Daniel Fernandez

All Publications

• Ion-selective conformational stabilization of a disordered repeats-in-toxin protein domain. Biophysical journal Gudinas, A. P., Shambharkar, G. M., Chang, M. P., Fernández, D., Matsui, T., Mai, D. J. 2025

  Abstract

  Ion-binding intrinsically disordered proteins (IDPs) recruit and bind to specific metal ions to perform critical biological functions. In proteins where ion binding and structural transitions are coupled, interactions with off-target toxic metals can dramatically disrupt protein structure and function, exemplified by lead and mercury poisoning. Understanding the complex mechanisms underlying how IDPs exclude or allow binding to different ionic species is crucial for addressing the origins of metal toxicity in biological systems. Here, we elucidate mechanisms of ion selectivity in an IDP that adopts a structure upon Ca2+ binding. We probed ion-induced conformational changes of a repeats-in-toxin (RTX) protein domain in the presence of different ion ligands-Mg2+, Ca2+, Sr2+, and Ba2+-with chemical similarities but drastically different ionic radii. RTX adopts ion-selective conformations measured by X-ray crystallography, small-angle X-ray scattering (SAXS), and circular dichroism (CD). High resolution X-ray structures reveal that Sr2+ induces a nearly identical RTX structure as natively binding Ca2+, enabled by the intrinsic flexibility and disorder of the protein. SAXS and CD indicate that smaller Mg2+ does not induce a significant conformational change in RTX, whereas larger Ba2+ induces a partially folded structure. These results highlight the importance of geometric constraints imposed by protein structure in determining metal ion selectivity, yielding insights into how off-target ion binding may result in protein misfolding and malfunction.

  View details for DOI 10.1016/j.bpj.2025.10.014

  View details for PubMedID 41088755

• Therapeutic targeting of the nuclear pore complex with molecular glue degraders in pancreatic cancer Yuan, L., Ji, W., Dwyer, B. G., Lu, J., Bian, J., Colombo, G. M., Martinez, M. J., Fernandez, D., Phillips, N. A., Tang, M. T., Zhou, C. W., Jones, H. M., Calla, N., Huancas, C., Eckart, M., Tran, J., Qiu, T., Doench, J. G., Rees, M. G., Roth, J. A., Cameron, M. D., Charville, G. W., Kuo, C. J., Dixon, S. J., Zhang, T., Hinshaw, S. M., Gray, N. S., Corsello, S. M. AMER ASSOC CANCER RESEARCH. 2025

  View details for DOI 10.1158/1538-7445.PANCREATIC25-B003

  View details for Web of Science ID 001588114400040

• Defining the antitumor mechanism of action of a clinical-stage compound as a selective degrader of the nuclear pore complex. Cancer discovery Yuan, L., Ji, W., Dwyer, B. G., Lu, J., Bian, J., Colombo, G. M., Martinez, M. J., Fernandez, D., Phillips, N. A., Tang, M. T., Zhou, C. W., Quispe Calla, N. E., Guzman Huancas, C., Eckart, M., Tran, J., Jones, H. M., Qiu, T., Doench, J. G., Rees, M. G., Roth, J. A., Cameron, M. D., Charville, G. W., Kuo, C. J., Dixon, S. J., Zhang, T., Hinshaw, S. M., Gray, N. S., Corsello, S. M. 2025

  Abstract

  Cancer cells are acutely dependent on nuclear transport due to elevated transcriptional activity, suggesting an unrealized opportunity for selective therapeutic inhibition of the nuclear pore complex. Through large-scale phenotypic profiling of cancer cell lines, genome-scale functional genomic modifier screens, and mass spectrometry-based proteomics, we discovered that the clinical drug PRLX-93936 is a molecular glue that binds and reprograms the TRIM21 ubiquitin ligase to degrade the nuclear pore complex. Upon compound-induced TRIM21 recruitment, the nuclear pore is ubiquitylated and degraded, resulting in the loss of short-lived cytoplasmic mRNA transcripts and induction of cancer cell apoptosis. Direct compound binding to TRIM21 was confirmed via surface plasmon resonance and x-ray crystallography, while compound-induced TRIM21-nucleoporin complex formation was demonstrated through multiple orthogonal approaches in cells and in vitro. Phenotype-guided optimization yielded compounds with 10-fold greater potency and drug-like properties with robust pharmacokinetics and efficacy against pancreatic cancer xenografts and patient-derived organoids.

