by shape complementarity within the Dpr-DIP binding interface. Each of the six dpr and DIP genes examined here is expressed by a unique subset of larval and pupal neurons. In the neuromuscular system, interactions between Dpr11 and DIP-γ affect presynaptic terminal development, trophic factor responses, and neurotransmission. In the visual system, dpr11 is selectively expressed by R7 photoreceptors that use Rh4 opsin (yR7s). Their primary synaptic targets, Dm8 amacrine neurons, express DIP-γ. In dpr11 or DIP-γ mutants, yR7 terminals extend beyond their normal termination zones in layer M6 of the medulla. DIP-γ is also required for Dm8 survival or differentiation. Our findings suggest that Dpr-DIP interactions are important determinants of synaptic connectivity.

    View details for DOI 10.1016/j.cell.2015.11.022

    View details for Web of Science ID 000366854200022

    View details for PubMedID 26687361

    View details for PubMedCentralID PMC4720259

  • Instructive roles for cytokine-receptor binding parameters in determining signaling and functional potency SCIENCE SIGNALING Moraga, I., Richter, D., Wilmes, S., Winkelmann, H., Jude, K., Thomas, C., Suhoski, M. M., Engleman, E. G., Piehler, J., Garcia, K. C. 2015; 8 (402)

    View details for DOI 10.1126/scisignal.aab2677

    View details for PubMedID 26554818

  • Rationally designed chemokine-based toxin targeting the viral G protein-coupled receptor US28 potently inhibits cytomegalovirus infection in vivo. Proceedings of the National Academy of Sciences of the United States of America Spiess, K., Jeppesen, M. G., Malmgaard-Clausen, M., Krzywkowski, K., Dulal, K., Cheng, T., Hjortø, G. M., Larsen, O., Burg, J. S., Jarvis, M. A., Christopher Garcia, K., Zhu, H., Kledal, T. N., Rosenkilde, M. M. 2015; 112 (27): 8427-8432


    The use of receptor-ligand interactions to direct toxins to kill diseased cells selectively has shown considerable promise for treatment of a number of cancers and, more recently, autoimmune disease. Here we move the fusion toxin protein (FTP) technology beyond cancer/autoimmune therapeutics to target the human viral pathogen, human cytomegalovirus (HCMV), on the basis of its expression of the 7TM G protein-coupled chemokine receptor US28. The virus origin of US28 provides an exceptional chemokine-binding profile with high selectivity and improved binding for the CX3C chemokine, CX3CL1. Moreover, US28 is constitutively internalizing by nature, providing highly effective FTP delivery. We designed a synthetic CX3CL1 variant engineered to have ultra-high affinity for US28 and greater specificity for US28 than the natural sole receptor for CX3CL1, CX3CR1, and we fused the synthetic variant with the cytotoxic domain of Pseudomonas Exotoxin A. This novel strategy of a rationally designed FTP provided unparalleled anti-HCMV efficacy and potency in vitro and in vivo.

    View details for DOI 10.1073/pnas.1509392112

    View details for PubMedID 26080445

    View details for PubMedCentralID PMC4500259

  • Antibodies to Interleukin-2 Elicit Selective T Cell Subset Potentiation through Distinct Conformational Mechanisms IMMUNITY Spangler, J. B., Tomala, J., Luca, V. C., Jude, K. M., Dong, S., Ring, A. M., Votavova, P., Pepper, M., Kovar, M., Garcia, K. C. 2015; 42 (5): 815-825


    Interleukin-2 (IL-2) is a pleiotropic cytokine that regulates immune cell homeostasis and has been used to treat a range of disorders including cancer and autoimmune disease. IL-2 signals via interleukin-2 receptor-β (IL-2Rβ):IL-2Rγ heterodimers on cells expressing high (regulatory T cells, Treg) or low (effector cells) amounts of IL-2Rα (CD25). When complexed with IL-2, certain anti-cytokine antibodies preferentially stimulate expansion of Treg (JES6-1) or effector (S4B6) cells, offering a strategy for targeted disease therapy. We found that JES6-1 sterically blocked the IL-2:IL-2Rβ and IL-2:IL-2Rγ interactions, but also allosterically lowered the IL-2:IL-2Rα affinity through a "triggered exchange" mechanism favoring IL-2Rα(hi) Treg cells, creating a positive feedback loop for IL-2Rα(hi) cell activation. Conversely, S4B6 sterically blocked the IL-2:IL-2Rα interaction, while also conformationally stabilizing the IL-2:IL-2Rβ interaction, thus stimulating all IL-2-responsive immune cells, particularly IL-2Rβ(hi) effector cells. These insights provide a molecular blueprint for engineering selectively potentiating therapeutic antibodies.

    View details for DOI 10.1016/j.immuni.2015.04.015

    View details for Web of Science ID 000354827400009

    View details for PubMedID 25992858

    View details for PubMedCentralID PMC4439582

  • Interleukin-2 Activity Can Be Fine Tuned with Engineered Receptor Signaling Clamps IMMUNITY Mitra, S., Ring, A. M., Amarnath, S., Spangler, J. B., Li, P., Ju, W., Fischer, S., Oh, J., Spolski, R., Weiskopf, K., Kohrt, H., Foley, J. E., Rajagopalan, S., Long, E. O., Fowler, D. H., Waldmann, T. A., Garcia, K. C., Leonard, W. J. 2015; 42 (5): 826-838


    Interleukin-2 (IL-2) regulates lymphocyte function by signaling through heterodimerization of the IL-2Rβ and γc receptor subunits. IL-2 is of considerable therapeutic interest, but harnessing its actions in a controllable manner remains a challenge. Previously, we have engineered an IL-2 "superkine" with enhanced affinity for IL-2Rβ. Here, we describe next-generation IL-2 variants that function as "receptor signaling clamps." They retained high affinity for IL-2Rβ, inhibiting binding of endogenous IL-2, but their interaction with γc was weakened, attenuating IL-2Rβ-γc heterodimerization. These IL-2 analogs acted as partial agonists and differentially affected lymphocytes poised at distinct activation thresholds. Moreover, one variant, H9-RETR, antagonized IL-2 and IL-15 better than blocking antibodies against IL-2Rα or IL-2Rβ. Furthermore, this mutein prolonged survival in a model of graft-versus-host disease and blocked spontaneous proliferation of smoldering adult T cell leukemia (ATL) T cells. This receptor-clamping approach might be a general mechanism-based strategy for engineering cytokine partial agonists for therapeutic immunomodulation.

    View details for DOI 10.1016/j.immuni.2015.04.018

    View details for PubMedID 25992859

  • "Velcro" engineering of high affinity CD47 ectodomain as signal regulatory protein a (SIRPa) antagonists that enhance antibody-dependent cellular phagocytosis. journal of biological chemistry Ho, C. C., Guo, N., Sockolosky, J. T., Ring, A. M., Weiskopf, K., Özkan, E., Mori, Y., Weissman, I. L., Garcia, K. C. 2015; 290 (20): 12650-12663


    CD47 is a cell surface protein that transmits an anti-phagocytic signal, known as the "don't-eat-me" signal, to macrophages upon engaging its receptor signal regulatory protein α (SIRPα). Molecules that antagonize the CD47-SIRPα interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRPα variant CV1, have been shown to facilitate macrophage-mediated anti-tumor responses. However, these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47. These factors reduce bioavailability and increase the risk of toxicity. Here, we present an alternative strategy to antagonize the CD47-SIRPα pathway by engineering high affinity CD47 variants that target SIRPα, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRPα. Therefore, we developed a novel engineering approach, whereby we augmented the existing contact interface via N-terminal peptide extension, coined "Velcro" engineering. The high affinity variant (Velcro-CD47) bound to the two most prominent human SIRPα alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRPα on human macrophages. Velcro-CD47 synergizes with tumor-specific monoclonal antibodies to enhance macrophage phagocytosis of tumor cells in vitro, with similar potency as CV1. Finally, Velcro-CD47 interacts specifically with a subset of myeloid-derived cells in human blood, whereas CV1 binds all myeloid, lymphoid, and erythroid populations interrogated. This is consistent with the restricted expression of SIRPα compared with CD47. Herein, we have demonstrated that "Velcro" engineering is a powerful protein-engineering tool with potential applications to other systems and that Velcro-CD47 could be an alternative adjuvant to CD47-targeting agents for cancer immunotherapy.

    View details for DOI 10.1074/jbc.M115.648220

    View details for PubMedID 25837251

    View details for PubMedCentralID PMC4432284

  • Wnt acylation and its functional implication in Wnt signalling regulation BIOCHEMICAL SOCIETY TRANSACTIONS Janda, C. Y., Garcia, K. C. 2015; 43: 211-216


    Wnt proteins are conserved signalling molecules that have an essential role in regulating diverse processes during embryogenesis and adult tissue homoeostasis. Wnts are post-translationally modified by palmitoylation, which is essential for Wnt secretion and function. Intriguingly, the crystal structure of XWnt8 in complex with the extracellular domain of the Frizzled 8 cysteine-rich domain (Fzd8-CRD) revealed that Wnts use the fatty acid as a 'hotspot' residue to engage its receptor, which is a unique mode of receptor-ligand recognition. In addition, there are several lines of evidence suggesting that Wnts engage several signalling modulators and alternative receptors by means of fatty acids as a critical contact residue. In the present article, we review our current understanding of Wnt acylation and its functional role in Wnt signalling regulation.

    View details for DOI 10.1042/BST20140249

    View details for Web of Science ID 000352293500014

    View details for PubMedID 25849919

  • Tuning Cytokine Receptor Signaling by Re-orienting Dimer Geometry with Surrogate Ligands CELL Moraga, I., Wernig, G., Wilmes, S., Gryshkova, V., Richter, C. P., Hong, W., Sinha, R., Guo, F., Fabionar, H., Wehrman, T. S., Krutzik, P., Demharter, S., Plo, I., Weissman, I. L., Minary, P., Majeti, R., Constantinescu, S. N., Piehler, J., Garcia, K. C. 2015; 160 (6): 1196-1208


    Most cell-surface receptors for cytokines and growth factors signal as dimers, but it is unclear whether remodeling receptor dimer topology is a viable strategy to "tune" signaling output. We utilized diabodies (DA) as surrogate ligands in a prototypical dimeric receptor-ligand system, the cytokine Erythropoietin (EPO) and its receptor (EpoR), to dimerize EpoR ectodomains in non-native architectures. Diabody-induced signaling amplitudes varied from full to minimal agonism, and structures of these DA/EpoR complexes differed in EpoR dimer orientation and proximity. Diabodies also elicited biased or differential activation of signaling pathways and gene expression profiles compared to EPO. Non-signaling diabodies inhibited proliferation of erythroid precursors from patients with a myeloproliferative neoplasm due to a constitutively active JAK2V617F mutation. Thus, intracellular oncogenic mutations causing ligand-independent receptor activation can be counteracted by extracellular ligands that re-orient receptors into inactive dimer topologies. This approach has broad applications for tuning signaling output for many dimeric receptor systems.

    View details for DOI 10.1016/j.cell.2015.02.011

    View details for PubMedID 25728669

  • Structural biology. Structural basis for chemokine recognition and activation of a viral G protein-coupled receptor. Science Burg, J. S., Ingram, J. R., Venkatakrishnan, A. J., Jude, K. M., Dukkipati, A., Feinberg, E. N., Angelini, A., Waghray, D., Dror, R. O., Ploegh, H. L., Garcia, K. C. 2015; 347 (6226): 1113-1117


    Chemokines are small proteins that function as immune modulators through activation of chemokine G protein-coupled receptors (GPCRs). Several viruses also encode chemokines and chemokine receptors to subvert the host immune response. How protein ligands activate GPCRs remains unknown. We report the crystal structure at 2.9 angstrom resolution of the human cytomegalovirus GPCR US28 in complex with the chemokine domain of human CX3CL1 (fractalkine). The globular body of CX3CL1 is perched on top of the US28 extracellular vestibule, whereas its amino terminus projects into the central core of US28. The transmembrane helices of US28 adopt an active-state-like conformation. Atomic-level simulations suggest that the agonist-independent activity of US28 may be due to an amino acid network evolved in the viral GPCR to destabilize the receptor's inactive state.

    View details for DOI 10.1126/science.aaa5026

    View details for PubMedID 25745166

    View details for PubMedCentralID PMC4445376

  • Structural biology. Structural basis for Notch1 engagement of Delta-like 4. Science Luca, V. C., Jude, K. M., Pierce, N. W., Nachury, M. V., Fischer, S., Garcia, K. C. 2015; 347 (6224): 847-853


    Notch receptors guide mammalian cell fate decisions by engaging the proteins Jagged and Delta-like (DLL). The 2.3 angstrom resolution crystal structure of the interacting regions of the Notch1-DLL4 complex reveals a two-site, antiparallel binding orientation assisted by Notch1 O-linked glycosylation. Notch1 epidermal growth factor-like repeats 11 and 12 interact with the DLL4 Delta/Serrate/Lag-2 (DSL) domain and module at the N-terminus of Notch ligands (MNNL) domains, respectively. Threonine and serine residues on Notch1 are functionalized with O-fucose and O-glucose, which act as surrogate amino acids by making specific, and essential, contacts to residues on DLL4. The elucidation of a direct chemical role for O-glycans in Notch1 ligand engagement demonstrates how, by relying on posttranslational modifications of their ligand binding sites, Notch proteins have linked their functional capacity to developmentally regulated biosynthetic pathways.

    View details for DOI 10.1126/science.1261093

    View details for PubMedID 25700513

  • Self-determination in the T cell repertoire. Immunity Birnbaum, M. E., Garcia, K. C. 2015; 42 (1): 8-10


    The number of T cells specific for various antigens can vary dramatically. In this issue of Immunity, Nelson et al. (2015) report that these differences might be, at least in part, set by the number of cross-reactive self peptides encountered by T cells during development.

    View details for DOI 10.1016/j.immuni.2014.12.025

    View details for PubMedID 25607452

  • Insights into cytokine-receptor interactions from cytokine engineering. Annual review of immunology Spangler, J. B., Moraga, I., Mendoza, J. L., Garcia, K. C. 2015; 33: 139-167


    Cytokines exert a vast array of immunoregulatory actions critical to human biology and disease. However, the desired immunotherapeutic effects of native cytokines are often mitigated by toxicity or lack of efficacy, either of which results from cytokine receptor pleiotropy and/or undesired activation of off-target cells. As our understanding of the structural principles of cytokine-receptor interactions has advanced, mechanism-based manipulation of cytokine signaling through protein engineering has become an increasingly feasible and powerful approach. Modified cytokines, both agonists and antagonists, have been engineered with narrowed target cell specificities, and they have also yielded important mechanistic insights into cytokine biology and signaling. Here we review the theory and practice of cytokine engineering and rationalize the mechanisms of several engineered cytokines in the context of structure. We discuss specific examples of how structure-based cytokine engineering has opened new opportunities for cytokines as drugs, with a focus on the immunotherapeutic cytokines interferon, interleukin-2, and interleukin-4.

    View details for DOI 10.1146/annurev-immunol-032713-120211

    View details for PubMedID 25493332

  • Molecular architecture of the alpha beta T cell receptor-CD3 complex PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Birnbaum, M. E., Berry, R., Hsiao, Y., Chen, Z., Shingu-Vazquez, M. A., Yu, X., Waghray, D., Fischer, S., McCluskey, J., Rossjohn, J., Walz, T., Garcia, K. C. 2014; 111 (49): 17576-17581


    αβ T-cell receptor (TCR) activation plays a crucial role for T-cell function. However, the TCR itself does not possess signaling domains. Instead, the TCR is noncovalently coupled to a conserved multisubunit signaling apparatus, the CD3 complex, that comprises the CD3εγ, CD3εδ, and CD3ζζ dimers. How antigen ligation by the TCR triggers CD3 activation and what structural role the CD3 extracellular domains (ECDs) play in the assembled TCR-CD3 complex remain unclear. Here, we use two complementary structural approaches to gain insight into the overall organization of the TCR-CD3 complex. Small-angle X-ray scattering of the soluble TCR-CD3εδ complex reveals the CD3εδ ECDs to sit underneath the TCR α-chain. The observed arrangement is consistent with EM images of the entire TCR-CD3 integral membrane complex, in which the CD3εδ and CD3εγ subunits were situated underneath the TCR α-chain and TCR β-chain, respectively. Interestingly, the TCR-CD3 transmembrane complex bound to peptide-MHC is a dimer in which two TCRs project outward from a central core composed of the CD3 ECDs and the TCR and CD3 transmembrane domains. This arrangement suggests a potential ligand-dependent dimerization mechanism for TCR signaling. Collectively, our data advance our understanding of the molecular organization of the TCR-CD3 complex, and provides a conceptual framework for the TCR activation mechanism.

    View details for DOI 10.1073/pnas.1420936111

    View details for Web of Science ID 000345921500055

    View details for PubMedID 25422432

    View details for PubMedCentralID PMC4267357

  • Screening and large-scale expression of membrane proteins in mammalian cells for structural studies NATURE PROTOCOLS Goehring, A., Lee, C., Wang, K. H., Michel, J. C., Claxton, D. P., Baconguis, I., Althoff, T., Fischer, S., Garcia, K. C., Gouaux, E. 2014; 9 (11): 2574-2585


    Structural, biochemical and biophysical studies of eukaryotic membrane proteins are often hampered by difficulties in overexpression of the candidate molecule. Baculovirus transduction of mammalian cells (BacMam), although a powerful method to heterologously express membrane proteins, can be cumbersome for screening and expression of multiple constructs. We therefore developed plasmid Eric Gouaux (pEG) BacMam, a vector optimized for use in screening assays, as well as for efficient production of baculovirus and robust expression of the target protein. In this protocol, we show how to use small-scale transient transfection and fluorescence-detection size-exclusion chromatography (FSEC) experiments using a GFP-His8-tagged candidate protein to screen for monodispersity and expression level. Once promising candidates are identified, we describe how to generate baculovirus, transduce HEK293S GnTI(-) (N-acetylglucosaminyltransferase I-negative) cells in suspension culture and overexpress the candidate protein. We have used these methods to prepare pure samples of chicken acid-sensing ion channel 1a (cASIC1) and Caenorhabditis elegans glutamate-gated chloride channel (GluCl) for X-ray crystallography, demonstrating how to rapidly and efficiently screen hundreds of constructs and accomplish large-scale expression in 4-6 weeks.

    View details for DOI 10.1038/nprot.2014.173

    View details for Web of Science ID 000344248300004

    View details for PubMedCentralID PMC4291175

  • Screening and large-scale expression of membrane proteins in mammalian cells for structural studies. Nature protocols Goehring, A., Lee, C., Wang, K. H., Michel, J. C., Claxton, D. P., Baconguis, I., Althoff, T., Fischer, S., Garcia, K. C., Gouaux, E. 2014; 9 (11): 2574-2585


    Structural, biochemical and biophysical studies of eukaryotic membrane proteins are often hampered by difficulties in overexpression of the candidate molecule. Baculovirus transduction of mammalian cells (BacMam), although a powerful method to heterologously express membrane proteins, can be cumbersome for screening and expression of multiple constructs. We therefore developed plasmid Eric Gouaux (pEG) BacMam, a vector optimized for use in screening assays, as well as for efficient production of baculovirus and robust expression of the target protein. In this protocol, we show how to use small-scale transient transfection and fluorescence-detection size-exclusion chromatography (FSEC) experiments using a GFP-His8-tagged candidate protein to screen for monodispersity and expression level. Once promising candidates are identified, we describe how to generate baculovirus, transduce HEK293S GnTI(-) (N-acetylglucosaminyltransferase I-negative) cells in suspension culture and overexpress the candidate protein. We have used these methods to prepare pure samples of chicken acid-sensing ion channel 1a (cASIC1) and Caenorhabditis elegans glutamate-gated chloride channel (GluCl) for X-ray crystallography, demonstrating how to rapidly and efficiently screen hundreds of constructs and accomplish large-scale expression in 4-6 weeks.

    View details for DOI 10.1038/nprot.2014.173

    View details for PubMedID 25299155

  • Deconstructing the Peptide-MHC Specificity of T Cell Recognition. Cell Birnbaum, M. E., Mendoza, J. L., Sethi, D. K., Dong, S., Glanville, J., Dobbins, J., Ozkan, E., Davis, M. M., Wucherpfennig, K. W., Garcia, K. C. 2014; 157 (5): 1073-1087


    In order to survey a universe of major histocompatibility complex (MHC)-presented peptide antigens whose numbers greatly exceed the diversity of the T cell repertoire, T cell receptors (TCRs) are thought to be cross-reactive. However, the nature and extent of TCR cross-reactivity has not been conclusively measured experimentally. We developed a system to identify MHC-presented peptide ligands by combining TCR selection of highly diverse yeast-displayed peptide-MHC libraries with deep sequencing. Although we identified hundreds of peptides reactive with each of five different mouse and human TCRs, the selected peptides possessed TCR recognition motifs that bore a close resemblance to their known antigens. This structural conservation of the TCR interaction surface allowed us to exploit deep-sequencing information to computationally identify activating microbial and self-ligands for human autoimmune TCRs. The mechanistic basis of TCR cross-reactivity described here enables effective surveillance of diverse self and foreign antigens without necessitating degenerate recognition of nonhomologous peptides.

    View details for DOI 10.1016/j.cell.2014.03.047

    View details for PubMedID 24855945

    View details for PubMedCentralID PMC4071348

  • Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein NATURE METHODS Chu, J., Haynes, R. D., Corbel, S. Y., Li, P., Gonzalez-Gonzalez, E., Burg, J. S., Ataie, N. J., Lam, A. J., Cranfill, P. J., Baird, M. A., Davidson, M. W., Ng, H., Garcia, K. C., Contag, C. H., Shen, K., Blau, H. M., Lin, M. Z. 2014; 11 (5): 572-578


    A method for non-invasive visualization of genetically labeled cells in animal disease models with micrometer-level resolution would greatly facilitate development of cell-based therapies. Imaging of fluorescent proteins (FPs) using red excitation light in the 'optical window' above 600 nm is one potential method for visualizing implanted cells. However, previous efforts to engineer FPs with peak excitation beyond 600 nm have resulted in undesirable reductions in brightness. Here we report three new red-excitable monomeric FPs obtained by structure-guided mutagenesis of mNeptune. Two of these, mNeptune2 and mNeptune2.5, demonstrate improved maturation and brighter fluorescence than mNeptune, whereas the third, mCardinal, has a red-shifted excitation spectrum without reduction in brightness. We show that mCardinal can be used to non-invasively and longitudinally visualize the differentiation of myoblasts into myocytes in living mice with high anatomical detail.

    View details for DOI 10.1038/NMETH.2888

    View details for Web of Science ID 000335873400026

    View details for PubMedID 24633408

    View details for PubMedCentralID PMC4008650

  • Extracellular Architecture of the SYG-1/SYG-2 Adhesion Complex Instructs Synaptogenesis. Cell Ozkan, E., Chia, P. H., Wang, R. R., Goriatcheva, N., Borek, D., Otwinowski, Z., Walz, T., Shen, K., Garcia, K. C. 2014; 156 (3): 482-494


    SYG-1 and SYG-2 are multipurpose cell adhesion molecules (CAMs) that have evolved across all major animal taxa to participate in diverse physiological functions, ranging from synapse formation to formation of the kidney filtration barrier. In the crystal structures of several SYG-1 and SYG-2 orthologs and their complexes, we find that SYG-1 orthologs homodimerize through a common, bispecific interface that similarly mediates an unusual orthogonal docking geometry in the heterophilic SYG-1/SYG-2 complex. C. elegans SYG-1's specification of proper synapse formation in vivo closely correlates with the heterophilic complex affinity, which appears to be tuned for optimal function. Furthermore, replacement of the interacting domains of SYG-1 and SYG-2 with those from CAM complexes that assume alternative docking geometries or the introduction of segmental flexibility compromised synaptic function. These results suggest that SYG extracellular complexes do not simply act as "molecular velcro" and that their distinct structural features are important in instructing synaptogenesis. PAPERFLICK:

    View details for DOI 10.1016/j.cell.2014.01.004

    View details for PubMedID 24485456

  • Multifarious determinants of cytokine receptor signaling specificity. Advances in immunology Moraga, I., Spangler, J., Mendoza, J. L., Garcia, K. C. 2014; 121: 1-39


    Cytokines play crucial roles in regulating immune homeostasis. Two important characteristics of most cytokines are pleiotropy, defined as the ability of one cytokine to exhibit diverse functionalities, and redundancy, defined as the ability of multiple cytokines to exert overlapping activities. Identifying the determinants for unique cellular responses to cytokines in the face of shared receptor usage, pleiotropy, and redundancy will be essential in order to harness the potential of cytokines as therapeutics. Here, we discuss the biophysical (ligand-receptor geometry and affinity) and cellular (receptor trafficking and intracellular abundance of signaling molecules) parameters that contribute to the specificity of cytokine bioactivities. Whereas the role of extracellular ternary complex geometry in cytokine-induced signaling is still not completely elucidated, cytokine-receptor affinity is known to impact signaling through modulation of the stability and kinetics of ternary complex formation. Receptor trafficking also plays an important and likely underappreciated role in the diversification of cytokine bioactivities but it has been challenging to experimentally probe trafficking effects. We also review recent efforts to quantify levels of intracellular signaling components, as second messenger abundance can affect cytokine-induced bioactivities both quantitatively and qualitatively. We conclude by discussing the application of protein engineering to develop therapeutically relevant cytokines with reduced pleiotropy and redirected biological functionalities.

    View details for DOI 10.1016/B978-0-12-800100-4.00001-5

    View details for PubMedID 24388212

  • Activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature Kruse, A. C., Ring, A. M., Manglik, A., Hu, J., Hu, K., Eitel, K., Hübner, H., Pardon, E., Valant, C., Sexton, P. M., Christopoulos, A., Felder, C. C., Gmeiner, P., Steyaert, J., Weis, W. I., Garcia, K. C., Wess, J., Kobilka, B. K. 2013; 504 (7478): 101-106


    Despite recent advances in crystallography and the availability of G-protein-coupled receptor (GPCR) structures, little is known about the mechanism of their activation process, as only the β2 adrenergic receptor (β2AR) and rhodopsin have been crystallized in fully active conformations. Here we report the structure of an agonist-bound, active state of the human M2 muscarinic acetylcholine receptor stabilized by a G-protein mimetic camelid antibody fragment isolated by conformational selection using yeast surface display. In addition to the expected changes in the intracellular surface, the structure reveals larger conformational changes in the extracellular region and orthosteric binding site than observed in the active states of the β2AR and rhodopsin. We also report the structure of the M2 receptor simultaneously bound to the orthosteric agonist iperoxo and the positive allosteric modulator LY2119620. This structure reveals that LY2119620 recognizes a largely pre-formed binding site in the extracellular vestibule of the iperoxo-bound receptor, inducing a slight contraction of this outer binding pocket. These structures offer important insights into the activation mechanism and allosteric modulation of muscarinic receptors.

    View details for DOI 10.1038/nature12735

    View details for PubMedID 24256733

  • Adrenaline-activated structure of ß2-adrenoceptor stabilized by an engineered nanobody. Nature Ring, A. M., Manglik, A., Kruse, A. C., Enos, M. D., Weis, W. I., Garcia, K. C., Kobilka, B. K. 2013; 502 (7472): 575-579


    G-protein-coupled receptors (GPCRs) are integral membrane proteins that have an essential role in human physiology, yet the molecular processes through which they bind to their endogenous agonists and activate effector proteins remain poorly understood. So far, it has not been possible to capture an active-state GPCR bound to its native neurotransmitter. Crystal structures of agonist-bound GPCRs have relied on the use of either exceptionally high-affinity agonists or receptor stabilization by mutagenesis. Many natural agonists such as adrenaline, which activates the β2-adrenoceptor (β2AR), bind with relatively low affinity, and they are often chemically unstable. Using directed evolution, we engineered a high-affinity camelid antibody fragment that stabilizes the active state of the β2AR, and used this to obtain crystal structures of the activated receptor bound to multiple ligands. Here we present structures of the active-state human β2AR bound to three chemically distinct agonists: the ultrahigh-affinity agonist BI167107, the high-affinity catecholamine agonist hydroxybenzyl isoproterenol, and the low-affinity endogenous agonist adrenaline. The crystal structures reveal a highly conserved overall ligand recognition and activation mode despite diverse ligand chemical structures and affinities that range from 100 nM to ∼80 pM. Overall, the adrenaline-bound receptor structure is similar to the others, but it has substantial rearrangements in extracellular loop three and the extracellular tip of transmembrane helix 6. These structures also reveal a water-mediated hydrogen bond between two conserved tyrosines, which appears to stabilize the active state of the β2AR and related GPCRs.

    View details for DOI 10.1038/nature12572

    View details for PubMedID 24056936

  • Human LilrB2 is a ß-amyloid receptor and its murine homolog PirB regulates synaptic plasticity in an Alzheimer's model. Science Kim, T., Vidal, G. S., Djurisic, M., William, C. M., Birnbaum, M. E., Garcia, K. C., Hyman, B. T., Shatz, C. J. 2013; 341 (6152): 1399-1404


    Soluble β-amyloid (Aβ) oligomers impair synaptic plasticity and cause synaptic loss associated with Alzheimer's disease (AD). We report that murine PirB (paired immunoglobulin-like receptor B) and its human ortholog LilrB2 (leukocyte immunoglobulin-like receptor B2), present in human brain, are receptors for Aβ oligomers, with nanomolar affinity. The first two extracellular immunoglobulin (Ig) domains of PirB and LilrB2 mediate this interaction, leading to enhanced cofilin signaling, also seen in human AD brains. In mice, the deleterious effect of Aβ oligomers on hippocampal long-term potentiation required PirB, and in a transgenic model of AD, PirB not only contributed to memory deficits present in adult mice, but also mediated loss of synaptic plasticity in juvenile visual cortex. These findings imply that LilrB2 contributes to human AD neuropathology and suggest therapeutic uses of blocking LilrB2 function.

    View details for DOI 10.1126/science.1242077

    View details for PubMedID 24052308

  • Improving macrophage responses to therapeutic antibodies by molecular engineering of SIRPα variants. Oncoimmunology Weiskopf, K., Ring, A. M., Schnorr, P. J., Volkmer, J. P., Volkmer, A. K., Weissman, I. L., Garcia, K. C. 2013; 2 (9): e25773


    CD47 transduces inhibitory signals through signal-regulatory protein α (SIRPα), a plasma membrane receptor expressed by macrophages. Many cancers upregulate CD47 to evade immunosurveillance. We have recently engineered SIRPα variants that potently antagonize CD47 for use as anticancer immunotherapeutics. These high-affinity SIRPα variants synergize with antineoplastic antibodies by lowering the threshold for macrophage-mediated destruction of malignant cells.

    View details for DOI 10.4161/onci.25773

    View details for PubMedID 24319639

    View details for PubMedCentralID PMC3850276

  • Engineered SIRPa variants as immunotherapeutic adjuvants to anticancer antibodies. Science Weiskopf, K., Ring, A. M., Ho, C. C., Volkmer, J., Levin, A. M., Volkmer, A. K., Ozkan, E., Fernhoff, N. B., van de Rijn, M., Weissman, I. L., Garcia, K. C. 2013; 341 (6141): 88-91


    CD47 is an antiphagocytic signal that cancer cells employ to inhibit macrophage-mediated destruction. Here, we modified the binding domain of human SIRPα, the receptor for CD47, for use as a CD47 antagonist. We engineered high-affinity SIRPα variants with approximately 50,000-fold increased affinity for human CD47 relative to wild-type SIRPα. As high-affinity SIRPα monomers, they potently antagonized CD47 on cancer cells but did not induce macrophage phagocytosis on their own. Instead, they exhibited remarkable synergy with all tumor-specific monoclonal antibodies tested by increasing phagocytosis in vitro and enhancing antitumor responses in vivo. This "one-two punch" directs immune responses against tumor cells while lowering the threshold for macrophage activation, thereby providing a universal method for augmenting the efficacy of therapeutic anticancer antibodies.

    View details for DOI 10.1126/science.1238856

    View details for PubMedID 23722425

  • An extracellular interactome of immunoglobulin and LRR proteins reveals receptor-ligand networks. Cell Özkan, E., Carrillo, R. A., Eastman, C. L., Weiszmann, R., Waghray, D., Johnson, K. G., Zinn, K., Celniker, S. E., Garcia, K. C. 2013; 154 (1): 228-39


    Extracellular domains of cell surface receptors and ligands mediate cell-cell communication, adhesion, and initiation of signaling events, but most existing protein-protein "interactome" data sets lack information for extracellular interactions. We probed interactions between receptor extracellular domains, focusing on a set of 202 proteins composed of the Drosophila melanogaster immunoglobulin superfamily (IgSF), fibronectin type III (FnIII), and leucine-rich repeat (LRR) families, which are known to be important in neuronal and developmental functions. Out of 20,503 candidate protein pairs tested, we observed 106 interactions, 83 of which were previously unknown. We "deorphanized" the 20 member subfamily of defective-in-proboscis-response IgSF proteins, showing that they selectively interact with an 11 member subfamily of previously uncharacterized IgSF proteins. Both subfamilies interact with a single common "orphan" LRR protein. We also observed interactions between Hedgehog and EGFR pathway components. Several of these interactions could be visualized in live-dissected embryos, demonstrating that this approach can identify physiologically relevant receptor-ligand pairs.

    View details for DOI 10.1016/j.cell.2013.06.006

    View details for PubMedID 23827685

    View details for PubMedCentralID PMC3756661

  • Plum, an Immunoglobulin Superfamily Protein, Regulates Axon Pruning by Facilitating TGF-ß Signaling. Neuron Yu, X. M., Gutman, I., Mosca, T. J., Iram, T., Ozkan, E., Garcia, K. C., Luo, L., Schuldiner, O. 2013; 78 (3): 456-468


    Axon pruning during development is essential for proper wiring of the mature nervous system, but its regulation remains poorly understood. We have identified an immunoglobulin superfamily (IgSF) transmembrane protein, Plum, that is cell autonomously required for axon pruning of mushroom body (MB) γ neurons and for ectopic synapse refinement at the developing neuromuscular junction in Drosophila. Plum promotes MB γ neuron axon pruning by regulating the expression of Ecdysone Receptor-B1, a key initiator of axon pruning. Genetic analyses indicate that Plum acts to facilitate signaling of Myoglianin, a glial-derived TGF-β, on MB γ neurons upstream of the type-I TGF-β receptor Baboon. Myoglianin, Baboon, and Ecdysone Receptor-B1 are also required for neuromuscular junction ectopic synapse refinement. Our study highlights both IgSF proteins and TGF-β facilitation as key promoters of developmental axon elimination and demonstrates a mechanistic conservation between MB axon pruning during metamorphosis and the refinement of ectopic larval neuromuscular connections.

    View details for DOI 10.1016/j.neuron.2013.03.004

    View details for PubMedID 23664613

  • Mechanistic and structural insight into the functional dichotomy between IL-2 and IL-15. Nature immunology Ring, A. M., Lin, J., Feng, D., Mitra, S., Rickert, M., Bowman, G. R., Pande, V. S., Li, P., Moraga, I., Spolski, R., Ozkan, E., Leonard, W. J., Garcia, K. C. 2012; 13 (12): 1187-1195


    Interleukin 15 (IL-15) and IL-2 have distinct immunological functions even though both signal through the receptor subunit IL-2Rβ and the common γ-chain (γ(c)). Here we found that in the structure of the IL-15-IL-15Rα-IL-2Rβ-γ(c) quaternary complex, IL-15 binds to IL-2Rβ and γ(c) in a heterodimer nearly indistinguishable from that of the IL-2-IL-2Rα-IL-2Rβ-γ(c) complex, despite their different receptor-binding chemistries. IL-15Rα substantially increased the affinity of IL-15 for IL-2Rβ, and this allostery was required for IL-15 trans signaling. Consistent with their identical IL-2Rβ-γ(c) dimer geometries, IL-2 and IL-15 showed similar signaling properties in lymphocytes, with any differences resulting from disparate receptor affinities. Thus, IL-15 and IL-2 induced similar signals, and the cytokine specificity of IL-2Rα versus IL-15Rα determined cellular responsiveness. Our results provide new insights for the development of specific immunotherapeutics based on IL-15 or IL-2.

    View details for DOI 10.1038/ni.2449

    View details for PubMedID 23104097

  • Redirecting cell-type specific cytokine responses with engineered interleukin-4 superkines NATURE CHEMICAL BIOLOGY Junttila, I. S., Creusot, R. J., Moraga, I., Bates, D. L., Wong, M. T., Alonso, M. N., Suhoski, M. M., Lupardus, P., Meier-Schellersheim, M., Engleman, E. G., Utz, P. J., Fathman, C. G., Paul, W. E., Garcia, K. C. 2012; 8 (12): 990-998


    Cytokines dimerize their receptors, with the binding of the 'second chain' triggering signaling. In the interleukin (IL)-4 and IL-13 system, different cell types express varying numbers of alternative second receptor chains (γc or IL-13Rα1), forming functionally distinct type I or type II complexes. We manipulated the affinity and specificity of second chain recruitment by human IL-4. A type I receptor-selective IL-4 'superkine' with 3,700-fold higher affinity for γc was three- to ten-fold more potent than wild-type IL-4. Conversely, a variant with high affinity for IL-13Rα1 more potently activated cells expressing the type II receptor and induced differentiation of dendritic cells from monocytes, implicating the type II receptor in this process. Superkines showed signaling advantages on cells with lower second chain numbers. Comparative transcriptional analysis reveals that the superkines induce largely redundant gene expression profiles. Variable second chain numbers can be exploited to redirect cytokines toward distinct cell subsets and elicit new actions, potentially improving the selectivity of cytokine therapy.

    View details for DOI 10.1038/NCHEMBIO.1096

    View details for Web of Science ID 000311491200014

    View details for PubMedID 23103943

    View details for PubMedCentralID PMC3508151

  • Insights into immune structure, recognition, and signaling. Immunological reviews Garcia, K. C. 2012; 250 (1): 5-9

    View details for DOI 10.1111/imr.12007

    View details for PubMedID 23046119

    View details for PubMedCentralID PMC3982141

  • Diversity-oriented approaches for interrogating T-cell receptor repertoire, ligand recognition, and function IMMUNOLOGICAL REVIEWS Birnbaum, M. E., Dong, S., Garcia, K. C. 2012; 250: 82-101


    Molecular diversity lies at the heart of adaptive immunity. T-cell receptors and peptide-major histocompatibility complex molecules utilize and rely upon an enormous degree of diversity at the levels of genetics, chemistry, and structure to engage one another and carry out their functions. This high level of diversity complicates the systematic study of important aspects of T-cell biology, but recent technical advances have allowed for the ability to study diversity in a comprehensive manner. In this review, we assess insights gained into T-cell receptor function and biology from our increasingly precise ability to assess the T-cell repertoire as a whole or to perturb individual receptors with engineered reagents. We conclude with a perspective on a new class of high-affinity, non-stimulatory peptide ligands we have recently discovered using diversity-oriented techniques that challenges notions for how we think about T-cell receptor signaling.

    View details for DOI 10.1111/imr.12006

    View details for Web of Science ID 000309752300006

    View details for PubMedID 23046124

    View details for PubMedCentralID PMC3474532

  • Reconciling views on T cell receptor germline bias for MHC. Trends in immunology Garcia, K. C. 2012; 33 (9): 429-36


    Whether MHC restriction by the T cell receptor (TCR) is a product of evolutionary pressures leading to germline-encoded 'rules of engagement' remains avidly debated. Structural results derived from analysis of TCR-peptide-MHC complexes appear to support a model of physical specificity between TCR germline V regions and MHC. Yet, some recent evidence suggests that thymic selection, and co-receptors may have misled us into thinking the TCR is exclusively MHC-specific, when in fact, TCRs can robustly engage non-MHC ligands when given the chance. Here, I propose that seemingly contradictory data and hypotheses for, and against, germline bias are, in fact, compatible and can be reconciled into a unifying model.

    View details for DOI 10.1016/

    View details for PubMedID 22771140

    View details for PubMedCentralID PMC3983780

  • Structural Architecture and Functional Evolution of Wnts DEVELOPMENTAL CELL Bazan, J. F., Janda, C. Y., Garcia, K. C. 2012; 23 (2): 227-232


    The crystal structure of a Wnt morphogen bound to its Frizzled receptor ectodomain provides insights into the evolutionary provenance of this complex fold and offers an explanation for why Wnts utilize both lipid- and protein-mediated contacts to engage Frizzleds.

    View details for DOI 10.1016/j.devcel.2012.07.011

    View details for Web of Science ID 000307695500001

    View details for PubMedID 22898770

    View details for PubMedCentralID PMC3458506

  • Structural Basis of Wnt Recognition by Frizzled SCIENCE Janda, C. Y., Waghray, D., Levin, A. M., Thomas, C., Garcia, K. C. 2012; 337 (6090): 59-64


    Wnts are lipid-modified morphogens that play critical roles in development principally through engagement of Frizzled receptors. The 3.25 angstrom structure of Xenopus Wnt8 (XWnt8) in complex with mouse Frizzled-8 (Fz8) cysteine-rich domain (CRD) reveals an unusual two-domain Wnt structure, not obviously related to known protein folds, resembling a "hand" with "thumb" and "index" fingers extended to grasp the Fz8-CRD at two distinct binding sites. One site is dominated by a palmitoleic acid lipid group projecting from serine 187 at the tip of Wnt's thumb into a deep groove in the Fz8-CRD. In the second binding site, the conserved tip of Wnt's "index finger" forms hydrophobic amino acid contacts with a depression on the opposite side of the Fz8-CRD. The conservation of amino acids in both interfaces appears to facilitate ligand-receptor cross-reactivity, which has important implications for understanding Wnt's functional pleiotropy and for developing Wnt-based drugs for cancer and regenerative medicine.

    View details for DOI 10.1126/science.1222879

    View details for PubMedID 22653731

  • Exploiting a natural conformational switch to engineer an interleukin-2 'superkine' NATURE Levin, A. M., Bates, D. L., Ring, A. M., Krieg, C., Lin, J. T., Su, L., Moraga, I., Raeber, M. E., Bowman, G. R., Novick, P., Pande, V. S., Fathman, C. G., Boyman, O., Garcia, K. C. 2012; 484 (7395): 529-U159


    The immunostimulatory cytokine interleukin-2 (IL-2) is a growth factor for a wide range of leukocytes, including T cells and natural killer (NK) cells. Considerable effort has been invested in using IL-2 as a therapeutic agent for a variety of immune disorders ranging from AIDS to cancer. However, adverse effects have limited its use in the clinic. On activated T cells, IL-2 signals through a quaternary 'high affinity' receptor complex consisting of IL-2, IL-2Rα (termed CD25), IL-2Rβ and IL-2Rγ. Naive T cells express only a low density of IL-2Rβ and IL-2Rγ, and are therefore relatively insensitive to IL-2, but acquire sensitivity after CD25 expression, which captures the cytokine and presents it to IL-2Rβ and IL-2Rγ. Here, using in vitro evolution, we eliminated the functional requirement of IL-2 for CD25 expression by engineering an IL-2 'superkine' (also called super-2) with increased binding affinity for IL-2Rβ. Crystal structures of the IL-2 superkine in free and receptor-bound forms showed that the evolved mutations are principally in the core of the cytokine, and molecular dynamics simulations indicated that the evolved mutations stabilized IL-2, reducing the flexibility of a helix in the IL-2Rβ binding site, into an optimized receptor-binding conformation resembling that when bound to CD25. The evolved mutations in the IL-2 superkine recapitulated the functional role of CD25 by eliciting potent phosphorylation of STAT5 and vigorous proliferation of T cells irrespective of CD25 expression. Compared to IL-2, the IL-2 superkine induced superior expansion of cytotoxic T cells, leading to improved antitumour responses in vivo, and elicited proportionally less expansion of T regulatory cells and reduced pulmonary oedema. Collectively, we show that in vitro evolution has mimicked the functional role of CD25 in enhancing IL-2 potency and regulating target cell specificity, which has implications for immunotherapy.

    View details for DOI 10.1038/nature10975

    View details for Web of Science ID 000303200400054

    View details for PubMedID 22446627

    View details for PubMedCentralID PMC3338870

  • Structure of the activating IL-1 receptor signaling complex NATURE STRUCTURAL & MOLECULAR BIOLOGY Thomas, C., Bazan, J. F., Garcia, K. C. 2012; 19 (4): 455-457


    Interleukin-1 (IL-1)-family cytokines are mediators of innate and adaptive immunity. They exert proinflammatory effects by binding a primary receptor that recruits a receptor accessory protein to form a signaling-competent heterotrimeric complex. Here we present the crystal structure of IL-1β bound to its primary receptor IL-1RI and its receptor accessory protein IL-1RAcP, providing insight into how IL-1-type cytokines initiate signaling and revealing an evolutionary relationship with the fibroblast growth factor receptor family.

    View details for DOI 10.1038/nsmb.2260

    View details for PubMedID 22426547

  • Secreted Semaphorins from Degenerating Larval ORN Axons Direct Adult Projection Neuron Dendrite Targeting NEURON Sweeney, L. B., Chou, Y., Wu, Z., Joo, W., Komiyama, T., Potter, C. J., Kolodkin, A. L., Garcia, K. C., Luo, L. 2011; 72 (5): 734-747


    During assembly of the Drosophila olfactory circuit, projection neuron (PN) dendrites prepattern the developing antennal lobe before the arrival of axons from their presynaptic partners, the adult olfactory receptor neurons (ORNs). We previously found that levels of transmembrane Semaphorin-1a, which acts as a receptor, instruct PN dendrite targeting along the dorsolateral-ventromedial axis. Here we show that two secreted semaphorins, Sema-2a and Sema-2b, provide spatial cues for PN dendrite targeting. Sema-2a and Sema-2b proteins are distributed in gradients opposing the Sema-1a protein gradient, and Sema-1a binds to Sema-2a-expressing cells. In Sema-2a and Sema-2b double mutants, PN dendrites that normally target dorsolaterally in the antennal lobe mistarget ventromedially, phenocopying cell-autonomous Sema-1a removal from these PNs. Cell ablation, cell-specific knockdown, and rescue experiments indicate that secreted semaphorins from degenerating larval ORN axons direct dendrite targeting. Thus, a degenerating brain structure instructs the wiring of a developing circuit through the repulsive action of secreted semaphorins.