  View details for DOI 10.1158/2159-8290.CD-25-0271

  View details for PubMedID 40891634

• ENPP1 inhibitor with ultralong drug-target residence time as an innate immune checkpoint blockade cancer therapy. Cell reports. Medicine Wang, S., Johnson, R. M., Carozza, J. A., Fernandez, D., Scicinski, J., Verity, N. A., Mardjuki, R., Cao, X., Guo, Y., Papkoff, J., Ray, N., Li, L. 2025: 102336

  Abstract

  Only one in five patients respond to immune checkpoint inhibitors, which primarily target adaptive immunity. Ectonucleotide pyrophosphatase/phophodiesterase 1 (ENPP1), the dominant hydrolase of 2'3'-cyclic-GMP-AMP (cGAMP) that suppresses downstream stimulator of interferon genes (STING) signaling, has emerged as a promising innate immunotherapy target. However, existing ENPP1 inhibitors have been optimized for prolonged systemic residence time rather than effective target inhibition within tumors. Here, we report the characterization of STF-1623, a highly potent ENPP1 inhibitor with an exceptionally long tumor residence time despite rapid systemic clearance, enabled by its high ENPP1 binding affinity and slow dissociation rate. We show that membrane-bound ENPP1 on tumor cells, not the abundant soluble ENPP1 in serum, drives tumor progression. Consequently, STF-1623 unleashes anti-tumor immunity to produce robust anti-tumor and anti-metastatic effects across multiple tumor models. Conceptually, this work establishes a noncovalent small-molecule inhibitor of ENPP1 with ultralong drug-target engagement as a safe and precise strategy to activate STING within tumors.

  View details for DOI 10.1016/j.xcrm.2025.102336

  View details for PubMedID 40914167

• ANGPTL3 orchestrates hepatic fructose sensing and metabolism. Cell reports Zhao, M., Linde-Garelli, K. Y., Zhang, Z., Toomer, D., Reghupaty, S. C., Jimenez, J. I., Coassolo, L., Wat, L. W., Fernandez, D., Svensson, K. J. 2025; 44 (7): 115962

  Abstract

  Fructose metabolism is linked to metabolic dysfunction-associated steatotic liver disease (MASLD), but the regulatory mechanisms governing fructose uptake remain poorly understood. Here, we demonstrate that MASLD livers exhibit increased uptake of fructose-derived carbons compared to healthy livers and identify that the MASLD hepatocyte secretome can increase fructose metabolism. By performing fractionation and untargeted proteomics, we uncover a role for Angiopoietin-like 3 (ANGPTL3) as a regulator of hepatic fructose metabolism, independent of its role as a lipoprotein lipase (LPL) inhibitor. Circulating ANGPTL3 levels increase in response to fructose exposure, consistent with an action as a fructose sensor. Angptl3 knockdown in the liver resulted in a significant reduction in the uptake of hepatic fructose metabolites in vivo and downregulation of the facilitative hepatic fructose transporter slc2a8 (GLUT8) and fructolysis enzymes. This work demonstrates the existence of extracellular control of hepatic fructose metabolism through ANGPTL3.

  View details for DOI 10.1016/j.celrep.2025.115962

  View details for PubMedID 40638391

• Utilizing Machine Learning to Improve Neutralization Potency of an HIV-1 Antibody Targeting the gp41 N-Heptad Repeat. ACS chemical biology Filsinger Interrante, M. V., Tang, S., Kim, S., Shanker, V. R., Hie, B. L., Bruun, T. U., Wu, W., Pak, J. E., Fernandez, D., Kim, P. S. 2025