    View details for DOI 10.1016/j.neuron.2011.09.026

    View details for PubMedID 22153371

  • T Cell Receptor Signaling Is Limited by Docking Geometry to Peptide-Major Histocompatibility Complex IMMUNITY Adams, J. J., Narayanan, S., Liu, B., Birnbaum, M. E., Kruse, A. C., Bowerman, N. A., Chen, W., Levin, A. M., Connolly, J. M., Zhu, C., Kranz, D. M., Garcia, K. C. 2011; 35 (5): 681-693


    T cell receptor (TCR) engagement of peptide-major histocompatibility complex (pMHC) is essential to adaptive immunity, but it is unknown whether TCR signaling responses are influenced by the binding topology of the TCR-peptide-MHC complex. We developed yeast-displayed pMHC libraries that enabled us to identify new peptide sequences reactive with a single TCR. Structural analysis showed that four peptides bound to the TCR with distinct 3D and 2D affinities using entirely different binding chemistries. Three of the peptides that shared a common docking mode, where key TCR-MHC germline interactions are preserved, induced TCR signaling. The fourth peptide failed to induce signaling and was recognized in a substantially different TCR-MHC binding mode that apparently exceeded geometric tolerances compatible with signaling. We suggest that the stereotypical TCR-MHC docking paradigm evolved from productive signaling geometries and that TCR signaling can be modulated by peptides that are recognized in alternative TCR-pMHC binding orientations.

    View details for DOI 10.1016/j.immuni.2011.09.013

    View details for Web of Science ID 000297390800009

    View details for PubMedID 22101157

    View details for PubMedCentralID PMC3253265

  • Structural Linkage between Ligand Discrimination and Receptor Activation by Type I Interferons CELL Thomas, C., Moraga, I., Levin, D., Krutzik, P. O., Podoplelova, Y., Trejo, A., Lee, C., Yarden, G., Vleck, S. E., Glenn, J. S., Nolan, G. P., Piehler, J., Schreiber, G., Garcia, K. C. 2011; 146 (4): 621-632


    Type I Interferons (IFNs) are important cytokines for innate immunity against viruses and cancer. Sixteen human type I IFN variants signal through the same cell-surface receptors, IFNAR1 and IFNAR2, yet they can evoke markedly different physiological effects. The crystal structures of two human type I IFN ternary signaling complexes containing IFNα2 and IFNω reveal recognition modes and heterotrimeric architectures that are unique among the cytokine receptor superfamily but conserved between different type I IFNs. Receptor-ligand cross-reactivity is enabled by conserved receptor-ligand "anchor points" interspersed among ligand-specific interactions that "tune" the relative IFN-binding affinities, in an apparent extracellular "ligand proofreading" mechanism that modulates biological activity. Functional differences between IFNs are linked to their respective receptor recognition chemistries, in concert with a ligand-induced conformational change in IFNAR1, that collectively control signal initiation and complex stability, ultimately regulating differential STAT phosphorylation profiles, receptor internalization rates, and downstream gene expression patterns.

    View details for DOI 10.1016/j.cell.2011.06.048

    View details for PubMedID 21854986

  • Structural Basis of Specificity and Cross-Reactivity in T Cell Receptors Specific for Cytochrome c-I-E-k JOURNAL OF IMMUNOLOGY Newell, E. W., Ely, L. K., Kruse, A. C., Reay, P. A., Rodriguez, S. N., Lin, A. E., Kuhns, M. S., Garcia, K. C., Davis, M. M. 2011; 186 (10): 5823-5832


    T cells specific for the cytochrome c Ag are widely used to investigate many aspects of TCR specificity and interactions with peptide-MHC, but structural information has long been elusive. In this study, we present structures for the well-studied 2B4 TCR, as well as a naturally occurring variant of the 5c.c7 TCR, 226, which is cross-reactive with more than half of possible substitutions at all three TCR-sensitive residues on the peptide Ag. These structures alone and in complex with peptide-MHC ligands allow us to reassess many prior mutagenesis results. In addition, the structure of 226 bound to one peptide variant, p5E, shows major changes in the CDR3 contacts compared with wild-type, yet the TCR V-region contacts with MHC are conserved. These and other data illustrate the ability of TCRs to accommodate large variations in CDR3 structure and peptide contacts within the constraints of highly conserved TCR-MHC interactions.

    View details for DOI 10.4049/jimmunol.1100197

    View details for Web of Science ID 000290150700034

    View details for PubMedID 21490152

  • Although Divergent in Residues of the Peptide Binding Site, Conserved Chimpanzee Patr-AL and Polymorphic Human HLA-A*02 Have Overlapping Peptide-Binding Repertoires JOURNAL OF IMMUNOLOGY Gleimer, M., Wahl, A. R., Hickman, H. D., Abi-Rached, L., Norman, P. J., Guethlein, L. A., Hammond, J. A., Draghi, M., Adams, E. J., Juo, S., Jalili, R., Gharizadeh, B., Ronaghi, M., Garcia, K. C., Hildebrand, W. H., Parham, P. 2011; 186 (3): 1575-1588


    Patr-AL is an expressed, non-polymorphic MHC class I gene carried by ∼50% of chimpanzee MHC haplotypes. Comparing Patr-AL(+) and Patr-AL(-) haplotypes showed Patr-AL defines a unique 125-kb genomic block flanked by blocks containing classical Patr-A and pseudogene Patr-H. Orthologous to Patr-AL are polymorphic orangutan Popy-A and the 5' part of human pseudogene HLA-Y, carried by ∼10% of HLA haplotypes. Thus, the AL gene alternatively evolved in these closely related species to become classical, nonclassical, and nonfunctional. Although differing by 30 aa substitutions in the peptide-binding α(1) and α(2) domains, Patr-AL and HLA-A*0201 bind overlapping repertoires of peptides; the overlap being comparable with that between the A*0201 and A*0207 subtypes differing by one substitution. Patr-AL thus has the A02 supertypic peptide-binding specificity. Patr-AL and HLA-A*0201 have similar three-dimensional structures, binding peptides in similar conformation. Although comparable in size and shape, the B and F specificity pockets of Patr-AL and HLA-A*0201 differ in both their constituent residues and contacts with peptide anchors. Uniquely shared by Patr-AL, HLA-A*0201, and other members of the A02 supertype are the absence of serine at position 9 in the B pocket and the presence of tyrosine at position 116 in the F pocket. Distinguishing Patr-AL from HLA-A*02 is an unusually electropositive upper face on the α(2) helix. Stimulating PBMCs from Patr-AL(-) chimpanzees with B cells expressing Patr-AL produced potent alloreactive CD8 T cells with specificity for Patr-AL and no cross-reactivity toward other MHC class I molecules, including HLA-A*02. In contrast, PBMCs from Patr-AL(+) chimpanzees are tolerant of Patr-AL.

    View details for DOI 10.4049/jimmunol.1002990

    View details for Web of Science ID 000286381200037

    View details for PubMedID 21209280

    View details for PubMedCentralID PMC3124313

  • Structural Snapshots of Full-Length Jak1, a Transmembrane gp130/IL-6/IL-6R alpha Cytokine Receptor Complex, and the Receptor-Jak1 Holocomplex STRUCTURE Lupardus, P. J., Skiniotis, G., Rice, A. J., Thomas, C., Fischer, S., Walz, T., Garcia, K. C. 2011; 19 (1): 45-55


    The shared cytokine receptor gp130 signals as a homodimer or heterodimer through activation of Janus kinases (Jaks) associated with the receptor intracellular domains. Here, we reconstitute, in parts and whole, the full-length gp130 homodimer in complex with the cytokine interleukin-6 (IL-6), its alpha receptor (IL-6Rα) and Jak1, for electron microscopy imaging. We find that the full-length gp130 homodimer complex has intimate interactions between the trans- and juxtamembrane segments of the two receptors, appearing to form a continuous connection between the extra- and intracellular regions. 2D averages and 3D reconstructions of full-length Jak1 reveal a three lobed structure comprising FERM-SH2, pseudokinase, and kinase modules possessing extensive intersegmental flexibility that likely facilitates allosteric activation. Single-particle imaging of the gp130/IL-6/IL-6Rα/Jak1 holocomplex shows Jak1 associated with the membrane proximal intracellular regions of gp130, abutting the would-be inner leaflet of the cell membrane. Jak1 association with gp130 is enhanced by the presence of a membrane environment.

    View details for DOI 10.1016/j.str.2010.10.010

    View details for PubMedID 21220115

  • Rational Design of Inhibitors and Activity-Based Probes Targeting Clostridium difficile Virulence Factor TcdB CHEMISTRY & BIOLOGY Puri, A. W., Lupardus, P. J., Deu, E., Albrow, V. E., Garcia, K. C., Bogyo, M., Shen, A. 2010; 17 (11): 1201-1211


    Clostridium difficile is a leading cause of nosocomial infections. The major virulence factors of this pathogen are the multi-domain toxins TcdA and TcdB. These toxins contain a cysteine protease domain (CPD) that autoproteolytically releases a cytotoxic effector domain upon binding intracellular inositol hexakisphosphate. Currently, there are no known inhibitors of this protease. Here, we describe the rational design of covalent small molecule inhibitors of TcdB CPD. We identified compounds that inactivate TcdB holotoxin function in cells and solved the structure of inhibitor-bound protease to 2.0 Å. This structure reveals the molecular basis of CPD substrate recognition and informed the synthesis of activity-based probes for this enzyme. The inhibitors presented will guide the development of therapeutics targeting C. difficile, and the probes will serve as tools for studying the unique activation mechanism of bacterial toxin CPDs.

    View details for DOI 10.1016/j.chembiol.2010.09.011

    View details for Web of Science ID 000285405000009

    View details for PubMedID 21095570

    View details for PubMedCentralID PMC3005307

  • Peptide-MHC heterodimers show that thymic positive selection requires a more restricted set of self-peptides than negative selection JOURNAL OF EXPERIMENTAL MEDICINE Juang, J., Ebert, P. J., Feng, D., Garcia, K. C., Krogsgaard, M., Davis, M. M. 2010; 207 (6): 1223-1234


    T cell selection and maturation in the thymus depends on the interactions between T cell receptors (TCRs) and different self-peptide-major histocompatibility complex (pMHC) molecules. We show that the affinity of the OT-I TCR for its endogenous positively selecting ligands, Catnb-H-2Kb and Cappa1-H-2Kb, is significantly lower than for previously reported positively selecting altered peptide ligands. To understand how these extremely weak endogenous ligands produce signals in maturing thymocytes, we generated soluble monomeric and dimeric peptide-H-2Kb ligands. Soluble monomeric ovalbumin (OVA)-Kb molecules elicited no detectable signaling in OT-I thymocytes, whereas heterodimers of OVA-Kb paired with positively selecting or nonselecting endogenous peptides, but not an engineered null peptide, induced deletion. In contrast, dimer-induced positive selection was much more sensitive to the identity of the partner peptide. Catnb-Kb-Catnb-Kb homodimers, but not heterodimers of Catnb-Kb paired with a nonselecting peptide-Kb, induced positive selection, even though both ligands bind the OT-I TCR with detectable affinity. Thus, both positive and negative selection can be driven by dimeric but not monomeric ligands. In addition, positive selection has much more stringent requirements for the partner self-pMHC.

    View details for DOI 10.1084/jem.20092170

    View details for Web of Science ID 000278554200011

    View details for PubMedID 20457759

    View details for PubMedCentralID PMC2882826

  • Molecular Basis for Shared Cytokine Recognition Revealed in the Structure of an Unusually High Affinity Complex between IL-13 and IL-13R alpha 2 STRUCTURE Lupardus, P. J., Birnbaum, M. E., Garcia, K. C. 2010; 18 (3): 332-342


    Interleukin-13 is a cytokine important for development of T helper cell type 2 (Th2) responses and plays a critical role in asthma and allergy. The IL-13 Receptor alpha2 (IL-13Ralpha2) is a receptor for IL-13 lacking canonical Jak/STAT signaling functions. Here we present the crystal structure along with a mutational and biophysical analysis of the IL-13/IL-13Ralpha2 complex. While retaining a similar mode of IL-13 binding to its related signaling receptor, IL-13Ralpha1, IL-13Ralpha2 uses peripheral receptor residues unused in the IL-13/IL-13Ralpha1 complex to generate a larger and more complementary interface for IL-13. This results in a four orders of magnitude increase in affinity, to the femtomolar level, compared to IL-13Ralpha1. Alanine scanning mutagenesis of the IL-13 interface reveals several common "hotspot" residues important for binding to both receptors, but also identifies a prominent IL-13Ralpha2-specific contact. These results provide a framework for development of receptor subtype-selective IL-13 antagonists and indicate a decoy function for IL-13Ralpha2.

    View details for DOI 10.1016/j.str.2010.01.003

    View details for Web of Science ID 000275492000010

    View details for PubMedID 20223216

    View details for PubMedCentralID PMC2850121

  • Molecular and Structural Insight into proNGF Engagement of p75NTR and Sortilin JOURNAL OF MOLECULAR BIOLOGY Feng, D., Kim, T., Ozkan, E., Light, M., Torkin, R., Teng, K. K., Hempstead, B. L., Garcia, K. C. 2010; 396 (4): 967-984


    Nerve growth factor (NGF) is initially synthesized as a precursor, proNGF, that is cleaved to release its C-terminal mature form. Recent studies suggested that proNGF is not an inactive precursor but acts as a signaling ligand distinct from its mature counterpart. proNGF and mature NGF initiate opposing biological responses by utilizing both distinct and shared receptor components. In this study, we carried out structural and biochemical characterization of proNGF interactions with p75NTR and sortilin. We crystallized proNGF complexed to p75NTR and present the structure at 3.75-A resolution. The structure reveals a 2:2 symmetric binding mode, as compared with the asymmetric structure of a previously reported crystal structure of mature NGF complexed to p75NTR and the 2:2 symmetric complex of neurotrophin-3 (NT-3) and p75NTR. Here, we discuss the possible origins and implications of the different stoichiometries. In the proNGF-p75NTR complex, the pro regions of proNGF are mostly disordered and two hairpin loops (loop 2) at the top of the NGF dimer have undergone conformational changes in comparison with mature NT structures, suggesting possible interactions with the propeptide. We further explored the binding characteristics of proNGF to sortilin using surface plasmon resonance and cell-based assays and determined that calcium ions promote the formation of a stable ternary complex of proNGF-sortilin-p75NTR. These results, together with those of previous structural and mechanistic studies of NT-receptor interactions, suggest the potential for distinct signaling activities through p75NTR mediated by different NT-induced conformational changes.

    View details for DOI 10.1016/j.jmb.2009.12.030

    View details for PubMedID 20036257

  • Structural basis of receptor sharing by interleukin 17 cytokines NATURE IMMUNOLOGY Ely, L. K., Fischer, S., Garcia, K. C. 2009; 10 (12): 1245-U3


    Interleukin 17 (IL-17)-producing helper T cells (T(H)-17 cells), together with their effector cytokines, including members of the IL-17 family, are emerging as key mediators of chronic inflammatory and autoimmune disorders. Here we present the crystal structure of a complex of IL-17 receptor A (IL-17RA) bound to IL-17F in a 1:2 stoichiometry. The mechanism of complex formation was unique for cytokines and involved the engagement of IL-17 by two fibronectin-type domains of IL-17RA in a groove between the IL-17 homodimer interface. Binding of the first receptor to the IL-17 cytokines modulated the affinity and specificity of the second receptor-binding event, thereby promoting heterodimeric versus homodimeric complex formation. IL-17RA used a common recognition strategy to bind to several members of the IL-17 family, which allows it to potentially act as a shared receptor in multiple different signaling complexes.

    View details for DOI 10.1038/ni.1813

    View details for Web of Science ID 000271872800007

    View details for PubMedID 19838198

    View details for PubMedCentralID PMC2783927

  • CPDadh: A new peptidase family homologous to the cysteine protease domain in bacterial MARTX toxins PROTEIN SCIENCE Pei, J., Lupardus, P. J., Garcia, K. C., Grishin, N. V. 2009; 18 (4): 856-862


    A cysteine protease domain (CPD) has been recently discovered in a group of multifunctional, autoprocessing RTX toxins (MARTX) and Clostridium difficile toxins A and B. These CPDs (referred to as CPDmartx) autocleave the toxins to release domains with toxic effects inside host cells. We report identification and computational analysis of CPDadh, a new cysteine peptidase family homologous to CPDmartx. CPDadh and CPDmartx share a Rossmann-like structural core and conserved catalytic residues. In bacteria, domains of the CPDadh family are present at the N-termini of a diverse group of putative cell-cell interaction proteins and at the C-termini of some RHS (recombination hot spot) proteins. In eukaryotes, catalytically inactive members of the CPDadh family are found in cell surface protein NELF (nasal embryonic LHRH factor) and some putative signaling proteins.

    View details for DOI 10.1002/pro.78

    View details for Web of Science ID 000264942000021

    View details for PubMedID 19309740

  • STIM1 Clusters and Activates CRAC Channels via Direct Binding of a Cytosolic Domain to Orai1 CELL Park, C. Y., Hoover, P. J., Mullins, F. M., Bachhawat, P., Covington, E. D., Raunser, S., Walz, T., Garcia, K. C., Dolmetsch, R. E., Lewis, R. S. 2009; 136 (5): 876-890


    Store-operated Ca(2+) channels activated by the depletion of Ca(2+) from the endoplasmic reticulum (ER) are a major Ca(2+) entry pathway in nonexcitable cells and are essential for T cell activation and adaptive immunity. After store depletion, the ER Ca(2+) sensor STIM1 and the CRAC channel protein Orai1 redistribute to ER-plasma membrane (PM) junctions, but the fundamental issue of how STIM1 activates the CRAC channel at these sites is unresolved. Here, we identify a minimal, highly conserved 107-aa CRAC activation domain (CAD) of STIM1 that binds directly to the N and C termini of Orai1 to open the CRAC channel. Purified CAD forms a tetramer that clusters CRAC channels, but analysis of STIM1 mutants reveals that channel clustering is not sufficient for channel activation. These studies establish a molecular mechanism for store-operated Ca(2+) entry in which the direct binding of STIM1 to Orai1 drives the accumulation and the activation of CRAC channels at ER-PM junctions.

    View details for DOI 10.1016/j.cell.2009.02.014

    View details for Web of Science ID 000263930900011

    View details for PubMedID 19249086

    View details for PubMedCentralID PMC2670439

  • The molecular basis of TCR germline bias for MHC is surprisingly simple NATURE IMMUNOLOGY Garcia, K. C., Adams, J. J., Feng, D., Ely, L. K. 2009; 10 (2): 143-147


    The elusive etiology of germline bias of the T cell receptor (TCR) for major histocompatibility complex (MHC) has been clarified by recent 'proof-of-concept' structural results demonstrating the conservation of specific TCR-MHC interfacial contacts in complexes bearing common variable segments and MHC allotypes. We suggest that each TCR variable-region gene product engages each type of MHC through a 'menu' of structurally coded recognition motifs that have arisen through coevolution. The requirement for MHC-restricted T cell recognition during thymic selection and peripheral surveillance has necessitated the existence of such a coded recognition system. Given these findings, a reconsideration of the TCR-peptide-MHC structural database shows that not only have the answers been there all along but also they were predictable by the first principles of physical chemistry.

    View details for DOI 10.1038/ni.f.219

    View details for Web of Science ID 000263178500007

    View details for PubMedID 19148199

    View details for PubMedCentralID PMC3982143

  • Structural Biology of Shared Cytokine Receptors ANNUAL REVIEW OF IMMUNOLOGY Wang, X., Lupardus, P., LaPorte, S. L., Garcia, K. C. 2009; 27: 29-60


    Recent structural information for complexes of cytokine receptor ectodomains bound to their ligands has significantly expanded our understanding of the macromolecular topology and ligand recognition mechanisms used by our three principal shared cytokine signaling receptors-gp130, gamma(c), and beta(c). The gp130 family receptors intricately coordinate three structurally unique cytokine-binding sites on their four-helix bundle cytokine ligands to assemble multimeric signaling complexes. These organizing principles serve as topological blueprints for the entire gp130 family of cytokines. Novel structures of gamma(c) and beta(c) complexes show us new twists, such as the use of a nonstandard sushi-type alpha receptors for IL-2 and IL-15 in assembling quaternary gamma(c) signaling complexes and an antiparallel interlocked dimer in the GM-CSF signaling complex with beta(c). Unlike gp130, which appears to recognize vastly different cytokine surfaces in chemically unique fashions for each ligand, the gamma(c)-dependent cytokines appear to seek out some semblance of a knobs-in-holes shape recognition code in order to engage gamma(c) in related fashions. We discuss the structural similarities and differences between these three shared cytokine receptors, as well as the implications for transmembrane signaling.

    View details for DOI 10.1146/annurev.immunol.24.021605.090616

    View details for Web of Science ID 000268071600002

    View details for PubMedID 18817510

  • BacMam system for high-level expression of recombinant soluble and membrane glycoproteins for structural studies PROTEIN EXPRESSION AND PURIFICATION Dukkipati, A., Park, H. H., Waghray, D., Fischer, S., Garcia, K. C. 2008; 62 (2): 160-170


    Baculovirus mediated gene transduction of mammalian cells (BacMam) is an emerging technique for rapid recombinant protein expression in mammalian cells. We constructed two baculovirus transfer vectors that incorporate several mammalian transcriptional regulatory elements necessary for high-level protein expression in mammalian cells. Using these vectors, we show that the BacMam system in combination with the 293 GnTI(-) cell line can be used for production of milligram quantities of soluble glycoproteins. Moreover, for crystallization trials, the purified glycoproteins are sensitive to EndoH treatment resulting in a loss of the bulk of the attached N-linked glycosylation. In addition, we also show that a combination of the BacMam system and 293 GnTI(-) cell line can be used for producing milligram quantities of a GPCR-protein ligand complex suitable for crystallization trials.

    View details for DOI 10.1016/j.pep.2008.08.004

    View details for Web of Science ID 000260598900004

    View details for PubMedID 18782620

    View details for PubMedCentralID PMC2637115

  • Different Thermodynamic Binding Mechanisms and Peptide Fine Specificities Associated with a Panel of Structurally Similar High-Affinity T Cell Receptors BIOCHEMISTRY Jones, L. L., Colf, L. A., Bankovich, A. J., Stone, J. D., Gao, Y., Chan, C. M., Huang, R. H., Garcia, K. C., Kranz, D. M. 2008; 47 (47): 12398-12408


    To understand the mechanisms that govern T cell receptor (TCR)-peptide MHC (pMHC) binding and the role that different regions of the TCR play in affinity and antigen specificity, we have studied the TCR from T cell clone 2C. High-affinity mutants of the 2C TCR that bind QL9-L(d) as a strong agonist were generated previously by site-directed mutagenesis of complementarity determining regions (CDRs) 1beta, 2alpha, 3alpha, or 3beta. We performed isothermal titration calorimetry to assess whether they use similar thermodynamic mechanisms to achieve high affinity for QL9-L(d). Four of the five TCRs examined bound to QL9-L(d) in an enthalpically driven, entropically unfavorable manner. In contrast, the high-affinity CDR1beta mutant resembled the wild-type 2C TCR interaction, with favorable entropy. To assess fine specificity, we measured the binding and kinetics of these mutants for both QL9-L(d) and a single amino acid peptide variant of QL9, called QL9-Y5-L(d). While 2C and most of the mutants had equal or higher affinity for the Y5 variant than for QL9, mutant CDR1beta exhibited 8-fold lower affinity for Y5 compared to QL9. To examine possible structural correlates of the thermodynamic and fine specificity signatures of the TCRs, the structure of unliganded QL9-L(d) was solved and compared to structures of the 2C TCR/QL9-L(d) complex and three high-affinity TCR/QL9-L(d) complexes. Our findings show that the QL9-L(d) complex does not undergo major conformational changes upon binding. Thus, subtle changes in individual CDRs account for the diverse thermodynamic and kinetic binding mechanisms and for the different peptide fine specificities.

    View details for DOI 10.1021/bi801349g

    View details for Web of Science ID 000261000100018

    View details for PubMedID 18973345

  • Distinct CDR3 Conformations in TCRs Determine the Level of Cross-Reactivity for Diverse Antigens, but Not the Docking Orientation JOURNAL OF IMMUNOLOGY Jones, L. L., Colf, L. A., Stone, J. D., Garcia, K. C., Kranz, D. M. 2008; 181 (9): 6255-6264


    T cells are known to cross-react with diverse peptide MHC Ags through their alphabeta TCR. To explore the basis of such cross-reactivity, we examined the 2C TCR that recognizes two structurally distinct ligands, SIY-K(b) and alloantigen QL9-L(d). In this study we characterized the cross-reactivity of several high-affinity 2C TCR variants that contained mutations only in the CDR3alpha loop. Two of the TCR lost their ability to cross-react with the reciprocal ligand (SIY-K(b)), whereas another TCR (m67) maintained reactivity with both ligands. Crystal structures of four of the TCRs in complex with QL9-L(d) showed that CDR1, CDR2, and CDR3beta conformations and docking orientations were remarkably similar. Although the CDR3alpha loop of TCR m67 conferred a 2000-fold higher affinity for SIY-K(b), the TCR maintained the same docking angle on QL9-L(d) as the 2C TCR. Thus, CDR3alpha dictated the affinity and level of cross-reactivity, yet it did so without affecting the conserved docking orientation.

    View details for Web of Science ID 000260659000051

    View details for PubMedID 18941216

  • The structure of interleukin-23 reveals the molecular basis of p40 subunit sharing with interleukin-12 JOURNAL OF MOLECULAR BIOLOGY Lupardus, P. J., Garcia, K. C. 2008; 382 (4): 931-941


    Interleukin (IL)-23 is a recently identified member of the IL-12 family of heterodimeric cytokines that modulate subpopulations of T helper cells, and both IL-12 and IL-23 are attractive targets for therapy of autoimmune diseases. IL-23 is a binary complex of a four-helix bundle cytokine (p19) and a soluble class I cytokine receptor p40. IL-12 and IL-23 share p40 as an alpha-receptor subunit, yet show only 15% sequence homology between their four-helix cytokines p19 and p35, respectively, and signal through different combinations of shared receptors. In order to elucidate the structural basis of p40 sharing, we have determined a 2.3-A crystal structure of IL-23 for comparison to the previously determined structure of IL-12. The docking mode of p19 to p40 is altered compared to p35, deviating by a 'tilt' and 'roll' that results in an altered footprint of p40 on the A and D helices of the respective cytokines. Binding of p19 to p40 is mediated primarily by an arginine residue on helix D of p19 that forms an extensive charge and hydrogen-bonding network with residues at the base of a pocket on p40. This 'arginine pocket' is lined with an inner shell of hydrophobic interactions that are ringed by an outer shell of polar interactions. Comparative analysis indicates that the IL-23 and IL-12 complexes 'mimic' the network of interactions constituting the central arginine pocket despite p19 and p35 having limited sequence homology. The majority of the structural epitopes in the two complexes are composed of unique p19 and p35 pairwise contacts with common residues on p40. Thus, while the critical hotspot is maintained in the two complexes, the majority of the interfaces are structurally distinct and, therefore, provide a basis for the therapeutic targeting of IL-12 versus IL-23 heterodimer formation despite their use of a common receptor subunit.

    View details for DOI 10.1016/j.jmb.2008.07.051

    View details for Web of Science ID 000260024500010

    View details for PubMedID 18680750

    View details for PubMedCentralID PMC2666310

  • Small molecule-induced allosteric activation of the Vibrio cholerae RTX cysteine protease domain SCIENCE Lupardus, P. J., Shen, A., Bogyo, M., Garcia, K. C. 2008; 322 (5899): 265-268


    Vibrio cholerae RTX (repeats in toxin) is an actin-disrupting toxin that is autoprocessed by an internal cysteine protease domain (CPD). The RTX CPD is efficiently activated by the eukaryote-specific small molecule inositol hexakisphosphate (InsP6), and we present the 2.1 angstrom structure of the RTX CPD in complex with InsP6. InsP6 binds to a conserved basic cleft that is distant from the protease active site. Biochemical and kinetic analyses of CPD mutants indicate that InsP6 binding induces an allosteric switch that leads to the autoprocessing and intracellular release of toxin-effector domains.

    View details for DOI 10.1126/science.1162403

    View details for Web of Science ID 000259902300050

    View details for PubMedID 18845756

    View details for PubMedCentralID PMC3272704

  • Structural organization of a full-length gp130/LIF-R cytokine receptor transmembrane complex MOLECULAR CELL Skiniotis, G., Lupardus, P. J., Martick, M., Walz, T., Garcia, K. C. 2008; 31 (5): 737-748


    gp130 is a shared receptor for at least nine cytokines and can signal either as a homodimer or as a heterodimer with Leukemia Inhibitory Factor Receptor (LIF-R). Here, we biophysically and structurally characterize the full-length, transmembrane form of a quaternary cytokine receptor complex consisting of gp130, LIF-R, the cytokine Ciliary Neurotrophic Factor (CNTF), and its alpha receptor (CNTF-Ralpha). Thermodynamic analysis indicates that, unlike the cooperative assembly of the symmetric gp130/Interleukin-6/IL-6Ralpha hexameric complex, CNTF/CNTF-Ralpha heterodimerizes gp130 and LIF-R via noncooperative energetics to form an asymmetric 1:1:1:1 complex. Single particle electron microscopic analysis of the full-length gp130/LIF-R/CNTF-Ralpha/CNTF quaternary complex elucidates an asymmetric structural arrangement, in which the receptor extracellular and transmembrane segments join as a continuous, rigid unit, poised to sensitively transduce ligand engagement to the membrane-proximal intracellular signaling regions. These studies also enumerate the organizing principles for assembly of the "tall" class of gp130 family cytokine receptor complexes including LIF, IL-27, IL-12, and others.

    View details for DOI 10.1016/j.molcel.2008.08.011

    View details for Web of Science ID 000259113800014

    View details for PubMedID 18775332

    View details for PubMedCentralID PMC2607196

  • An autonomous CDR3 delta is sufficient for recognition of the nonclassical MHC class I molecules T10 and T22 by gamma delta T cells NATURE IMMUNOLOGY Adams, E. J., Strop, P., Shin, S., Chien, Y., Garcia, K. C. 2008; 9 (7): 777-784


    It remains unclear whether gammadelta T cell antigen receptors (TCRs) detect antigens in a way similar to antibodies or alphabeta TCRs. Here we show that reactivity between the G8 and KN6 gammadelta TCRs and the major histocompatibility complex class Ib molecule T22 could be recapitulated, with retention of wild-type ligand affinity, in an alphabeta TCR after grafting of a G8 or KN6 complementarity-determining region 3-delta (CDR3delta) loop in place of the CDR3alpha loop of an alphabeta TCR. We also found that a shared sequence motif in CDR3delta loops of all T22-reactive gammadelta TCRs bound T22 in energetically distinct ways, and that T10(d), which bound G8 with weak affinity, was converted into a high-affinity ligand by a single point mutation. Our results demonstrate unprecedented autonomy of a single CDR3 loop in antigen recognition.

    View details for DOI 10.1038/ni.1620

    View details for Web of Science ID 000256904900016

    View details for PubMedID 18516039

  • Predictors of mucoid Pseudomonas colonization in cystic fibrosis patients PEDIATRIC PULMONOLOGY Levy, H., Kalish, L. A., Cannon, C. L., Garcia, K. C., Gerard, C., Goldmann, D., Pier, G. B., Weiss, S. T., Colin, A. A. 2008; 43 (5): 463-471


    Chronic mucoid Pseudomonas aeruginosa within the airway in cystic fibrosis (CF) patients can determine prognosis. Understanding the risk factors of mucoid P. aeruginosa acquisition may change how we deliver care. This study aims to evaluate whether presence of risk factors reported to predict disease severity including gender, CFTR genotype, bacterial organisms in airway cultures, and serum levels of vitamins A and E, albumin, C-reactive protein, alpha 1-antitrypsin, and immunoglobulins increased the risk of mucoid P. aeruginosa acquisition.Primary endpoint was age at first transition from negative to positive culture for mucoid P. aeruginosa. Cox proportional hazards regression with time-dependent covariates examined development of mucoid P. aeruginosa infection and its association with longitudinally measured serum biomarkers, pulmonary function, and culture results for other organisms.Median ages at CF diagnosis and at first culture were 0.55 and 5.7 years, respectively. Median number of cultures/patient was 17. Of the 323 subjects, 150 developed mucoid P. aeruginosa during a median 8.1 years' follow-up. In multivariate analysis, gender (relative hazard [RH] 0.55 for male vs. female, P = 0.001), number of DF508 alleles (RH 1.66 for 1 or 2 vs. 0, P = 0.04), FEV(1) % (RH 1.16 for 10% decrease, P = 0.008), and most recent Staphylococcus aureus status (RH 0.24 for positive vs. negative, P < 0.0001) remained statistically significant.Female gender, number of DF508 alleles, decreased lung function, and lack of S. aureus on recent sputum culture are important risk factors for early detection of mucoid P. aeruginosa.

    View details for DOI 10.1002/ppul.20794

    View details for Web of Science ID 000255823800007

    View details for PubMedID 18361452

  • Molecular and structural basis of cytokine receptor pleiotropy in the interleukin-4/13 system CELL LaPorte, S. L., Juo, Z. S., Vaclavikova, J., Colf, L. A., Qi, X., Heller, N. M., Keegan, A. D., Garcia, K. C. 2008; 132 (2): 259-272


    Interleukin-4 and Interleukin-13 are cytokines critical to the development of T cell-mediated humoral immune responses, which are associated with allergy and asthma, and exert their actions through three different combinations of shared receptors. Here we present the crystal structures of the complete set of type I (IL-4R alpha/gamma(c)/IL-4) and type II (IL-4R alpha/IL-13R alpha1/IL-4, IL-4R alpha/IL-13R alpha1/IL-13) ternary signaling complexes. The type I complex reveals a structural basis for gamma(c)'s ability to recognize six different gamma(c)-cytokines. The two type II complexes utilize an unusual top-mounted Ig-like domain on IL-13R alpha1 for a novel mode of cytokine engagement that contributes to a reversal in the IL-4 versus IL-13 ternary complex assembly sequences, which are mediated through substantially different recognition chemistries. We also show that the type II receptor heterodimer signals with different potencies in response to IL-4 versus IL-13 and suggest that the extracellular cytokine-receptor interactions are modulating intracellular membrane-proximal signaling events.

    View details for DOI 10.1016/j.cell.2007.12.030

    View details for Web of Science ID 000253427700012

    View details for PubMedID 18243101

    View details for PubMedCentralID PMC2265076

  • Structure of the measles virus hemagglutinin NATURE STRUCTURAL & MOLECULAR BIOLOGY Colf, L. A., Juo, Z. S., Garcia, K. C. 2007; 14 (12): 1227-1228


    Measles virus is a highly pathogenic virus that infects roughly 20 million people per year. We report here the crystal structure of the measles virus hemagglutinin, the surface glycoprotein responsible for the binding of measles virus to its host cell receptors. Although the protein lacks neuraminidase activity, its structure resembles a 'dead' neuraminidase fold, presenting spatially distinct receptor-binding sites for its receptors CD46 and SLAM.

    View details for DOI 10.1038/nsmb1342

    View details for Web of Science ID 000251416500020

    View details for PubMedID 18026116

  • Posttranslational regulation of I-E-d by affinity for CLIP JOURNAL OF IMMUNOLOGY Rinderknecht, C. H., Belmares, M. P., Catanzarite, T. L., Bankovich, A. J., Holmes, T. H., Garcia, K. C., Nanda, N. K., Busch, R., Kovats, S., Mellins, E. D. 2007; 179 (9): 5907-5915


    Several MHC class II alleles linked with autoimmune diseases form unusually low stability complexes with CLIP, leading us to hypothesize that this is an important feature contributing to autoimmune pathogenesis. To investigate cellular consequences of altering class II/CLIP affinity, we evaluated invariant chain (Ii) mutants with varying CLIP affinity for a mouse class II allele, I-E(d), which has low affinity for wild-type CLIP and is associated with a mouse model of spontaneous, autoimmune joint inflammation. Increasing CLIP affinity for I-E(d) resulted in increased cell surface and total cellular abundance and half-life of I-E(d). This reveals a post-endoplasmic reticulum chaperoning capacity of Ii via its CLIP peptides. Quantitative effects on I-E(d) were less pronounced in DM-expressing cells, suggesting complementary chaperoning effects mediated by Ii and DM, and implying that the impact of allelic variation in CLIP affinity on immune responses will be highest in cells with limited DM activity. Differences in the ability of cell lines expressing wild-type or high-CLIP-affinity mutant Ii to present Ag to T cells suggest a model in which increased CLIP affinity for class II serves to restrict peptide loading to DM-containing compartments, ensuring proper editing of antigenic peptides.

    View details for Web of Science ID 000250388000035

    View details for PubMedID 17947664

  • Structural evidence for a germline-encoded T cell receptor-major histocompatibility complex interaction 'codon' NATURE IMMUNOLOGY Feng, D., Bond, C. J., Ely, L. K., Maynard, J., Garcia, K. C. 2007; 8 (9): 975-983


    All complexes of T cell receptors (TCRs) bound to peptide-major histocompatibility complex (pMHC) molecules assume a stereotyped binding 'polarity', despite wide variations in TCR-pMHC docking angles. However, existing TCR-pMHC crystal structures have failed to show broadly conserved pairwise interaction motifs. Here we determined the crystal structures of two TCRs encoded by the variable beta-chain 8.2 (V(beta)8.2), each bound to the MHC class II molecule I-A(u), and did energetic mapping of V(alpha) and V(beta) contacts with I-A(u). Together with two previously solved structures of V(beta)8.2-containing TCR-MHC complexes, we found four TCR-I-A complexes with structurally superimposable interactions between the V(beta) loops and the I-A alpha-helix. This examination of a narrow 'slice' of the TCR-MHC repertoire demonstrates what is probably one of many germline-derived TCR-MHC interaction 'codons'.

    View details for DOI 10.1038/ni1502

    View details for Web of Science ID 000248918200021

    View details for PubMedID 17694060

  • Elucidation of the interleukin-15 binding site on its alpha receptor by NMR BIOCHEMISTRY Hanick, N. A., Rickert, M., Varani, L., Bankovich, A. J., Cochran, J. R., Kim, D. M., Surh, C. D., Garcia, K. C. 2007; 46 (33): 9453-9461


    The cytokine interleukin-15 (IL-15) signals through the formation of a quaternary receptor complex composed of an IL-15-specific alpha receptor, together with beta and gammac receptors that are shared with interleukin-2 (IL-2). The initiating step in the formation of this signaling complex is the interaction between IL-15 and IL-15Ralpha, which is a single sushi domain bearing strong structural homology to one of the two sushi domains of IL-2Ralpha. The crystal structure of the IL2-Ralpha/IL-2 complex has been determined, however little is known about the analogous IL-15Ralpha/IL-15 binding interaction. Here we show that recombinant IL-15 can be overexpressed as a stable complex in the presence of its high affinity receptor, IL-15Ralpha. We find that this complex is 10-fold more active than IL-15 alone in stimulating proliferation and survival of memory phenotype CD8 T cells. To probe the ligand/receptor interface, we used solution NMR to map chemical shifts on 15N-labeled IL-15Ralpha in complex with unlabeled IL-15. Our results predict that the binding surface on IL-15Ralpha involves strands C and D, similar to IL-2Ralpha. The interface, as predicted here, leaves open the possibility of trans-presentation of IL-15 by IL-15Ralpha on an opposing cell.

    View details for DOI 10.1021/bi700652f

    View details for Web of Science ID 000248692400010

    View details for PubMedID 17655329

  • Solution mapping of T cell receptor docking footprints on peptide-MHC PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Varani, L., Bankovich, A. J., Liu, C. W., Colf, L. A., Jones, L. L., Kranz, D. M., Puglisi, J. D., Garcia, K. C. 2007; 104 (32): 13080-13085


    T cell receptor (TCR) recognition of peptide-MHC (pMHC) is central to the cellular immune response. A large database of TCR-pMHC structures is needed to reveal general structural principles, such as whether the repertoire of TCR/MHC docking modes is dictated by a "recognition code" between conserved elements of the TCR and MHC genes. Although approximately 17 cocrystal structures of unique TCR-pMHC complexes have been determined, cocrystallization of soluble TCR and pMHC remains a major technical obstacle in the field. Here we demonstrate a strategy, based on NMR chemical shift mapping, that permits rapid and reliable analysis of the solution footprint made by a TCR when binding onto the pMHC surface. We mapped the 2C TCR binding interaction with its allogeneic ligand H-2Ld-QL9 and identified a group of NMR-shifted residues that delineated a clear surface of the MHC that we defined as the TCR footprint. We subsequently found that the docking footprint described by NMR shifts was highly accurate compared with a recently determined high-resolution crystal structure of the same complex. The same NMR footprint analysis was done on a high-affinity mutant of the TCR. The current work serves as a foundation to explore the molecular dynamics of pMHC complexes and to rapidly determine the footprints of many Ld-specific TCRs.

    View details for DOI 10.1073/pnas.0703702104

    View details for PubMedID 17670943

  • Polyspecificity of T cell and B cell receptor recognition SEMINARS IN IMMUNOLOGY Wucherpfennig, K. W., Allen, P. M., Celada, F., Cohen, I. R., De Boer, R., Garcia, K. C., Goldstein, B., Greenspan, R., Hafler, D., Hodgkin, P., Huseby, E. S., Krakauer, D. C., Nemazee, D., Perelson, A. S., Pinilla, C., Strong, R. K., Sercarz, E. E. 2007; 19 (4): 216-224


    A recent workshop discussed the recognition of multiple distinct ligands by individual T cell and B cell receptors and the implications of this discovery for lymphocyte biology. The workshop recommends general use of the term polyspecificity because it emphasizes two fundamental aspects, the inherent specificity of receptor recognition and the ability to recognize multiple ligands. Many different examples of polyspecificity and the structural mechanisms were discussed, and the group concluded that polyspecificity is a general, inherent feature of TCR and antibody recognition. This review summarizes the relevance of polyspecificity for lymphocyte development, activation and disease processes.

    View details for DOI 10.1016/j.simm.2007.02.012

    View details for Web of Science ID 000248722400002

    View details for PubMedID 17398114

  • Structural elucidation of the m157 mouse cytomegalovirus ligand for Ly49 natural killer cell receptors PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Adams, E. J., Juo, Z. S., Venook, R. T., Boulanger, M. J., Arase, H., Garcia, K. C. 2007; 104 (24): 10128-10133


    Natural killer (NK) cells express activating and inhibitory receptors that, in concert, survey cells for proper expression of cell surface major histocompatibility complex (MHC) class I molecules. The mouse cytomegalovirus encodes an MHC-like protein, m157, which is the only known viral antigen to date capable of engaging both activating (Ly49H) and inhibitory (Ly49I) NK cell receptors. We have determined the 3D structure of m157 and studied its biochemical and cellular interactions with the Ly49H and Ly49I receptors. m157 has a characteristic MHC-fold, yet possesses several unique structural features not found in other MHC class I-like molecules. m157 does not bind peptides or other small ligands, nor does it associate with beta(2)-microglobulin. Instead, m157 engages in extensive intra- and intermolecular interactions within and between its domains to generate a compact minimal MHC-like molecule. m157's binding affinity for Ly49I (K(d) approximately 0.2 microM) is significantly higher than that of classical inhibitory Ly49-MHC interactions. Analysis of viral escape mutations on m157 that render it resistant to NK killing reveals that it is likely to be recognized by Ly49H in a binding mode that differs from Ly49/MHC-I. In addition, Ly49H+ NK cells can efficiently lyse RMA cells expressing m157, despite the presence of native MHC class I. Collectively, our results show that m157 represents a structurally divergent form of MHC class I-like proteins that directly engage Ly49 receptors with appreciable affinity in a noncanonical fashion.

    View details for DOI 10.1073/pnas.0703735104

    View details for Web of Science ID 000247363000039

    View details for PubMedID 17537914

    View details for PubMedCentralID PMC1891256

  • Structural insight into pre-B cell receptor function SCIENCE Bankovich, A. J., Raunser, S., Juo, Z. S., Walz, T., Davis, M. M., Garcia, K. C. 2007; 316 (5822): 291-294


    The pre-B cell receptor (pre-BCR) serves as a checkpoint in B cell development. In the 2.7 angstrom structure of a human pre-BCR Fab-like fragment, consisting of an antibody heavy chain (HC) paired with the surrogate light chain, the "unique regions" of VpreB and lambda5 replace the complementarity-determining region 3 (CDR3) loop of an antibody light chain and appear to "probe" the HC CDR3, potentially influencing the selection of the antibody repertoire. Biochemical analysis indicates that the pre-BCR is impaired in its ability to recognize antigen, which, together with electron microscopic visualization of a pre-BCR dimer, suggests ligand-independent oligomerization as the likely signaling mechanism.

    View details for DOI 10.1126/science.1139412

    View details for Web of Science ID 000245654500055

    View details for PubMedID 17431183

  • How a single T cell receptor recognizes both self and foreign MHC CELL Colf, L. A., Bankovich, A. J., Hanick, N. A., Bowerman, N. A., Jones, L. L., Kranz, D. M., Garcia, K. C. 2007; 129 (1): 135-146


    alphabeta T cell receptors (TCRs) can crossreact with both self- and foreign- major histocompatibility complex (MHC) proteins in an enigmatic phenomenon termed alloreactivity. Here we present the 2.35 A structure of the 2C TCR complexed with its foreign ligand H-2L(d)-QL9. Surprisingly, we find that this TCR utilizes a different strategy to engage the foreign pMHC in comparison to the manner in which it recognizes a self ligand H-2K(b)-dEV8. 2C engages both shared and polymorphic residues on L(d) and K(b), as well as the unrelated QL9 and dEV8 peptide antigens, in unique pair-wise contacts, resulting in greater structural complementarity with the L(d)-QL9 complex. In the structure of an engineered, high-affinity 2C TCR variant bound to H-2L(d)-QL9, the "wild-type" TCR-MHC binding orientation persists despite modified TCR-CDR3alpha interactions with peptide. Thus, a single TCR recognizes two globally similar, but distinct ligands by divergent mechanisms, indicating that receptor-ligand crossreactivity can occur in the absence of molecular mimicry.