  Abstract

  The N-heptad repeat (NHR) of the HIV-1 gp41 prehairpin intermediate (PHI) is an attractive potential vaccine target with high sequence conservation across diverse strains. However, despite the potency of NHR-targeting peptides and clinical efficacy of the NHR-targeting entry inhibitor enfuvirtide, no potently neutralizing NHR-directed monoclonal antibodies (mAbs) nor antisera have been identified or elicited to date. The lack of potent NHR-binding mAbs both dampens enthusiasm for vaccine development efforts at this target and presents a barrier to performing passive immunization experiments with NHR-targeting antibodies. To address this challenge, we previously developed an improved variant of the NHR-directed mAb D5, called D5_AR, which is capable of neutralizing diverse tier-2 viruses. Building on that work, here we present the 2.7Å-crystal structure of D5_AR bound to NHR mimetic peptide IQN17. We then utilize protein language models and supervised machine learning to generate small (n < 100) libraries of D5_AR variants that are subsequently screened for improved neutralization potency. We identify a variant with 5-fold improved neutralization potency, D5_FI, which is the most potent NHR-directed monoclonal antibody characterized to date and exhibits broad neutralization of tier-2 and -3 pseudoviruses as well as replicating R5 and X4 challenge strains. Additionally, our work highlights the ability of protein language models to efficiently identify improved mAb variants from relatively small libraries.

  View details for DOI 10.1021/acschembio.5c00035

  View details for PubMedID 40540236

• Crystal structure and ligand-binding specificity of the Borrelia burgdorferi proteins BmpA and BmpB Sharaf, N., Liu, Q., Fernandez, D. ELSEVIER. 2025

  View details for DOI 10.1016/j.jbc.2025.109939

  View details for Web of Science ID 001530451700093

• ENPP1 inhibitor with ultralong drug-target residence time as an innate immune checkpoint blockade cancer therapy. bioRxiv : the preprint server for biology Johnson, R. M., Wang, S., Carozza, J. A., Fernandez, D., Scicinski, J., Verity, N. A., Mardjuki, R., Cao, X., Papkoff, J., Ray, N., Li, L. 2025

  Abstract

  Only one in five patients is estimated to respond to immune checkpoint inhibitors, which primarily target adaptive immunity. To date, no FDA-approved immunotherapies directly activate the innate anti-cancer immunity-an essential driver of lymphocyte recruitment and potentiator of responses to existing cancer immunotherapies. ENPP1, the dominant hydrolase that degrades extracellular cGAMP and suppresses downstream STING-mediated innate immune signaling, has emerged as a promising therapeutic target. However, existing ENPP1 inhibitors have been optimized for prolonged systemic residence time rather than effective target inhibition within tumors. Here, we report the characterization of STF-1623, a highly potent ENPP1 inhibitor with an exceptionally long tumor residence time despite rapid systemic clearance, enabled by its high ENPP1 binding affinity and slow dissociation rate. We show that membrane-bound ENPP1 on tumor cells, not the abundant soluble ENPP1 in serum, drives tumor progression. Consequently, STF-1623 unleashes anti-tumor immunity and synergizes with ionizing radiation, anti-PD-L1 and anti-PD-1, and a DNA damaging agent to produce robust anti-tumor and anti-metastatic effects across multiple syngeneic mouse tumor models, all without detectable toxicity. Conceptually, this work establishes that a noncovalent small molecule inhibitor of ENPP1 with ultralong drug-target engagement offers a safe and precise strategy to activate STING within tumors, fulfilling an unmet need of innate immunotherapies in cancer.

  View details for DOI 10.1101/2025.05.18.654655

  View details for PubMedID 40475652

  View details for PubMedCentralID PMC12139726

• A Bivalent Molecular Glue Linking Lysine Acetyltransferases to Oncogene-induced Cell Death. bioRxiv : the preprint server for biology Nix, M. N., Gourisankar, S., Sarott, R. C., Dwyer, B. G., Nettles, S. A., Martinez, M. M., Abuzaid, H., Yang, H., Wang, Y., Simanauskaite, J. M., Romero, B. A., Jones, H. M., Krokhotin, A., Lowensohn, T. N., Chen, L., Low, C., Davis, M. M., Fernandez, D., Zhang, T., Green, M. R., Hinshaw, S. M., Gray, N. S., Crabtree, G. R. 2025