    View details for DOI 10.1016/j.cell.2007.01.048

    View details for Web of Science ID 000245661100017

    View details for PubMedID 17418792

  • Graded expression of Semaphorin-1a cell-autonomously directs dendritic targeting of olfactory projection neurons CELL Komiyama, T., Sweeney, L. B., Schuldiner, O., Garcia, K. C., Luo, L. 2007; 128 (2): 399-410


    Gradients of axon guidance molecules instruct the formation of continuous neural maps, such as the retinotopic map in the vertebrate visual system. Here we show that molecular gradients can also instruct the formation of a discrete neural map. In the fly olfactory system, axons of 50 classes of olfactory receptor neurons (ORNs) and dendrites of 50 classes of projection neurons (PNs) form one-to-one connections at discrete units called glomeruli. We provide expression, loss- and gain-of-function data to demonstrate that the levels of transmembrane Semaphorin-1a (Sema-1a), acting cell-autonomously as a receptor or part of a receptor complex, direct the dendritic targeting of PNs along the dorsolateral to ventromedial axis of the antennal lobe. Sema-1a also regulates PN axon targeting in higher olfactory centers. Thus, graded expression of Sema-1a contributes to connection specificity from ORNs to PNs and then to higher brain centers, ensuring proper representation of olfactory information in the brain.

    View details for DOI 10.1016/j.cell.2006.12.028

    View details for PubMedID 17254975

  • Structural and mechanistic insights into nerve growth factor interactions with the TrkA and p75 receptors NEURON Wehrman, T., He, X., Raab, B., Dukipatti, A., Blau, H., Garcia, K. C. 2007; 53 (1): 25-38


    Nerve growth factor engages two structurally distinct transmembrane receptors, TrkA and p75, which have been proposed to create a "high-affinity" NGF binding site through formation of a ternary TrkA/NGF/p75 complex. To define a structural basis for the high-affinity site, we have determined the three-dimensional structure of a complete extracellular domain of TrkA complexed with NGF. The complex reveals a crab-shaped homodimeric TrkA structure, but a mechanism for p75 coordination is not obvious. We investigated the heterodimerization of membrane-bound TrkA and p75, on intact mammalian cells, using a beta-gal protein-protein interaction system. We find that NGF dimerizes TrkA and that p75 exists on the cell surface as a preformed oligomer that is not dissociated by NGF. We find no evidence for a direct TrkA/p75 interaction. We propose that TrkA and p75 likely communicate through convergence of downstream signaling pathways and/or shared adaptor molecules, rather than through direct extracellular interactions.

    View details for DOI 10.1016/j.neuron.2006.09.034

    View details for Web of Science ID 000245126500006

    View details for PubMedID 17196528

  • In vitro reconstitution and preparative purification of complexes between the chemokine receptor CXCR4 and its ligands SDF-1 alpha, gp120-CD4 and AMD3100 PROTEIN EXPRESSION AND PURIFICATION Dukkipati, A., Vaclavikova, J., Waghray, D., Garcia, K. C. 2006; 50 (2): 203-214


    CXCR4 belongs to the family of G protein-coupled receptors and mediates the various developmental and regulatory effects of the chemokine SDF-1alpha. In addition, CXCR4 acts as a co-receptor along with CD4 for the HIV-1 viral glycoprotein gp120. Recently, there has also been a small molecule described that antagonizes both SDF-1 and gp120 binding to CXCR4. The structural and mechanistic basis for this dual recognition ability of CXCR4 is unknown largely due to the technical challenges of biochemically producing the components of the various complexes. We expressed the human CXCR4 receptor using a modified baculovirus expression vector that facilitates a single step antibody affinity purification of CXCR4 to >80% purity from Hi5 cells. The recombinant receptor undergoes N-linked glycosylation, tyrosine sulfation and is recognized by the 12G5 conformation specific antibody against human CXCR4. We are able to purify CXCR4 alone as well as complexed with its endogenous ligand SDF-1, its viral ligand gp120, and a small molecule antagonist AMD3100 by ion-exchange chromatography. We anticipate that the expression and purification scheme described in this paper will facilitate structure-function studies aimed at elucidating the molecular basis for CXCR4 recognition of its endogenous chemokine and viral ligands.

    View details for DOI 10.1016/j.pep.2006.07.016

    View details for Web of Science ID 000242783600009

    View details for PubMedID 16962791

  • Engineering and characterization of a stabilized alpha 1/alpha 2 module of the class I major histocompatibility complex product L-d JOURNAL OF BIOLOGICAL CHEMISTRY Jones, L. L., Brophy, S. E., Bankovich, A. J., Colf, L. A., Hanick, N. A., Garcia, K. C., Kranz, D. M. 2006; 281 (35): 25734-25744


    The major histocompatibility complex (MHC) is the most polymorphic locus known, with thousands of allelic variants. There is considerable interest in understanding the diversity of structures and peptide-binding features represented by this class of proteins. Although many MHC proteins have been crystallized, others have not been amenable to structural or biochemical studies due to problems with expression or stability. In the present study, yeast display was used to engineer stabilizing mutations into the class I MHC molecule, Ld. The approach was based on previous studies that showed surface levels of yeast-displayed fusion proteins are directly correlated with protein stability. To engineer a more stable Ld, we selected Ld mutants with increased surface expression from randomly mutated yeast display libraries using anti-Ld antibodies or high affinity, soluble T-cell receptors (TCRs). The most stable Ld mutant, Ld-m31, consisted of a single-chain MHC module containing only the alpha1 and alpha2 domains. The enhanced stability was in part due to a single mutation (Trp-97 --> Arg), shown previously to be present in the allele Lq. Mutant Ld-m31 could bind to Ld peptides, and the specific peptide.Ld-m31 complex (QL9.Ld-m31) was recognized by alloreactive TCR 2C. A soluble form of the Ld-m31 protein was expressed in Escherichia coli and refolded from inclusion bodies at high yields. Surface plasmon resonance showed that TCRs bound to peptide.Ld-m31 complexes with affinities similar to those of native full-length Ld. The TCR and QL9.Ld-m31 formed complexes that could be resolved by native gel electrophoresis, suggesting that stabilized alpha1/alpha2 class I platforms may enable various structural studies.

    View details for DOI 10.1074/jbc.M604343200

    View details for Web of Science ID 000240031300076

    View details for PubMedID 16815841

  • Structural determinants of natriuretic peptide receptor specificity and degeneracy JOURNAL OF MOLECULAR BIOLOGY He, X., Dukkipati, A., Garcia, K. C. 2006; 361 (4): 698-714


    Cardiovascular homeostasis and blood pressure regulation are reliant, in part, on interactions between natriuretic peptide (NP) hormones and natriuretic peptide receptors (NPR). The C-type NPR (NPR-C) is responsible for clearance of NP hormones from the circulation, and displays a cross-reactivity for all NP hormones (ANP, BNP, and CNP), in contrast to other NPRs, which are more restricted in their specificity. In order to elucidate the structural determinants for the binding specificity and cross-reactivity of NPR-C with NP hormones, we have determined the crystal structures of the complexes of NPR-C with atrial natriuretic peptide (ANP), and with brain natriuretic peptide (BNP). A structural comparison of these complexes, with the previous structure of the NPR-C/CNP complex, reveals that NPR-C uses a conformationally inflexible surface to bind three different, highly flexible, NP ligands. The complex structures support a mechanism of rigid promiscuity rather than conformational plasticity by the receptor. While ANP and BNP appear to adopt similar receptor-bound conformations, the CNP structure diverges, yet shares sets of common receptor contacts with the other ligands. The degenerate versus selective hormone recognition properties of different NPRs appears to derive largely from two cavities on the receptor surfaces, pocket I and pocket II, that serve as anchoring sites for hormone side-chains and modulate receptor selectivity.

    View details for DOI 10.1016/j.jmb.2006.06.060

    View details for Web of Science ID 000240233700008

    View details for PubMedID 16870210

  • Structure of artemin complexed with its receptor GFR alpha 3: Convergent recognition of glial cell line-derived neurotrophic factors STRUCTURE Wang, X., Baloh, R. H., Milbrandt, J., Garcia, K. C. 2006; 14 (6): 1083-1092


    Artemin (ARTN) is a member of the glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) which regulate the development and maintenance of many neuronal populations in the mammalian nervous system. Here we report the 1.92 A crystal structure of the complex formed between ARTN and its receptor GFRalpha3, which is the initiating step in the formation of a ternary signaling complex containing the shared RET receptor. It represents a new receptor-ligand interaction mode for the TGF-beta superfamily that reveals both conserved and specificity-determining anchor points for all GFL-GFRalpha pairs. In tandem with the complex structure, cellular studies using receptor chimeras implicate dyad-symmetric composite interfaces for recruitment and dimerization of RET, leading to intracellular signaling. These studies should facilitate the functional dissection of the specific versus pleiotropic roles of this system in neurobiology, as well as its exploitation for therapeutic applications.

    View details for DOI 10.1016/j.str.2006.05.010

    View details for Web of Science ID 000238730300016

    View details for PubMedID 16765900

  • Deconstructing the form and function of the TCR/CD3 complex IMMUNITY Kuhns, M. S., DAVIS, M. M., Garcia, K. C. 2006; 24 (2): 133-139


    When T cells encounter antigens via the T cell antigen receptor (TCR), information about the quantity and quality of antigen engagement is relayed to the intracellular signal transduction machinery. This process is poorly understood. The TCR itself lacks a significant intracellular domain. Instead, it is associated with CD3 molecules that contain intracellular signaling domains that couple the TCR/CD3 complex to the downstream signaling machinery. The earliest events in TCR signaling must involve the transfer of information from the antigen binding TCR subunit to the CD3 signaling subunits of the TCR/CD3 complex. Elucidating the structural organization of the TCR with the associated CD3 signaling molecules is necessary for understanding the mechanism by which TCR engagement is coupled to activation. Here, we review the current state of our understanding of the structure and organization of the TCR/CD3 complex.

    View details for DOI 10.1016/j.immuni.2006.01.006

    View details for Web of Science ID 000235479000005

    View details for PubMedID 16473826

  • High-level bacterial secretion of single-chain alpha beta T-cell receptors JOURNAL OF IMMUNOLOGICAL METHODS Maynard, J., Adams, E. J., Krogsgaard, M., Petersson, K., Liu, C. W., Garcia, K. C. 2005; 306 (1-2): 51-67


    While numerous antibody-antigen systems have been structurally characterized, studies of structurally analogous T-cell receptor MHC systems have lagged behind largely due to the lack of a general TCR expression system. Efforts to develop bacterial systems have resulted in low yields (< 0.5 mg/l) of active material which is prone to proteolysis and aggregation. Here we report a strategy to secrete folded, soluble single chain T-cell receptors (scTCR) in the Escherichia coli periplasm using three representative alphabeta TCRs (172.10, 1934.4/c19 and 2B4). Shake flask yields between 0.5 and 30 mg/l active, purified material were attained for all TCRs studied and found to depend on the introduction of solubility-increasing amino acid substitutions, skp chaperone co-expression and C-terminal fusion to a human kappa constant domain in the context of a tightly regulated expression vector. This system will greatly enable crystallographic, thermodynamic and other biophysical analyses of TCRs which require large quantities of homogeneous material.

    View details for DOI 10.1016/j.jim.2005.07.022

    View details for Web of Science ID 000234174500005

    View details for PubMedID 16198365

  • Structure of the quaternary complex of interleukin-2 with its alpha, beta, and gamma(c) receptors SCIENCE Wang, X. Q., Rickert, M., Garcia, K. C. 2005; 310 (5751): 1159-1163


    Interleukin-2 (IL-2) is an immunoregulatory cytokine that acts through a quaternary receptor signaling complex containing alpha (IL-2Ralpha), beta (IL-2Rbeta), and common gamma chain (gc) receptors. In the structure of the quaternary ectodomain complex as visualized at a resolution of 2.3 angstroms, the binding of IL-2Ralpha to IL-2 stabilizes a secondary binding site for presentation to IL-2Rbeta. gammac is then recruited to the composite surface formed by the IL-2/IL-2Rbeta complex. Consistent with its role as a shared receptor for IL-4, IL-7, IL-9, IL-15, and IL-21, gammac forms degenerate contacts with IL-2. The structure of gammac provides a rationale for loss-of-function mutations found in patients with X-linked severe combined immunodeficiency diseases (X-SCID). This complex structure provides a framework for other gammac-dependent cytokine-receptor interactions and for the engineering of improved IL-2 therapeutics.

    View details for DOI 10.1126/science.1117893

    View details for Web of Science ID 000233437300038

    View details for PubMedID 16293754

  • How the T cell receptor sees antigen - A structural view CELL Garcia, K. C., Adams, E. J. 2005; 122 (3): 333-336


    Structures of many of the cell surface receptor-ligand complexes mediating the interactions between T cells and target cells have been determined in the past ten years. While snapshots of T cell receptors bound to their peptide-MHC ligands appear to have defined a general interaction or "docking" solution, many of the most fundamental structural questions in antigen recognition lack detailed answers and thus pose exciting experimental challenges for the future.

    View details for DOI 10.1016/j.cell.2005.07.015

    View details for Web of Science ID 000231254400006

    View details for PubMedID 16096054

  • The structure of interleukin-2 complexed with its alpha receptor SCIENCE Rickert, M., Wang, X. Q., Boulanger, M. J., Goriatcheva, N., Garcia, K. C. 2005; 308 (5727): 1477-1480


    Interleukin-2 (IL-2) is an immunoregulatory cytokine that binds sequentially to the alpha (IL-2Ralpha), beta (IL-2Rbeta), and common gamma chain (gammac) receptor subunits. Here we present the 2.8 angstrom crystal structure of a complex between human IL-2 and IL-2Ralpha, which interact in a docking mode distinct from that of other cytokine receptor complexes. IL-2Ralpha is composed of strand-swapped "sushi-like" domains, unlike the classical cytokine receptor fold. As a result of this domain swap, IL-2Ralpha uses a composite surface to dock into a groove on IL-2 that also serves as a binding site for antagonist drugs. With this complex, we now have representative structures for each class of hematopoietic cytokine receptor-docking modules.

    View details for DOI 10.1126/science.1109745

    View details for Web of Science ID 000229619300056

    View details for PubMedID 15933202

  • Signaling conformations of the tall cytokine receptor gp130 when in complex with IL-6 and IL-6 receptor NATURE STRUCTURAL & MOLECULAR BIOLOGY Skiniotis, G., Boulanger, M. J., Garcia, K. C., Walz, T. 2005; 12 (6): 545-551


    gp130 is a shared cytokine signaling receptor and the founding member of the 'tall' class of cytokine receptors. A crystal structure of the ligand-binding domains of gp130 in complex with human interleukin-6 (IL-6) and its a-receptor (IL-6Ralpha) revealed a hexameric architecture in which the gp130 membrane-distal regions were approximately 100 A apart, in contrast to the close apposition seen between short cytokine receptor complexes. Here we used single-particle EM to visualize the entire extracellular hexameric IL-6-IL-6Ralpha-gp130 complex, containing all six gp130 domains. The structure reveals that gp130 is bent such that the membrane-proximal domains of gp130 are close together at the cell surface, enabling activation of intracellular signaling. Variation in the receptor bend angles suggests a possible conformational transition from open to closed states upon ligand binding; this transition is probably representative of the other tall cytokine receptors.

    View details for DOI 10.1038/nsmb941

    View details for Web of Science ID 000229533800016

    View details for PubMedID 15895091

  • A new paradigm for hormone recognition and allosteric receptor activation revealed from structural studies of NPR-C PEPTIDES He, X. L., Dukkipati, A., Wang, X. Q., Garcia, K. C. 2005; 26 (6): 1035-1043


    The natriuretic peptide system of hormones and receptors poses an abundance of interesting biophysical questions regarding receptor structure, hormone recognition, and receptor activation. Functional and biochemical data have implicated a series of conformational changes as the mechanism by which NP receptor activation is achieved. We have explored the structural basis of hormone recognition by the NP clearance receptor, termed NPR-C. While NPR-C does not contain the classical guanylyl-cyclase activity in its intracellular domains, its extracellular domain is highly similar to the GC-coupled members of this family. The 1:2 stoichiometry of hormone binding to NPR-C is also used by NPR-A and -B to bind hormones. The structure of NPR-C in both quiescent and hormone-bound forms reveals the hormone intercalates within the interface of a receptor dimer, inducing a large-scale conformational change in the membrane proximal regions. This mechanism of hormone recognition will be conserved across the entire NPR family. The allosteric response of the NPR-C ectodomain to ligand binding is likely a glimpse of the general activation signal of these receptors, despite their differing downstream signaling cascades. In this review, we discuss our results on NPR-C and their relevance to the NPR family as a whole, as well as its place as a basic new paradigm for receptor activation.

    View details for DOI 10.1016/j.peptides.2004.08.035

    View details for Web of Science ID 000229622200011

    View details for PubMedID 15911071

  • Antigen recognition determinants of gamma delta T cell receptors SCIENCE Shin, S., El-Diwany, R., Schaffert, S., Adams, E. J., Garcia, K. C., Pereira, P., Chien, Y. H. 2005; 308 (5719): 252-255


    The molecular basis of gammadelta T cell receptor (TCR) recognition is poorly understood. Here, we analyze the TCR sequences of a natural gammadelta T cell population specific for the major histocompatibility complex class Ib molecule T22. We find that T22 recognition correlates strongly with a somatically recombined TCRdelta complementarity-determining region 3 (CDR3) motif derived from germ line-encoded residues. Sequence diversity around these residues modulates TCR ligand-binding affinities, whereas V gene usage correlates mainly with tissue origin. These results show how an antigen-specific gammadelta TCR repertoire can be generated at a high frequency and suggest that gammadelta T cells recognize a limited number of antigens.

    View details for DOI 10.1126/science.1106480

    View details for Web of Science ID 000228273700053

    View details for PubMedID 15821090

  • Structure of a gamma delta T cell receptor in complex with the nonclassical MHC T22 SCIENCE Adams, E. J., Chien, Y. H., Garcia, K. C. 2005; 308 (5719): 227-231


    Gammadelta T cell receptors (TCRs), alphabeta TCRs, and antibodies are the three lineages of somatically recombined antigen receptors. The structural basis for ligand recognition is well defined for alphabeta TCR and antibodies but is lacking for gammadelta TCRs. We present the 3.4 A structure of the murine gammadelta TCR G8 bound to its major histocompatibility complex (MHC) class Ib ligand, T22. G8 predominantly uses germline-encoded residues of its delta chain complementarity-determining region 3 (CDR3) loop to bind T22 in an orientation substantially different from that seen in alphabeta TCR/peptide-MHC. That junctionally encoded G8 residues play an ancillary role in binding suggests a fusion of innate and adaptive recognition strategies.

    View details for DOI 10.1126/science.1106885

    View details for Web of Science ID 000228273700045

    View details for PubMedID 15821084

  • A TNF receptor family member, TROY, is a coreceptor with Nogo receptor in mediating the inhibitory activity of myelin inhibitors NEURON Park, J. B., Yiu, G., Kaneko, S., Wang, J., Chang, J. F., He, Z. G. 2005; 45 (3): 345-351


    A major obstacle for successful axon regeneration in the adult central nervous system (CNS) arises from inhibitory molecules in CNS myelin, which signal through a common receptor complex on neurons consisting of the ligand-binding Nogo-66 receptor (NgR) and two transmembrane coreceptors, p75 and LINGO-1. However, p75 expression is only detectable in subpopulations of mature neurons, raising the question of how these inhibitory signals are transduced in neurons lacking p75. In this study, we demonstrate that TROY (also known as TAJ), a TNF receptor family member selectively expressed in the adult nervous system, can form a functional receptor complex with NgR and LINGO-1 to mediate cellular responses to myelin inhibitors. Also, both overexpressing a dominant-negative TROY or presence of a soluble TROY protein can efficiently block neuronal response to myelin inhibitors. Our results implicate TROY in mediating myelin inhibition, offering new insights into the molecular mechanisms of regeneration failure in the adult nervous system.

    View details for DOI 10.1016/j.neuron.2004.12.040

    View details for Web of Science ID 000226787700007

    View details for PubMedID 15694321

  • Genetic deletion of the Nogo receptor does not reduce neurite inhibition in vitro or promote corticospinal tract regeneration in vivo PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zheng, B. H., Atwal, J., Ho, C., Case, L., He, X. L., Garcia, K. C., Steward, O., Tessier-Lavigne, M. 2005; 102 (4): 1205-1210


    Axon regeneration failure in the adult mammalian CNS is attributed in part to the inhibitory nature of CNS myelin. Three myelin-associated, structurally distinct proteins, Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein, have been implicated in this inhibition. Neuronal Nogo receptor (NgR) binds to each of the three inhibitors and has been proposed to mediate their inhibitory signals by complexing with a signal-transducing coreceptor, the neurotrophin receptor p75(NTR). To assess the contribution of NgR to mediating myelin inhibitory signals and regeneration failure in vivo, we generated and characterized NgR-deficient mice. Nogo transcripts are up-regulated in NgR mutants, indicating that NgR regulates Nogo in vivo. However, neurite outgrowth from NgR-deficient postnatal dorsal root ganglion or cerebellar granule neurons is inhibited by myelin and by a Nogo-66 substrate to the same extent as is from wild-type neurons, whereas p75(NTR)-deficient neurons are less inhibited. The NgR ligand-binding domain promotes neurite outgrowth on Nogo-66, regardless of the genotype of the neurons, indicating that the NgR ligand-binding domain can act independent of NgR. Thus, NgR is not essential for mediating inhibitory signals from CNS myelin, at least in the neurons tested, whereas p75(NTR) plays a central role in this response. Neither NgR-nor p75(NTR)-deficient mice showed enhanced regeneration of corticospinal tract axons in comparison with wild-type controls after spinal dorsal hemisection. Our results thus fail to support a central role for NgR in axonal growth inhibition in vitro or in corticospinal tract regeneration block in vivo.

    View details for DOI 10.1073/pnas.0409026102

    View details for Web of Science ID 000226617900045

    View details for PubMedID 15647357

    View details for PubMedCentralID PMC544342

  • Structure of an autoimmune T cell receptor complexed with class II peptide-MHC: Insights into MHC bias and antigen specificity IMMUNITY Maynard, J., Petersson, K., Wilson, D. H., Adams, E. J., Blondelle, S. E., Boulanger, M. J., Wilson, D. B., Garcia, K. C. 2005; 22 (1): 81-92


    T cell receptor crossreactivity with different peptide ligands and biased recognition of MHC are coupled features of antigen recognition that are necessary for the T cell's diverse functional repertoire. In the crystal structure between an autoreactive, EAE T cell clone 172.10 and myelin basic protein (1-11) presented by class II MHC I-Au, recognition of the MHC is dominated by the Vbeta domain of the TCR, which interacts with the MHC alpha chain in a manner suggestive of a germline-encoded TCR/MHC "anchor point." Strikingly, there are few specific contacts between the TCR CDR3 loops and the MBP peptide. We also find that over 1,000,000 different peptides derived from combinatorial libraries can activate 172.10, yet the TCR strongly prefers the native MBP contact residues. We suggest that while TCR scanning of pMHC may be degenerate due to the TCR germline bias for MHC, recognition of structurally distinct agonist peptides is not indicative of TCR promiscuity, but rather highly specific alternative solutions to TCR engagement.

    View details for DOI 10.1016/j.immuni.2004.11.015

    View details for PubMedID 15664161

  • Structure of a human A-type potassium channel interacting protein DPPX, a member of the dipeptidyl aminopeptidase family JOURNAL OF MOLECULAR BIOLOGY Strop, P., Bankovich, A. J., Hansen, K. C., Garcia, K. C., Brunger, A. T. 2004; 343 (4): 1055-1065


    It has recently been reported that dipeptidyl aminopeptidase X (DPPX) interacts with the voltage-gated potassium channel Kv4 and that co-expression of DPPX together with Kv4 pore forming alpha-subunits, and potassium channel interacting proteins (KChIPs), reconstitutes properties of native A-type potassium channels in vitro. Here we report the X-ray crystal structure of the extracellular domain of human DPPX determined at 3.0A resolution. This structure reveals the potential for a surface electrostatic change based on the protonation state of histidine. Subtle changes in extracellular pH might modulate the interaction of DPPX with Kv4.2 and possibly with other proteins. We propose models of DPPX interaction with the voltage-gated potassium channel complex. The dimeric structure of DPPX is highly homologous to the related protein DPP-IV. Comparison of the active sites of DPPX and DPP-IV reveals loss of the catalytic serine residue but the presence of an additional serine near the "active" site. However, the arrangement of residues is inconsistent with that of canonical serine proteases and DPPX is unlikely to function as a protease (dipeptidyl aminopeptidase).

    View details for DOI 10.1016/j.jmb.2004.09.003

    View details for Web of Science ID 000224656100021

    View details for PubMedID 15476821

  • Compensatory energetic mechanisms mediating the assembly of signaling complexes between interleukin-2 and its alpha, beta, and gamma(c) receptors JOURNAL OF MOLECULAR BIOLOGY Rickert, M., Boulanger, M. J., Goriatcheva, N., Garcia, K. C. 2004; 339 (5): 1115-1128


    Interleukin-2 is a key immuno-regulatory cytokine whose actions are mediated by three different cell surface receptors: the alpha, beta and the "common gamma" (gamma(c)) chains. We have undertaken a complete thermodynamic characterization of the stepwise assembly cycle for multiple possible combinations of the receptor-ligand, and receptor-receptor interactions that are necessary for formation of the high-affinity IL-2/alphabetagamma(c) signaling complex. We find an entropically favorable high affinity interaction between IL-2 and its alpha receptor, a moderately entropically favorable low affinity interaction between IL-2 and its beta receptor, and no interaction between IL-2 and the shared receptor, gamma(c). Formation of the stable intermediate trimolecular complexes of IL-2 with alpha and beta receptors, as well as IL-2 with beta and gamma(c) receptors proceeds through enthalpy-entropy compensation mechanisms. Surprisingly, we see a moderate affinity interaction between the unliganded receptor alpha and beta chains, suggesting that a preformed alphabeta complex may serve as the initial interaction complex for IL-2. Reconstitution of the IL-2/Ralphabetagamma(c) high-affinity quaternary signaling complex shows it to be assembled through cooperative energetics to form a 1:1:1:1 assembly. Collectively, the favorable entropy of the bimolecular interactions appears to be offset by the loss in rigid body entropy of the receptor components in the higher-order complexes, but overcome by the formation of increasingly enthalpically favorable composite interfaces. This enthalpic mechanism utilized by gamma(c) contrasts with the favorable entropic mechanism utilized by gp130 for degenerate cytokine interaction. In conclusion, we find that several energetically redundant pathways exist for formation of IL-2 receptor signaling complexes, suggesting a more complex equilibrium on the cell surface than has been previously appreciated.

    View details for DOI 10.1016/j.jmb.2004.04.038

    View details for Web of Science ID 000221982400008

    View details for PubMedID 15178252

  • Structure of nerve growth factor complexed with the shared neurotrophin receptor p75 SCIENCE He, X. L., Garcia, K. C. 2004; 304 (5672): 870-875


    Neurotrophins are secreted growth factors critical for the development and maintenance of the vertebrate nervous system. Neurotrophins activate two types of cell surface receptors, the Trk receptor tyrosine kinases and the shared p75 neurotrophin receptor. We have determined the 2.4 A crystal structure of the prototypic neurotrophin, nerve growth factor (NGF), complexed with the extracellular domain of p75. Surprisingly, the complex is composed of an NGF homodimer asymmetrically bound to a single p75. p75 binds along the homodimeric interface of NGF, which disables NGF's symmetry-related second p75 binding site through an allosteric conformational change. Thus, neurotrophin signaling through p75 may occur by disassembly of p75 dimers and assembly of asymmetric 2:1 neurotrophin/p75 complexes, which could potentially engage a Trk receptor to form a trimolecular signaling complex.

    View details for Web of Science ID 000221243000044

    View details for PubMedID 15131306

  • Specificity and degeneracy of T cells MOLECULAR IMMUNOLOGY Wilson, D. B., Wilson, D. H., Schroder, K., Pinilla, C., Blondelle, S., Houghten, R. A., Garcia, K. C. 2004; 40 (14-15): 1047-1055


    The extent of T cell cross-reactivity is significant, even to the point where the terms "promiscuous" and "degenerate" apply. Degeneracy is an important feature of the immune response mechanism that permits effective T cell responses to a vast number of potential peptide sequences complexed to MHC molecules with specificity sufficient to distinguish between self and foreign peptides and thus to avoid autoimmune disease. Degeneracy at the clonal level of T cells also permits the use of very large combinatorial peptide libraries to identify and optimize peptide sequences that might be used for the treatment of T cell mediated autoimmune disease and for the design of vaccines for treatment of infectious diseases and cancer. Here we explore some recent findings derived from studies involving library scans of T cell lines and clones having clinically relevant specificities. In particular, we discuss two new insights: degeneracy among CD8 T cells appears to be substantially less than among CD4 T cells, and T cell clones characterized by a high affinity TCR interaction with a given pMHC complex are less degenerate than lower avidity T cells that require higher levels of pMHC complex for their activation.

    View details for DOI 10.1016/j.molimm.2003.11.022

    View details for Web of Science ID 000220002800011

    View details for PubMedID 15036909

  • Peptide register shifting within the MHC groove: theory becomes reality MOLECULAR IMMUNOLOGY Bankovich, A. J., Girvin, A. T., Moesta, A. K., Garcia, K. C. 2004; 40 (14-15): 1033-1039


    Degeneracy in immune recognition is usually thought of in terms of the astonishing ability of the T cell receptor to recognize an enormously diverse array of peptides bound to major histocompatibility complex (MHC) molecules. However, in this essay we discuss an alternative aspect of degeneracy in T cell recognition: the notion that peptides can assume different "registers" in the groove of a single MHC molecule, as first suggested and demonstrated by Sercarz and co-workers (reviewed in [J. autoimmun. 16 (2001) 201]). There is now abundant evidence, derived from functional, biochemical and structural studies, that single peptides can assume alternative, unpredictable binding registers by frameshifting within the MHC groove [Nat. Immunol. 3 (2002) 175;; J. Exp. Med. 187 (1998) 1505; J. Mol. Biol. 304 (2000) 177; Biochemistry 38 (1999) 16663; J. Exp. Med. 197 (2003) 1391; Eur. J. Immunol. 19 (1989) 681]. Hence, register shifting adds an additional dimension to the concept of degeneracy. In fact, the possibility of register shifting multiplies the universe of peptide-MHC (pMHC) surfaces that a TCR must recognize by an unknown, perhaps enormous factor. Register shifting also has profound implication for autoimmunity: (1) as a mechanism to "mask" autoantigenic epitopes during thymic education [Immunol. Rev. 169 (1999) 147; Immunity 17 (2002) 83]; and (2) as a possible source for pMHC complexes capable of molecular mimicry.

    View details for DOI 10.1016/j.molimm.2003.11.016

    View details for Web of Science ID 000220002800009

    View details for PubMedID 15036907

  • Molecular mechanisms for viral mimicry of a human cytokine: Activation of gp130 by HHV-8 interleukin-6 JOURNAL OF MOLECULAR BIOLOGY Boulanger, M. J., Chow, D. C., Brevnova, E., Martick, M., SANDFORD, G., Nicholas, J., Garcia, K. C. 2004; 335 (2): 641-654


    Kaposi's sarcoma-associated herpesvirus (KSHV, or HHV-8) encodes a pathogenic viral homologue of human interleukin-6 (IL-6). In contrast to human IL-6 (hIL-6), viral IL-6 (vIL-6) binds directly to, and activates, the shared human cytokine signaling receptor gp130 without the requirement for pre-complexation to a specific alpha-receptor. Here, we dissect the biochemical and functional basis of vIL-6 mimicry of hIL-6. We find that, in addition to the "alpha-receptor-independent" tetrameric vIL-6/gp130 complex, the viral cytokine can engage the human alpha-receptor (IL-6Ralpha) to form a hexameric vIL-6/IL-6Ralpha/gp130 complex with enhanced signaling potency. In contrast to the assembly sequence of the hIL-6 hexamer, the preformed vIL-6/gp130 tetramer can be decorated with IL-6Ralpha, post facto, in a "vIL-6-dependent" fashion. A detailed comparison of the viral and human cytokine/gp130 interfaces indicates that vIL-6 has evolved a unique molecular strategy to interact with gp130, as revealed by an almost entirely divergent structural makeup of its receptor binding sites. Viral IL-6 appears to utilize an elegant combination of both convergent, and unexpectedly divergent, molecular strategies to oligomerize gp130 and activate similar downstream signaling cascades as its human counterpart.

    View details for DOI 10.1016/j.jmb.2003.10.070

    View details for Web of Science ID 000188164000022

    View details for PubMedID 14672670

  • Shared cytokine signaling receptors: Structural insights from the GP130 system CELL SURFACE RECEPTORS Boulanger, M. J., Garcia, K. C. 2004; 68: 107-?


    The vast majority of cytokine signaling is mediated by "shared" receptors that form central signaling components of higher-order complexes incorporating ligand-specific receptors. These include the common gamma chain (gamma(c)), common beta chain (beta(c)), and gp130, as well as others. These receptors have the dual tasks of cross-reactive cytokine recognition, and formation of precisely oriented multimeric signaling assemblies. Currently, detailed structural information on a shared receptor complex exists only for gp130, which is a highly pleiotropic shared cytokine signaling receptor essential for mammalian cell growth and homeostasis. To date, more than 10 different four-helix bundle ligands have been identified that incorporate gp130, or one of its close relatives such as LIF receptor, into functional oligomeric signaling complexes. In this review we summarize our current knowledge of shared receptor recognition and activation, with a focus on gp130. We discuss recent structural and functional information to analyze overall architectural assemblies of gp130 cytokine complexes and probe the basis for the extreme cross-reactivity of gp130 for its multiple cytokine ligands.

    View details for Web of Science ID 000225856800004

    View details for PubMedID 15500860

  • Evidence that structural rearrangements and/or flexibility during TCR binding can contribute to T cell activation MOLECULAR CELL Krogsgaard, M., Prado, N., Adams, E. J., He, X. L., Chow, D. C., Wilson, D. B., Garcia, K. C., Davis, M. M. 2003; 12 (6): 1367-1378


    While in many cases the half-life of T cell receptor (TCR) binding to a particular ligand is a good predictor of activation potential, numerous exceptions suggest that other physical parameter(s) must also play a role. Accordingly, we analyzed the thermodynamics of TCR binding to a series of peptide-MHC ligands, three of which are more stimulatory than their stability of binding would predict. Strikingly, we find that during TCR binding these outliers show anomalously large changes in heat capacity, an indicator of conformational change or flexibility in a binding interaction. By combining the values for heat capacity (DeltaCp) and the half-life of TCR binding (t(1/2)), we find that we can accurately predict the degree of T cell stimulation. Structural analysis shows significant changes in the central TCR contact residue of the peptide-MHC, indicating that structural rearrangements within the TCR-peptide-MHC interface can contribute to T cell activation.

    View details for Web of Science ID 000187511600007

    View details for PubMedID 14690592

  • Convergent mechanisms for recognition of divergent cytokines by the shared signaling receptor gp130 MOLECULAR CELL Boulanger, M. J., Bankovich, A. J., Kortemme, T., Baker, D., Garcia, K. C. 2003; 12 (3): 577-589


    Gp130 is a shared cell-surface signaling receptor for at least ten different hematopoietic cytokines, but the basis of its degenerate recognition properties is unknown. We have determined the crystal structure of human leukemia inhibitory factor (LIF) bound to the cytokine binding region (CHR) of gp130 at 2.5 A resolution. Strikingly, we find that the shared binding site on gp130 has an entirely rigid core, while the LIF binding interface diverges sharply in structure and chemistry from that of other gp130 ligands. Dissection of the LIF-gp130 interface, along with comparative studies of other gp130 cytokines, reveal that gp130 has evolved a "thermodynamic plasticity" that is relatively insensitive to ligand structure, to enable crossreactivity. These observations reveal a novel and alternative mechanism for degenerate recognition from that of structural plasticity.

    View details for Web of Science ID 000185613800008

    View details for PubMedID 14527405

  • Hexameric structure and assembly of the interleukin-6/IL-6 alpha-receptor/gp130 complex SCIENCE Boulanger, M. J., Chow, D. C., Brevnova, E. E., Garcia, K. C. 2003; 300 (5628): 2101-2104


    Interleukin-6 (IL-6) is an immunoregulatory cytokine that activates a cell-surface signaling assembly composed of IL-6, the IL-6 alpha-receptor (IL-6Ralpha), and the shared signaling receptor gp130. The 3.65 angstrom-resolution structure of the extracellular signaling complex reveals a hexameric, interlocking assembly mediated by a total of 10 symmetry-related, thermodynamically coupled interfaces. Assembly of the hexameric complex occurs sequentially: IL-6 is first engaged by IL-6Ralpha and then presented to gp130in the proper geometry to facilitate a cooperative transition into the high-affinity, signaling-competent hexamer. The quaternary structures of other IL-6/IL-12 family signaling complexes are likely constructed by means of a similar topological blueprint.

    View details for Web of Science ID 000183774900049

    View details for PubMedID 12829785

  • Not just any T cell receptor will do IMMUNITY Bankovich, A. J., Garcia, K. C. 2003; 18 (1): 7-11


    Although our structural understanding of T cell recognition has rapidly evolved due to recent crystallographic results, the reality is that detailed answers to many of the most fundamental questions still remain elusive. In this issue, high-resolution insight into the phenomenon of TCR chain bias takes down another brick from the wall.

    View details for Web of Science ID 000180860600002

    View details for PubMedID 12530971

  • A structural template for gp130-cytokine signaling assemblies BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH Chow, D. C., Brevnova, L., He, X. L., Martick, M. M., Bankovich, A., Garcia, K. C. 2002; 1592 (3): 225-235


    The gp130-cytokine system has been fertile ground for protein structure-function studies aimed at elucidating the basis of ligand recognition and receptor activation. A number of longstanding questions involve the mechanism of the stepwise assembly of the active signaling complexes, as well as the structure of the gp130-cytokine complexes. It has been clear from functional studies that the paradigm of gp130-cyokine recognition will differ substantially from the classical homo-dimeric systems, typified by human growth hormone (hGH) and its receptor. Recently, a crystal structure of a viral interleukin-6 (vIL-6), complexed with the D1D2D3 domains of the gp130 extracellular domain, has resolved many of these questions, and reconciled much of the functional and mutagenesis data which have existed for a variety of gp130-cytokines. In this review, we discuss the structure of the vIL-6/gp130 complex in some detail and suggest that the geometry of this complex will be a common structural template utilized by other gp130-cytokines, as well as cytokines from distinct signaling systems.

    View details for Web of Science ID 000179178500003

    View details for PubMedID 12421668

  • A T cell receptor goes public STRUCTURE Adams, E. J., Garcia, K. C. 2002; 10 (11): 1468-1469


    The crystal structure of a human T cell receptor, which is used almost exclusively in the immune response to an Epstein-Barr virus protein, highlights the importance of noncontact residues in antigen recognition.

    View details for Web of Science ID 000179175300003

    View details for PubMedID 12429085

  • Two-step binding mechanism for T-cell receptor recognition of peptide-MHC NATURE Wu, L. C., Tuot, D. S., Lyons, D. S., Garcia, K. C., Davis, M. M. 2002; 418 (6897): 552-556


    T cells probe a diverse milieu of peptides presented by molecules of the major histocompatibility complex (MHC) by using the T-cell receptor (TCR) to scan these ligands with high sensitivity and specificity. Here we describe a physical basis for this scanning process by studying the residues involved in both the initial association and the stable binding of TCR to peptide-MHC, using the well-characterized TCR and peptide-MHC pair of 2B4 and MCC-IE(k) (moth cytochrome c, residues 88 103). We show that MHC contacts dictate the initial association, guiding TCR docking in a way that is mainly independent of the peptide. Subsequently, MCC-IE(k) peptide contacts dominate stabilization, imparting specificity and influencing T-cell activation by modulating the duration of binding. This functional subdivision of the peptide-MHC ligand suggests that a two-step process for TCR recognition facilitates the efficient scanning of diverse peptide-MHC complexes on the surface of cells and also makes TCRs inherently crossreactive towards different peptides bound by the same MHC.

    View details for DOI 10.1038/nature00920

    View details for Web of Science ID 000177162800045

    View details for PubMedID 12152083

  • Structure of the immunodominant surface antigen from the Toxoplasma gondii SRS superfamily NATURE STRUCTURAL BIOLOGY He, X. L., Grigg, M. E., Boothroyd, J. C., Garcia, K. C. 2002; 9 (8): 606-611


    Toxoplasma gondii is a persistent protozoan parasite capable of infecting almost any warm-blooded vertebrate. The surface of Toxoplasma is coated with a family of developmentally regulated glycosylphosphatidylinositol (GPI)-linked proteins (SRSs), of which SAG1 is the prototypic member. SRS proteins mediate attachment to host cells and interface with the host immune response to regulate the virulence of the parasite. The 1.7 A structure of the immunodominant SAG1 antigen reveals a homodimeric configuration in which the dimeric interface is mediated by an extended beta-sheet that forms a deep groove lined with positively charged amino acids. This basic groove seems to be conserved among SRS proteins and potentially serves as a sulfated proteoglycan-binding site on target cell surfaces, thus rationalizing the promiscuous attachment properties of Toxoplasma to a broad range of host cell types.

    View details for DOI 10.1038/nsb819

    View details for Web of Science ID 000177214200014

    View details for PubMedID 12091874

  • The V alpha 14 NKT cell TCR exhibits high-affinity binding to a glycolipid/CD1d complex JOURNAL OF IMMUNOLOGY Sidobre, S., Naidenko, O. V., Sim, B. C., Gascoigne, N. R., Garcia, K. C., Kronenberg, M. 2002; 169 (3): 1340-1348


    Most CD1d-dependent NKT cells in mice have a canonical V alpha 14J alpha 18 TCR rearrangement. However, relatively little is known concerning the molecular basis for their reactivity to glycolipid Ags presented by CD1d. Using glycolipid Ags, soluble forms of a V alpha 14 NKT cell-derived TCR, and mutant and wild-type CD1d molecules, we probed the TCR/CD1d interaction by surface plasmon resonance, tetramer equilibrium staining, and tetramer staining decay experiments. By these methods, several CD1d alpha-helical amino acids could be defined that do not greatly alter lipid binding, but that affect the interaction with the TCR. Binding of the V alpha 14(+) TCR to CD1d requires the agonist alpha-galactosylceramide (alpha-GalCer), as opposed to the nonantigenic beta-galactosylceramide, although both Ags bind to CD1d, indicating that the carbohydrate moiety of the CD1d-bound Ag plays a major role in the TCR interaction. The TCR has a relatively high-affinity binding to the alpha-GalCer/CD1d complex, with a particularly slow off rate. These unique properties are consistent with the coreceptor-independent action of the V alpha 14 TCR and may be related to the intense response to alpha-GalCer by NKT cells in vivo.

    View details for Web of Science ID 000177025100025

    View details for PubMedID 12133957

  • Structural snapshot of aberrant antigen presentation linked to autoimmunity: The immunodominant epitope of MBP complexed with I-A(u) IMMUNITY He, X. L., Radu, C., Sidney, J., Sette, A., Ward, E. S., Garcia, K. C. 2002; 17 (1): 83-94


    Murine experimental allergic encephalomyelitis (EAE) is a useful model for the demyelinating, autoimmune disease multiple sclerosis. In the EAE system, the immunodominant N-terminal epitope of myelin basic protein (MBP) is an unusually short, weakly binding peptide antigen which elicits highly biased TCR chain usage. In the 2.2 A crystal structure of I-A(u)/MBP1-11 complex, only MBP residues 1-7 are bound toward one end of the peptide binding cleft. The fourth residue of MBP1-11 is located in an incompatible p6 pocket of I-A(u), thus explaining the short half-life of I-A(u) complexed with Ac1-11. MBP peptides extended at the C terminus of Ac1-11 result in dramatic affinity increases, likely attributed to register shifting to a higher affinity cryptic epitope, which could potentially mask the presentation of the immunodominant MBP1-11 peptide during thymic education.

    View details for Web of Science ID 000177187500009

    View details for PubMedID 12150894

  • Structural comparison of allogeneic and syngeneic T cell receptor-peptide-major histocompatibility complex complexes: A buried alloreactive mutation subtly alters peptide presentation substantially increasing V-beta interactions JOURNAL OF EXPERIMENTAL MEDICINE Luz, J. G., Huang, M. D., Garcia, C., Rudolph, M. G., Apostolopoulos, V., Teyton, L., Wilson, I. A. 2002; 195 (9): 1175-1186


    The crystal structures of the 2C/H-2K(bm3)-dEV8 allogeneic complex at 2.4 A and H-2K(bm3)-dEV8 at 2.15 A, when compared with their syngeneic counterparts, elucidate structural changes that induce an alloresponse. The Asp77Ser mutation that imbues H-2K(bm3)-dEV8 with its alloreactive properties is located beneath the peptide and does not directly contact the T cell receptor (TCR). However, the buried mutation induces local rearrangement of the peptide itself to preserve hydrogen bonding interactions between the peptide and the alpha(1) 77 residue. The COOH terminus of the peptide main chain is tugged toward the alpha(1)-helix such that its presentation to the TCR is altered. These changes increase the stability of the allogeneic peptide-major histocompatibility complex (pMHC) complex and increase complementarity in the TCR-pMHC interface, placing greater emphasis on recognition of the pMHC by the TCR beta-chain, evinced by an increase in shape complementarity, buried surface area, and number of TCR-pMHC contacting residues. A nearly fourfold increase in the number of beta-chain-pMHC contacts is accompanied by a concomitant 64% increase in beta-chain-pMHC shape complementarity. Thus, the allogeneic mutation causes the same peptide to be presented differently, temporally and spatially, by the allogeneic and syngeneic MHCs.

    View details for DOI 10.1084/jem.20011644

    View details for Web of Science ID 000176110700009

    View details for PubMedID 11994422

  • Stabilization of soluble, low-affinity HLA-DM/HLA-DR1 complexes by leucine zippers JOURNAL OF IMMUNOLOGICAL METHODS Busch, R., Paschine, A., Garcia, K. C., Mellins, E. D. 2002; 263 (1-2): 111-121


    The ectodomains of interacting membrane-bound proteins, when expressed as recombinant soluble molecules, often have low affinities for each other, hampering studies of their interaction. We reasoned that stabilization of unstable protein-protein complexes should aid our understanding of the structural and functional consequences of complex formation. Here, we have used fusion with leucine zipper (LZ) domains to stabilize a complex formed between the class II major histocompatibility complex (MHC-II) protein, HLA-DR1 (which binds peptides for presentation to CD4+ T cells) and HLA-DM (which catalyzes peptide exchange of MHC-II molecules). To this end, the DM beta chain ectodomains were fused to acidic LZ domains (AcidP1 or Fos); similarly, the DR1 beta chain ectodomains were fused to basic LZ domains (BaseP1 or Jun). We expressed LZ-modified soluble DM or DR1 alphabeta dimers, or both, in insect cells and purified the secreted sDM-AcidP1 and sDR1-BaseP1 molecules as well as the complex. LZ modification greatly enhanced DM-catalyzed peptide binding to DR1 compared to unmodified soluble DM and DR1. We readily detected LZ-modified DM/DR complexes on native PAGE gels and by coimmunoprecipitation. Thus, fusion with artificial LZ domains can stabilize unstable protein-protein complexes for biochemical and structural studies of interactions within the complex.

    View details for Web of Science ID 000176076300010

    View details for PubMedID 12009208

  • Novel factor VC2-domain mutation (R2074H) in two families with factor V deficiency and bleeding THROMBOSIS AND HAEMOSTASIS Schrijver, I., Houissa-Kastally, R., Jones, C. D., Garcia, K. C., Zehnder, J. L. 2002; 87 (2): 294-299


    The molecular basis of Factor V deficiency has been defined in few patients only. We report a homozygous nucleotide change (G6395A) in two Tunisian probands with Factor V deficiency and bleeding episodes. This substitution results in the replacement of an arginine (R) by a histidine (H) in amino acid position 2074, located in the Factor V C2-domain. Mutations in this protein domain have not previously been described. Several lines of evidence support that this sequence variant is indeed disease causing: 1) Crystal structures of Factor V and molecular C2-domain modeling studies of H2074 suggest that the conserved R2074 is required for correct folding; 2) Structure-function studies of selective Factor V mutants (R2074A) demonstrate the importance of R2074 for structural stability of the Factor V C2-domain and for cofactor activity (1); 3) In Factor VIII, point mutations in codon 2209, which corresponds to position 2074 in Factor V, cause hemophilia A.

    View details for Web of Science ID 000173869300020

    View details for PubMedID 11858490

  • Promiscuous antigen presentation by the nonclassical MHC lb Qa-2 is enabled by a shallow, hydrophobic groove and self-stabilized peptide conformation STRUCTURE He, X. L., Tabaczewski, P., Ho, J., Stroynowski, I., Garcia, K. C. 2001; 9 (12): 1213-1224


    Qa-2 is a nonclassical MHC Ib antigen, which has been implicated in both innate and adaptive immune responses, as well as embryonic development. Qa-2 has an unusual peptide binding specificity in that it requires two dominant C-terminal anchor residues and is capable of associating with a substantially more diverse array of peptide sequences than other nonclassical MHC.We have determined the crystal structure, to 2.3 A, of the Q9 gene of murine Qa-2 complexed with a self-peptide derived from the L19 ribosomal protein, which is abundant in the pool of peptides eluted from the Q9 groove. The 9 amino acid peptide is bound high in a shallow, hydrophobic binding groove of Q9, which is missing a C pocket. The peptide makes few specific contacts and exhibits extremely poor shape complementarity to the MHC groove, which facilitates the presentation of a degenerate array of sequences. The L19 peptide is in a centrally bulged conformation that is stabilized by intramolecular interactions from the invariant P7 histidine anchor residue and by a hydrophobic core of preferred secondary anchor residues that have minimal interaction with the MHC.Unexpectedly, the preferred secondary peptide residues that exhibit tenuous contact with Q9 contribute significantly to the overall stability of the peptide-MHC complex. The structure of this complex implies a "conformational" selection by Q9 for peptide residues that optimally stabilize the large bulge rather than making intimate contact with the MHC pockets.

    View details for Web of Science ID 000172658700009

    View details for PubMedID 11738047

  • Immunomodulation of experimental autoimmune encephalomyelitis with ordered peptides based on MHC-TCR binding motifs JOURNAL OF IMMUNOLOGY Ruiz, P. J., DeVoss, J. J., Nguyen, L. V., Fontoura, P. P., Hirschberg, D. L., Mitchell, D. J., Garcia, K. C., Steinman, L. 2001; 167 (5): 2688-2693


    T cell-mediated destruction of the myelin sheath causes inflammatory damage of the CNS in multiple sclerosis (MS). The major T and B cell responses in MS patients who are HLA-DR2 (about two-thirds of MS patients) react to a region between residues 84 and 103 of myelin basic protein (1 ). The crystal structure of HLA-DR2 complexed with myelin basic protein(84-102) confirmed that Lys(91) is the major TCR contact site, whereas Phe(90) is a major anchor to MHC and binds the hydrophobic P4 pocket (2 ). We have tested peptides containing repetitive 4-aa sequences designed to bind critical MHC pockets and to interfere with T cell activation. One such sequence, EYYKEYYKEYYK, ameliorates experimental autoimmune encephalomyelitis in Lewis rats, an animal model of MS.

    View details for Web of Science ID 000172391800034

    View details for PubMedID 11509612

  • Allosteric activation of a spring-loaded natriuretic peptide receptor dimer by hormone SCIENCE He, X. L., Chow, D. C., Martick, M. M., Garcia, K. C. 2001; 293 (5535): 1657-1662


    Natriuretic peptides (NPs) are vasoactive cyclic-peptide hormones important in blood pressure regulation through interaction with natriuretic cell-surface receptors. We report the hormone-binding thermodynamics and crystal structures at 2.9 and 2.0 angstroms, respectively, of the extracellular domain of the unliganded human NP receptor (NPR-C) and its complex with CNP, a 22-amino acid NP. A single CNP molecule is bound in the interface of an NPR-C dimer, resulting in asymmetric interactions between the hormone and the symmetrically related receptors. Hormone binding induces a 20 angstrom closure between the membrane-proximal domains of the dimer. In each monomer, the opening of an interdomain cleft, which is tethered together by a linker peptide acting as a molecular spring, is likely a conserved allosteric trigger for intracellular signaling by the natriuretic receptor family.

    View details for Web of Science ID 000170802900063

    View details for PubMedID 11533490

  • Norepinephrine prevents the adverse effects of lidocaine upon the heart. An experimental study in isolated guinea-pig hearts PHARMACOLOGICAL RESEARCH Singi, G., Garcia, K. C., Coelho, A. E., Gazola, R. 2001; 44 (2): 129-134


    The adverse effects of local anesthetics associated with catecholamines upon the cardiovascular system, such as arterial pressure alterations and cardiac arrhythmias, have been highly discussed in pharmacological literature. When present in the blood circulation, the anesthetic salt promotes depressing effects at the cardiovascular system level, whilst the catecholaminic vasoconstrictors promote opposite effects upon the cardiovascular system. This paper aimed to verify which component is responsible for the initial effect upon the isolated heart of the guinea-pig by comparing the same anesthetic with and without vasoconstrictor. Guinea-pig hearts perfused using the Langendorff method received separate injections of lidocaine hydrochloride, without vasoconstrictor (LC), and with the vasoconstrictor norepinephrine at 1 : 50,000 (LCN) in doses of 3, 4 and 5 mg. LC was seen to significantly reduce the contraction force (CF) and the cardiac rate (CR) at all injected doses and at all times registered. LCN, though, significantly reduced CF in all doses only at 15 s after administration. At 30 and 60 s, LCN elevated CF to values close to those of the control. LCN in its 3 mg dose significantly increased CR at 30 and 60 s; with the 4 mg dose, significant alterations did not occur in CR and with the 5 mg dose CR was reduced significantly at 15 and 30 s, returning to values close to those of the control at 60 s. The electrocardiographic traces with LCN were similar to those with LC with a reduction occurring in the automatism of the sinus node, prolongation of the PR interval and widening of QRS, with all doses utilized at 15 s. Soon after the administration of LC and LCN ventricular asystole occurred, having a larger duration with LC. This duration was also increased when higher doses of anesthetics were used. The authors concluded that the initial cardio-depression effect of local anesthetics, with the vasoconstrictor norepinephrine, in the isolated guinea-pig heart is due to the anesthetic salt and that the vasoconstrictor exerts a protecting action against this effect.

    View details for Web of Science ID 000169979700011

    View details for PubMedID 11516263

  • In vitro reconstitution of recognition and activation complexes between interleukin-6 and gp130 BIOCHEMISTRY Chow, D. C., Ho, J., Pham, T. L., Rose-John, S., Garcia, K. C. 2001; 40 (25): 7593-7603


    Gp130 is a shared signal-transducing receptor for a family of four-helix cytokines, of which interleukin-6 is a prototypic member. IL-6-type cytokines activate gp130 to elicit downstream intracellular JAK/STAT signaling cascades through formation of hetero-oligomeric receptor complexes. Interleukin-6 must first complex with its specific alpha-receptor (Ralpha) in order to bind and activate gp130. We have dissected the extracellular activation pathway of human gp130 by human IL-6 through reconstitution of soluble complexes representing intermediate and final states in the hierarchical assembly of the IL-6/IL-6Ralpha/gp130 signaling complex. To isolate these hetero-complexes, we have applied a protein engineering strategy of covalently linking IL-6 to its Ralpha, which results in a "hyperactive" single-chain complex (hyper-IL-6) which we express in both Escherichia coli and insect cells. We have determined that IL-6/IL-Ralpha and the cytokine-binding homology region (CHR) of gp130 (D2D3) form a stable trimolecular "recognition" complex (trimer) consisting of 1IL-6,1 IL-6Ralpha, and 1 gp130-CHR. Addition of the N-terminal (D1) Ig-like domain (IGD) of gp130 to the CHR results in a transition to a hexameric "activation" complex containing 2 IL-6, 2IL-6Ralpha, and 2 gp130. These results clearly demonstrate that the recognition and activation complexes are disparate hetero-oligomeric molecular species linked by the recruitment of the gp130 IGD by the unique site III epitope present on all gp130-class cytokines. The results of these studies are relevant to other members of the IL-6 family of gp130-cytokines and address a longstanding question concerning the respective roles of the gp130 CHR and IGD in assembly of the active signaling oligomer.

    View details for Web of Science ID 000169536300025

    View details for PubMedID 11412113

  • Kinetics and thermodynamics of T cell receptor-autoantigen interactions in murine experimental autoimmune encephalomyelitis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Garcia, K. C., Radu, C. G., Ho, J., Ober, R. J., Ward, E. S. 2001; 98 (12): 6818-6823


    In the current study, cellular and molecular approaches have been used to analyze the biophysical nature of T cell receptor (TCR)-peptide MHC (pMHC) interactions for two autoreactive TCRs. These two TCRs recognize the N-terminal epitope of myelin basic protein (MBP1-11) bound to the MHC class II protein, I-A(u), and are associated with murine experimental autoimmune encephalomyelitis. Mice transgenic for the TCRs have been generated and characterized in other laboratories. These analyses indicate that the mice either develop encephalomyelitis spontaneously (172.10 TCR) or only if immunized with autoantigen in adjuvant (1934.4 TCR). Here, we show that the 172.10 TCR binds MBP1-11:I-A(u) with a 4-5-fold higher affinity than the 1934.4 TCR. Consistent with the higher affinity, 172.10 T hybridoma cells are significantly more responsive to autoantigen than 1934.4 cells. The interaction of the 172.10 TCR with cognate ligand is more entropically unfavorable than that of the 1934.4 TCR, indicating that the 172.10 TCR undergoes greater conformational rearrangements upon ligand binding. The studies therefore suggest a correlation between the strength and plasticity of a TCR-pMHC interaction and the frequency of spontaneous disease in the corresponding TCR transgenic mice. The comparative analysis of these two TCRs has implications for understanding autoreactive T cell recognition and activation.

    View details for Web of Science ID 000169151500053

    View details for PubMedID 11391002

  • Structure of an extracellular gp130 cytokine receptor signaling complex SCIENCE Chow, D. C., He, X. L., Snow, A. L., Rose-John, S., Garcia, K. C. 2001; 291 (5511): 2150-2155


    The activation of gp130, a shared signal-transducing receptor for a family of cytokines, is initiated by recognition of ligand followed by oligomerization into a higher order signaling complex. Kaposi's sarcoma-associated herpesvirus encodes a functional homolog of human interleukin-6 (IL-6) that activates human gp130. In the 2.4 angstrom crystal structure of the extracellular signaling assembly between viral IL-6 and human gp130, two complexes are cross-linked into a tetramer through direct interactions between the immunoglobulin domain of gp130 and site III of viral IL-6, which is necessary for receptor activation. Unlike human IL-6 (which uses many hydrophilic residues), the viral cytokine largely uses hydrophobic amino acids to contact gp130, which enhances the complementarity of the viral IL-6-gp130 binding interfaces. The cross-reactivity of gp130 is apparently due to a chemical plasticity evident in the amphipathic gp130 cytokine-binding sites.

    View details for Web of Science ID 000167563800040

    View details for PubMedID 11251120

  • The interaction of neuropilin-1 with vascular endothelial growth factor and its receptor Flt-1 JOURNAL OF BIOLOGICAL CHEMISTRY Fuh, G., Garcia, K. C., De Vos, A. M. 2000; 275 (35): 26690-26695


    Neuropilin-1 (NP-1) was first identified as a semaphorin receptor involved in neuron guidance. Subsequent studies demonstrated that NP-1 also binds an isoform of vascular endothelial growth factor (VEGF) as well as several VEGF homologs, suggesting that NP-1 may also function in angiogenesis. Here we report in vitro binding experiments that shed light on the interaction between VEGF165 and NP-1, as well as a previously unknown interaction between NP-1 and one of the VEGF receptor tyrosine kinases, VEGFR1 or Flt-1. BIAcore analysis demonstrated that, with the extracellular domain (ECD) of NP-1 immobilized at low density, VEGF165 bound with low affinity (K(d) = 2 microm) and fast kinetics. The interaction was dependent on the heparin-binding domain of VEGF165 and increased the affinity of VEGF165 for its signaling receptor VEGFR2 or kinase insert domain-containing receptor. The affinity of VEGF165 for the NP-1 ECD was greatly enhanced either by increasing the density of immobilized NP-1 (K(d) = 113 nm) or by the addition of heparin (K(d) = 25 nm). We attribute these affinity enhancements to avidity effects mediated by the bivalent VEGF165 homodimer or multivalent heparin. We also show that the NP-1 ECD binds with high affinity (K(d) = 1.8 nm) to domains 3 and 4 of Flt-1 and that this interaction inhibits the binding of NP-1 to VEGF165. Based on these results, we propose that NP-1 acts as a coreceptor for various ligands and that these functions are dependent on the density of NP-1 on the cell membrane. Furthermore, Flt-1 may function as a negative regulator of angiogenesis by competing for NP-1.

    View details for Web of Science ID 000089144800005

    View details for PubMedID 10842181

  • A structural framework for deciphering the link between I-A(g7) and autoimmune diabetes SCIENCE Corper, A. L., Stratmann, T., Apostolopoulos, V., Scott, C. A., Garcia, K. C., Kang, A. S., Wilson, I. A., Teyton, L. 2000; 288 (5465): 505-511


    Susceptibility to murine and human insulin-dependent diabetes mellitus correlates strongly with major histocompatibility complex (MHC) class II I-A or HLA-DQ alleles that lack an aspartic acid at position beta57. I-Ag7 lacks this aspartate and is the only class II allele expressed by the nonobese diabetic mouse. The crystal structure of I-Ag7 was determined at 2.6 angstrom resolution as a complex with a high-affinity peptide from the autoantigen glutamic acid decarboxylase (GAD) 65. I-Ag7 has a substantially wider peptide-binding groove around beta57, which accounts for distinct peptide preferences compared with other MHC class II alleles. Loss of Asp(beta57) leads to an oxyanion hole in I-Ag7 that can be filled by peptide carboxyl residues or, perhaps, through interaction with the T cell receptor.

    View details for Web of Science ID 000086626000043

    View details for PubMedID 10775108

  • A functional hot spot for antigen recognition in a superagonist TCR/MHC complex IMMUNITY Degano, M., Garcia, K. C., Apostolopoulos, V., Rudolph, M. G., Teyton, L., Wilson, I. A. 2000; 12 (3): 251-261


    A longstanding question in T cell receptor signaling is how structurally similar ligands, with similar affinities, can have substantially different biological activity. The crystal structure of the 2C TCR complex of H-2Kb with superagonist peptide SIYR at 2.8 A elucidates a structural basis for TCR discrimination of altered peptide ligands. The difference in antigen potency is modulated by two cavities in the TCR combining site, formed mainly by CDRs 3alpha, 3beta, and 1beta, that complement centrally located peptide residues. This "functional hot spot" allows the TCR to finely discriminate amongst energetically similar interactions within different ligands for those in which the peptide appropriately stabilizes the TCR/pMHC complex and provides a new structural perspective for understanding differential signaling resulting from T cell cross-reactivity.

    View details for Web of Science ID 000086292100003

    View details for PubMedID 10755612

  • Molecular interactions between extracellular components of the T-cell receptor signaling complex IMMUNOLOGICAL REVIEWS Garcia, K. C. 1999; 172: 73-85


    The structural and biochemical basis of antigen recognition by the T-cell receptor (TCR)-CD3 signaling complex has been illuminated greatly over the past few years. Structural biology has contributed enormously to this understanding through the determination of crystal structures of many of the individual components of this complex, and some of the complexes. A number of general principles can be derived for the structure of the alpha beta TCR and its interaction with peptide-major histocompatibility complex (pMHC) in class I systems, as well as interaction of the CD8 co-receptor with MHC. Large buried surface areas within the protein-protein interfaces, and varying degrees of shape complementarity appear critical for modulating the stability of the multicomponent, low-affinity macromolecular complexes consisting of TCR, pMHC, CD8 or CD4, and CD3 gamma, delta, epsilon and zeta. Significant structural alterations in TCR and pMHC, upon complex formation, hint at an as yet unclear role for conformational change in both recognition and activation. Subtle chemical alterations in key peptide residues which contact the TCR can have dramatic agonist or antagonist effects on receptor activation, which correlate only loosely with the TCR/pMHC complex affinity, implying an ability of the signaling complex to "sense" fine differences in the interface. The stoichiometry of an activated TCR signaling complex is still an unresolved issue, as is the structure and disposition of the CD3 components. However, functional experiments are bridging this gap and providing us with preliminary working models of the multimeric assemblies.

    View details for Web of Science ID 000084432700007

    View details for PubMedID 10631938

  • Structural basis of T cell recognition ANNUAL REVIEW OF IMMUNOLOGY Garcia, K. C., Teyton, L., Wilson, L. A. 1999; 17: 369-?


    Exciting breakthroughs in the last two years have begun to elucidate the structural basis of cellular immune recognition. Crystal structures have been determined for full-length and truncated forms of alpha beta T cell receptor (TCR) heterodimers, both alone and in complex with their peptide-MHC (pMHC) ligands or with anti-TCR antibodies. In addition, a truncated CD8 coreceptor has been visualized with a pMHC. Aided in large part by the substantial body of knowledge accumulated over the last 25 years on antibody structure, a number of general conclusions about TCR structure and its recognition of antigen can already be derived from the relatively few TCR structures that have been determined. Small, but important, variations between TCR and antibody structures bear on their functional differences as well as on their specific antigen recognition requirements. As observed in antibodies, canonical CDR loop structures are already emerging for some of the TCR CDR loops. Highly similar docking orientations of the TCR V alpha domains in the TCR/pMHC complex appear to play a primary role in dictating orientation, but the V beta positions diverge widely. Similar TCR contact positions, but whose exact amino acid content can vary, coupled with relatively poor interface shape complementarity, may explain the flexibility and short half-lives of many TCR interactions with pMHC. Here we summarize the current state of this field, and suggest that the knowledge gap between the three-dimensional structure and the signaling function of the TCR can be bridged through a synthesis of molecular biological and biophysical techniques.

    View details for Web of Science ID 000080436600013

    View details for PubMedID 10358763

  • Emerging principles for T cell receptor recognition of antigen in cellular immunity. Reviews in immunogenetics Garcia, K. C., Degano, M., Speir, J. A., Wilson, I. A. 1999; 1 (1): 75-90


    The structural basis of antigen recognition in cellular immunity has been elucidated through the determination of crystal structures of major histocompatibility complex (MHC) molecules bound to antigenic peptides, T cell receptors (TCR), CD8 and CD4 co-receptors and, most recently, TCRs in complex with peptide-MHC (pMHC). The mechanisms that generate the diversity of the immune response to invading microorganisms were first realized at a genetic level and are necessary in order to cope with the enormous number of potential antigens. This diversity is manifested in the protein products of the genes which code for the components of the TCR signalling complex. The structure of the TCR reveals both striking similarities with and fundamental differences from its functional counterpart, the antibody, in the humoral immune system. The conserved manner in which the TCR recognizes and interacts with its peptide-MHC ligand allows the TCR great latitude in its potential to form productive interactions with antigen-presenting cells that bear numerous ligands to which the TCR has not been previously exposed. This phenomenon of cross-, or alloreactivity arises from a combination of conserved structural features across all MHC molecules, both self and foreign, and some degree of molecular mimicry. Non-classical MHC ligands presenting either modified or specialized peptides, lipids, carbohydrates, or no ligand at all, are now thought to play increasingly important roles in cellular immunity. We review some of the recent structural results and our current state of knowledge about TCR structure, and how this relates to its function.

    View details for PubMedID 11256574

  • Probing the activation requirements for naive CD8(+) T cells with Drosophila cell transfectants as antigen presenting cells IMMUNOLOGICAL REVIEWS Cai, Z. L., Brunmark, A. B., Luxembourg, A. T., Garcia, K. C., Degano, M., Teyton, L., Wilson, I., PETERSON, P. A., Sprent, J., Jackson, M. R. 1998; 165: 249-265


    Activation of T cells involves multiple receptor-ligand interactions between T cells and antigen presenting cells (APC). At least two signals are required for T-cell activation: Signal 1 results from recognition of MHC/peptide complexes on the APC by cell surface T-cell receptors (TCR), whereas Signal 2 is induced by the interactions of co-stimulatory molecules on APC with their complementary receptors on T cells. This review focuses on our attempts to understand these various signals in a model system involving the 2C TCR. The structural basis of Signal 1 was investigated by determining the crystal structure of 2C TCR alone and in complex with MHC/peptide. Analysis of these structures has provided some basic rules for how TCR and MHC/peptide interact; however, the critical question of how this interaction transduces Signal 1 to T cells remains unclear. The effects of Signal 1 and Signal 2 on T-cell activation were examined with naive T cells from the 2C TCR transgenic mice, defined peptides as antigen and transfected Drosophila cells as APC. The results suggest that, except under extreme conditions, Signal 1 alone is unable to activate naive CD8 T cells despite the induction of marked TCR downregulation. Either B7 or intercellular adhesion molecule (ICAM)-1 can provide the second signal for CD8 T-cell activation. However, especially at low MHC/peptide densities, optimal activation and differentiation of CD8 T cells required interaction with both B7 and ICAM-1 on the same APC. Thus, the data suggest that at least two qualitatively different co-stimulation signals are required for full activation of CD8 T cells under physiological conditions.

    View details for Web of Science ID 000077188400019

    View details for PubMedID 9850865

  • T-cell receptor peptide-MHC interactions: biological lessons from structural studies CURRENT OPINION IN BIOTECHNOLOGY Garcia, K. C., Teyton, L. 1998; 9 (4): 338-343


    Fifteen years have passed since T-cell receptor (TCR) genes were identified (reviewed in [1]). Unlike the situation for antibodies, no direct structural information on the TCR proteins has been available for most of this time. Recently, however, the crystal structures of isolated alpha and beta chains were determined, shortly followed by the determination of the structure of an alpha beta heterodimer. Subsequently, the structures of two TCR peptide-MHC (pMHC) complexes have been reported. The windfall of this, and other more recent structural information, has elucidated some generalizations for TCR binding and recognition of pMHC. The crystal structures have, however, given us very little insight into the mechanisms of signal transduction by the TCR complex and the subsequent events which lead to activation of a T cell. Ultimately, the crystallographio results will be reconciled with experiments from other disciplines for a complete understanding of the molecular events of T cell activation.

    View details for Web of Science ID 000075406800002

    View details for PubMedID 9720260

  • Structural basis of 2C TCR allorecognition of H-2L(d) peptide complexes IMMUNITY Speir, J. A., Garcia, K. C., Brunmark, A., Degano, M., PETERSON, P. A., Teyton, L., Wilson, I. A. 1998; 8 (5): 553-562


    MHC class I H-2Ld complexed with peptide QL9 (or p2Ca) is a high-affinity alloantigen for the 2C TCR. We used the crystal structure of H-2Ld with a mixture of bound peptides at 3.1 A to construct a model of the allogeneic 2C-Ld/QL9 complex for comparison with the syngeneic 2C-Kb/dEV8 structure. A prominent ridge on the floor of the Ld peptide-binding groove, not present in Kb, creates a C-terminal bulge in Ld peptides that greatly increases interactions with the 2C beta-chain. Furthermore, weak electrostatic complementarity between Asp77 on the alpha1 helix of Kb and 2C is enhanced in the allogeneic complex by closer proximity of QL9 peptide residue AspP8 to the 2C HV4 loop.

    View details for Web of Science ID 000073885400003

    View details for PubMedID 9620676

  • Alanine scanning mutagenesis of an alpha beta T cell receptor: Mapping the energy of antigen recognition IMMUNITY Manning, T. C., Schlueter, C. J., Brodnicki, T. C., Parke, E. A., Speir, J. A., Garcia, K. C., Teyton, L., Wilson, I. A., Kranz, D. M. 1998; 8 (4): 413-425


    The T cell receptor (TCR) from the alloreactive T lymphocyte 2C recognizes a nonamer peptide QL9 complexed with the MHC class I molecule H2-Ld. Forty-two single-site alanine substitutions of the 2C TCR were analyzed for binding to QL9/Ld and anti-TCR antibodies. The results provided a detailed energy map of T cell antigen recognition and indicated that the pMHC and clonotypic antibody epitopes on the TCR were similar. Although residues in each Valpha and Vbeta CDR are important in binding pMHC, the most significant energy for the TCR/QL9/Ld interaction was contributed by CDRs 1 and 2 of both alpha and beta chains. The extent to which the individual energy contributions are directed at class I helices or peptide was also assessed.

    View details for Web of Science ID 000073348000004

    View details for PubMedID 9586632

  • Structural basis of plasticity in T cell receptor recognition of a self peptide MHC antigen SCIENCE Garcia, K. C., Degano, M., Pease, L. R., Huang, M. D., PETERSON, P. A., Teyton, L., Wilson, I. A. 1998; 279 (5354): 1166-1172


    The T cell receptor (TCR) inherently has dual specificity. T cells must recognize self-antigens in the thymus during maturation and then discriminate between foreign pathogens in the periphery. A molecular basis for this cross-reactivity is elucidated by the crystal structure of the alloreactive 2C TCR bound to self peptide-major histocompatibility complex (pMHC) antigen H-2Kb-dEV8 refined against anisotropic 3.0 angstrom resolution x-ray data. The interface between peptide and TCR exhibits extremely poor shape complementarity, and the TCR beta chain complementarity-determining region 3 (CDR3) has minimal interaction with the dEV8 peptide. Large conformational changes in three of the TCR CDR loops are induced upon binding, providing a mechanism of structural plasticity to accommodate a variety of different peptide antigens. Extensive TCR interaction with the pMHC alpha helices suggests a generalized orientation that is mediated by the Valpha domain of the TCR and rationalizes how TCRs can effectively "scan" different peptides bound within a large, low-affinity MHC structural framework for those that provide the slight additional kinetic stabilization required for signaling.

    View details for Web of Science ID 000072115200031

    View details for PubMedID 9469799

  • Engineering protein for X-ray crystallography: The murine Major Histocompatibility Complex class II molecule I-A(d) PROTEIN SCIENCE Scott, C. A., Garcia, K. C., Stura, E. A., PETERSON, P. A., Wilson, I. A., Teyton, L. 1998; 7 (2): 413-418


    Class II Major Histocompatibility (MHC) molecules are cell surface heterodimeric glycoproteins that play a central role in the immune response by presenting peptide antigens for surveillance by T cells. Due to the inherent instability of the class II MHC heterodimer, and its dependence on bound peptide for proper assembly, the production of electrophoretically pure samples of class II MHC proteins in complex with specific peptides has been problematic. A soluble form of the murine class II MHC molecule, I-Ad, with a leucine zipper tail added to each chain to enhance dimer assembly and secretion, has been produced in Drosophila melanogaster SC2 cells. To facilitate peptide loading, a high affinity ovalbumin peptide was covalently engineered to be attached by a six-residue linker to the amino terminus of the I-Adbeta chain. This modified I-Ad molecule was purified using preparative IEF and one fraction, after removal of the leucine zipper tails, produced crystals suitable for X-ray crystallographic analysis. The protein engineering and purification methods described here should be of general value for the expression of I-A and other class II MHC-peptide complexes.

    View details for Web of Science ID 000072056400022

    View details for PubMedID 9521118

  • alpha beta T cell receptor interactions with syngeneic and allogeneic ligands: Affinity measurements and crystallization PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Garcia, K. C., Tallquist, M. D., Pease, L. R., Brunmark, A., Scott, C. A., Degano, M., Stura, E. A., PETERSON, P. A., Wilson, I. A., Teyton, L. 1997; 94 (25): 13838-13843


    Cellular immunity is mediated by the interaction of an alphabeta T cell receptor (TCR) with a peptide presented within the context of a major histocompatibility complex (MHC) molecule. Alloreactive T cells have alphabeta TCRs that can recognize both self- and foreign peptide-MHC (pMHC) complexes, implying that the TCR has significant complementarity with different pMHC. To characterize the molecular basis for alloreactive TCR recognition of pMHC, we have produced a soluble, recombinant form of an alloreactive alphabeta T cell receptor in Drosophila melanogaster cells. This recombinant TCR, 2C, is expressed as a correctly paired alphabeta heterodimer, with the chains covalently connected via a disulfide bond in the C-terminal region. The native conformation of the 2C TCR was probed by surface plasmon resonance (SPR) analysis by using conformation-specific monoclonal antibodies, as well as syngeneic and allogeneic pMHC ligands. The 2C interaction with H-2Kb-dEV8, H-2Kbm3-dEV8, H-2Kb-SIYR, and H-2Ld-p2Ca spans a range of affinities from Kd = 10(-4) to 10(-6)M for the syngeneic (H-2Kb) and allogeneic (H-2Kbm3, H-2Ld) ligands. In general, the syngeneic ligands bind with weaker affinities than the allogeneic ligands, consistent with current threshold models of thymic selection and T cell activation. Crystallization of the 2C TCR required proteolytic trimming of the C-terminal residues of the alpha and beta chains. X-ray quality crystals of complexes of 2C with H-2Kb-dEV8, H-2Kbm3-dEV8 and H-2Kb-SIYR have been grown.

    View details for Web of Science ID A1997YK82500087

    View details for PubMedID 9391114

  • T-cell receptor structure and TCR complexes CURRENT OPINION IN STRUCTURAL BIOLOGY Wilson, I. A., Garcia, K. C. 1997; 7 (6): 839-848


    The first crystal structures of intact T-cell receptors (TCRs) and their complexes with MHC peptide antigens (pMHC) were reported during the past year, along with those of a single-chain TCR Fv fragment and a beta-chain complexed with two different bacterial superantigens. These structures have shown the similarities and differences in the architecture of the antigen-binding regions of TCRs and antibodies, and how the TCR interacts with pMHC ligands as well as with superantigens.

    View details for Web of Science ID 000071220100013

    View details for PubMedID 9434905

  • CD8 enhances formation of stable T-cell receptor MHC class I molecule complexes NATURE Garcia, K. C., Scott, C. A., Brunmark, A., Carbone, F. R., PETERSON, P. A., Wilson, I. A., Teyton, L. 1996; 384 (6609): 577-581


    T-cell antigen receptors (TCR) generally interact with moderate affinity with the complex formed by major histocompatibility complex (MHC) molecules and foreign peptides. MHC/TCR recognition is followed by the generation of a signal to the T cell through a monomorphic multicomponent system that includes the CD3 complex and accessory molecules such as CD4 and CD8. The interaction between the extracellular domains of MHC and TCR molecules, and the interaction of MHC and CD4/CD8 molecules, have been considered to occur independently of one another. We report here that the affinity of CD8 dimers for MHC class I molecules is independent of haplotype and peptide content, and that the affinity of the TCR for its specific ligand is enhanced through a reduced 'off' rate in the presence of either CD8alpha alpha homo- or CD8alpha beta heterodimers. Moreover, CD8 seems to help recognition of the specific MHC-peptide complex either by guiding an energetically favourable docking of TCR onto MHC, or by inducing conformational changes in the MHC complex that can augment the TCR/MHC-peptide interaction. CD8 should therefore be considered as an active participant in the T-cell recognition complex, rather than simply as an accessory molecule.

    View details for Web of Science ID A1996VX76900063

    View details for PubMedID 8955273

  • An alpha beta T cell receptor structure at 2.5 angstrom and its orientation in the TCR-MHC complex SCIENCE Garcia, K. C., Degano, M., Stanfield, R. L., Brunmark, A., Jackson, M. R., PETERSON, P. A., Teyton, L., Wilson, I. A. 1996; 274 (5285): 209-219


    The central event in the cellular immune response to invading microorganisms is the specific recognition of foreign peptides bound to major histocompatibility complex (MHC) molecules by the alphabeta T cell receptor (TCR). The x-ray structure of the complete extracellular fragment of a glycosylated alphabeta TCR was determined at 2.5 angstroms, and its orientation bound to a class I MHC-peptide (pMHC) complex was elucidated from crystals of the TCR-pMHC complex. The TCR resembles an antibody in the variable Valpha and Vbeta domains but deviates in the constant Calpha domain and in the interdomain pairing of Calpha with Cbeta. Four of seven possible asparagine-linked glycosylation sites have ordered carbohydrate moieties, one of which lies in the Calpha-Cbeta interface. The TCR combining site is relatively flat except for a deep hydrophobic cavity between the hypervariable CDR3s (complementarity-determining regions) of the alpha and beta chains. The 2C TCR covers the class I MHC H-2Kb binding groove so that the Valpha CDRs 1 and 2 are positioned over the amino-terminal region of the bound dEV8 peptide, the Vbeta chain CDRs 1 and 2 are over the carboxyl-terminal region of the peptide, and the Valpha and Vbeta CDR3s straddle the peptide between the helices around the central position of the peptide.

    View details for Web of Science ID A1996VM67100032

    View details for PubMedID 8824178

  • Role of chain pairing for the production of functional soluble IA major histocompatibility complex class II molecules JOURNAL OF EXPERIMENTAL MEDICINE Scott, C. A., Garcia, K. C., Carbone, F. R., Wilson, I. A., Teyton, L. 1996; 183 (5): 2087-2095


    Structural studies of cellular receptor molecules involved in immune recognition require the production of large quantities of the extracellular domains of these glycoproteins. The murine major histocompatibility complex (MHC) class II-restricted response has been extensively studied by functional means, but the engineering and purification of the native, empty form of the most-studied murine MHC class II molecule, IA, has been difficult to achieve. IA molecules, which are the murine equivalent of human histocompatibility leukocyte antigen-DQ molecules, have a low efficiency of chain pairing, which results in poor transport to the cell surface and in the appearance of mixed isotype pairs. We have engineered soluble IA molecules whose pairing has been forced by the addition of leucine zipper peptide dimers at their COOH-terminus. The molecules are secreted "empty" into the extracellular medium and can be loaded with single peptide after purification. These IA molecules have been expressed in milligram quantity for crystallization as well as for activation of T cells and measurement of MHC class II-T cell receptor interactions.

    View details for Web of Science ID A1996UM42400017

    View details for PubMedID 8642319



    Guanylin is a 15 amino acid mammalian hormone containing two disulfide bonds. Guanylin shares sequence similarity with the bacterial heat-stable enterotoxin (STa) and is capable of binding to and stimulating the STa guanylyl cyclase receptor. Biologically active peptides have been prepared by two methods: (1) enzymatic treatment of a 99 residue proprotein (denoted proguanylin) expressed in Escherichia coli and (2) solid-phase chemical synthesis. Although both sources yield material that is pure by high-performance liquid chromatography and mass spectrometry, analysis by nuclear magnetic resonance (NMR) indicates that peptides from both sources contain two conformationally distinct species present in a 1:1 ratio. The chemical shift differences between the two species are large, allowing unambiguous sequential NMR assignments to be made for both sets of resonances. Exchange between the two forms was not observed even at 70 degrees C. Structural restraints have been generated from nuclear Overhauser effects and scalar coupling constants and used to calculate structures for both forms using distance geometry and restrained energy minimization. The resulting structures for the first isoform are well defined (root-mean-square deviation from the average structure for backbone atoms of 0.47 A) and adopt a right-handed spiral conformation, similar to that observed for heat stable enterotoxin. The second isoform is less well defined (root-mean-square deviation from the average structure for backbone atoms of 1.07 A) but clearly adopts a very different fold consisting of a left-hand spiral. The differences in structure suggest that the two forms may have very different affinities toward the STa receptor. The observation of such isomerism has important implications for the common practice of introducing multiple disulfide bonds into small peptides to limit conformational flexibility and enhance bioactivity.

    View details for Web of Science ID A1994PT48500010

    View details for PubMedID 7947768

  • DO ANTIIDIOTYPIC ANTIBODIES MIMIC ANTIGEN RESEARCH IN IMMUNOLOGY Amzel, L. M., Garcia, K. C., Desiderio, S. 1994; 145 (1): 53-55

    View details for Web of Science ID A1994NB68400009

    View details for PubMedID 7516565



    Guanylin is a 15-amino acid peptide hormone that was originally isolated from the jejunum of the rat small intestine and shown to be an endogenous activator of the intestinal heat-stable enterotoxin receptor-guanylyl cyclase. Guanylin is synthesized as a 115-amino acid prohormone, proguanylin, which is processed at a site yet to be determined, into a C-terminal bioactive fragment(s). In order to examine the processing of proguanylin in vitro, we have generated large quantities of the properly folded prohormone by constructing an expression vector that directs its secretion into the periplasmic space of Escherichia coli. The bacterially expressed human proguanylin was then processed to smaller C-terminal fragments by protease digestion. Digestion with trypsin or lysine-C generated C-terminal peptides of different length, which have been purified and characterized. Guanylin-22 and guanylin-32 have binding affinities and biological activities similar to guanylin-15, while guanylin-63 and the entire proguanylin have only minimal bioactivity. Circular dichroism spectroscopy reveals that proguanylin is a stably folded protein containing mostly beta-sheet and beta-turn structure.

    View details for Web of Science ID A1993MD34800034

    View details for PubMedID 7901199



    The ability to predict the strength of the association of peptide hormones or other ligands with their protein receptors is of fundamental importance in the fields of protein engineering and rational drug design. To form a tight complex between a flexible peptide hormone and its receptor, the largeloss of configurational entropy must be overcome. Recently, the crystallographic structure of the complex between angiotensin II and the Fab fragment of a high affinity monoclonal antibody has been determined (Garcia, K.C., Ronco, P.M., Verroust, P.J., Brünger, A.T., Amzel, L.M. Three-dimensional structure of an angiotensin II-Fab complex at 3 A: Hormone recognition by an anti-idiotypic antibody. Science 257:502-507, 1992). In this paper we present a study of the thermodynamics of the association by high sensitivity isothermal titration calorimetry. The results of the experiments indicate that at 30 degrees C the binding is characterized by (1) a delta H of -8.9 +/- 0.7 kcal mol-1, (2) a delta Cp of -240 +/- 20 cal K-1 mol-1, and (3) the release of 1.1 +/- 0.1 protons per binding site in the pH range 6.0-7.3. Using these values and the previously determined binding constant in phosphate buffer, delta G at 30 degrees C is estimated as -11 kcal mol-1 and delta S as 6.9 cal K-1 mol-1. The calorimetric data indicate that binding is favored both enthalpically and entropically. These results have been complemented by structural thermodynamic calculations. The calculated and experimentally determined thermodynamic quantities are in good agreement.(ABSTRACT TRUNCATED AT 250 WORDS)

    View details for Web of Science ID A1993KJ87500001

    View details for PubMedID 8441749



    Genetic and sequence information are reported for an angiotensin II-reactive antibody (Ab1, MAb 110) and an anti--anti-idiotypic antibody (Ab3, MAb 131) that have identical antigen binding properties and that are related by an anti-idiotypic antibody (Ab2-beta) that satisfies accepted biochemical criteria for an internal image-bearing antibody. The sequences of the variable regions of the Ab3 and of the Ab1 are nearly identical, even though the Ab1 is an antibody to a peptide and the Ab3 is an antibody to a globular protein. Significantly, amino acid residues that make critical contacts with antigen in the crystal structure of the Ab3-antigen complex are highly conserved in Ab1, suggesting that the epitopes of the Ab2-beta recognized by the Ab3 do indeed resemble the bound structure of the antigen.

    View details for Web of Science ID A1992JE75500028

    View details for PubMedID 1636087

  • Three-dimensional structure of an angiotensin II-Fab complex at 3 A: hormone recognition by an anti-idiotypic antibody. Science Garcia, K. C., RONCO, P. M., VERROUST, P. J., Brünger, A. T., Amzel, L. M. 1992; 257 (5069): 502-507


    The elucidation of bioactive conformations of small peptide hormones remains an elusive goal to structural chemists because of the inherent flexibility of these molecules. Angiotensin II (AII), the major effector of the renin-angiotensin system, is an octapeptide hormone for which no clear structural models exist. Peptide hormones such as AII share the property that they bind to their receptors with high affinities, in spite of the fact that they must overcome an extremely large conformational entropy barrier to bind in one conformation. A "surrogate system" that consists of a high-affinity monoclonal antibody (MAb) and AII has been used to study a bound conformation of AII. The crystallographic structure of the complex reveals a structure of AII that is compatible with predicted bioactive conformations of AII derived from structure-activity studies and theoretical calculations. In the complex, the deeply bound hormone is folded into a compact structure in which two turns bring the amino and carboxyl termini close together. The antibody of this complex (MAb 131) has the unusual property that it was not generated against AII, but rather against an anti-idiotypic antibody reactive with a MAb to AII, which renders this antibody an anti-anti-idiotypic antibody. The high affinity for AII of the original MAb to AII was passed on to MAb 131 through a structural determinant on the anti-idiotypic antibody. Strikingly, the conformation of AII in this complex is highly similar to complementarity determining region loops of antibodies, possibly indicating that a true molecular mimic of bound AII was present on the anti-idiotypic antibody against which MAb 131 was elicited.

    View details for PubMedID 1636085



    The basic-helix-loop-helix-zipper (bHLH-Zip) motif is a conserved region of approximately 70 amino acids that mediates both sequence-specific DNA binding and protein dimerization. This motif is found in protein sequences from many eukaryotic organisms and is contained in the protein sequence of the oncogene myc and its partner max, and a shortened version of the motif (bHLH) is found in the muscle determination factor myoD and its partner E12. An evaluation of the conserved amino acids that define the motif coupled with the published mutagenic studies of this region has led to our formulation of a molecular model for the binding of this motif as a dimer to specific sequences of DNA. This model has the dimeric protein interacting with an abutted, dyad-symmetric DNA sequence. Helix 2 of each monomer is modeled as a coiled-coil extension of the C-terminal "leucine zipper." Helix 1 does not interact with helix 1 from its partner in the dimer but with the hydrophobic surface created when the helix 2 regions of the dimer interact with each other as a coiled-coil. Sequence-specific interactions are proposed between the basic region and the invariant cis elements that all bHLH-Zip proteins bind.

    View details for Web of Science ID A1992JR31200017

    View details for PubMedID 1622934



    mAb-131 is a monoclonal antibody that binds with high affinity (K alpha = 7.4 x 10(9) M-1) to the 8-residue peptide hormone angiotensin II, the major effector of the renin/angiotensin system. mAb-131 is a member of a well characterized idiotypic antibody network since it was raised as an anti-anti-idiotype of an antibody raised against angiotensin II. mAb-131 Fabs prepared with papain contain four major charge isoforms that can be separated by pH gradient elution from an anion-exchange column. Diffraction quality isomorphous crystals of two of the isoforms and of the Fab.peptide complexes have been grown. The crystals diffract to 3.5 A resolution, are tetragonal, space group P4(1) (or P4(3] with cell dimensions a = b = 78.6 A, c = 125.2 A, and have two Fab molecules per asymmetric unit. By using a different buffer, a second crystal form has been grown which diffracts to 3.3 A. It also belongs to space group P4(1) (or P4(3] but has cell dimensions of a = b = 109.6 A and c = 125.2 A. Knowledge of the three-dimensional structure of this Fab and of the peptide.Fab complex will give insight into two problems: 1) the recognition of small peptide hormones (which exist as random coils in solution) with high affinity by proteins, and 2) the nature of conservation of antibody combining sites in idiotypic networks.

    View details for Web of Science ID A1989CB33400049

    View details for PubMedID 2584225