  Abstract

  Developing cancer therapies that induce robust death of the malignant cell is critical to prevent relapse. Highly effective strategies, such as immunotherapy, exemplify this observation. Here we provide the structural and molecular underpinnings for an approach that leverages chemical induced proximity to produce specific cell killing of diffuse large B cell lymphoma, the most common non-Hodgkin's lymphoma. We develop KAT-TCIPs (lysine acetyltransferase transcriptional/epigenetic chemical inducers of proximity) that redirect p300 and CBP to activate programmed cell death genes normally repressed by the oncogenic driver, BCL6. Acute treatment rapidly reprograms the epigenome to initiate apoptosis and repress c-MYC. The crystal structure of the chemically induced p300-BCL6 complex reveals how chance interactions between the two proteins can be systematically exploited to produce the exquisite potency and selectivity of KAT-TCIPs. Thus, the malignant function of an oncogenic driver can be co-opted to activate robust cell death, with implications for precision epigenetic therapies.

  View details for DOI 10.1101/2025.03.14.643404

  View details for PubMedID 40166243

  View details for PubMedCentralID PMC11956963

• Author Correction: Targeting colorectal cancer with small-molecule inhibitors of ALDH1B1. Nature chemical biology Feng, Z., Hom, M. E., Bearrood, T. E., Rosenthal, Z. C., Fernández, D., Ondrus, A. E., Gu, Y., McCormick, A. K., Tomaske, M. G., Marshall, C. R., Kline, T., Chen, C. H., Mochly-Rosen, D., Kuo, C. J., Chen, J. K. 2024

  View details for DOI 10.1038/s41589-024-01810-2

  View details for PubMedID 39653787

• Expression, Purification, and Characterization of Escherichia Coli Diacylated Lipoprotein Ycjn Sharaf, N., Trevino, M. A., Amankwah, K., Fernandez, D., Weston, S., Stewart, C. J., Gallardo, J. M. WILEY. 2024: 118-119

  View details for Web of Science ID 001437110900163

• Basic Science and Pathogenesis. Alzheimer's & dementia : the journal of the Alzheimer's Association Ho, W. H., Djomehri, S. I., Alford, V. M., Chen, E. Y., Reinitz, F., Gottlieb, S., Igboabuchi, F. I., Chung, G. Y., Fernandez, D., Montabana, E., Singal, B., Qi, Z., Kuo, A. H., Qian, D., Clarke, M. F. 2024; 20 Suppl 1: e093300

  Abstract

  Hallmark pathologies of Alzheimer's Disease (AD) include the accumulation of both extracellular amyloid and intracellular tau proteins. While a significant body of knowledge exists surrounding the role of the protein aggregates in the context of AD, research supporting these as targets for therapeutic development have yielded inconsistent findings. One significant barrier is the inability to restore cognitive function despite the successful clearance of these proteins. In our recently published paper, we discuss a novel target for AD, USP16, which has been shown to promote ubiquitination of histones allowing for transcription of CDKN2A, a cell cycle regulator. We demonstrated that knockdown of USP16 improved neurosphere initiating capacity in mouse neural stem cells. Moreover, reduction of USP16 expression via heterozygosity resulted in improved cognition in 12-month-old mice. From these findings, our lab aims to identify the full-length protein structure of USP16 using cryogenic electron microscopy (cryo-EM) and develop a small molecule drug to inhibit its activity for end-use as a viable AD therapeutic option.Truncated regions corresponding to the active and zinc-finger (ZnF) domains of USP16 were expressed in both HEK293T and SF9 cells. Whole cell lysates were purified using affinity chromatography and identity was verified using Western blot. The purified protein products were submitted for cryo-EM to determine their respective structures. Focusing on the ZnF domain, we utilized both a previously published proton NMR-derived structure as well as AlphaFold-predicted USP16 protein structure to serve as model scaffolding onto which we could perform in silico small molecule drug screening. Libraries were obtained from Schrodinger, MilliporeSigma, Emanine, MolPort, Thermo Fisher, MedChemExpress, Life Chemicals, ChemDiv, and Mcule.Chromatogram from affinity chromatography and Western blot imaging together indicate successful isolation of truncated USP16 domains expressed in HEK293T and SF9 cells. Preliminary findings from cryo-EM on the WT SF9 construct recapitulates the major domains predicted by AlphaFold. Over 9.5 million small molecule hit interactions against the ZnF domain have been identified.Cryo-EM was successful in generating a course model of USP16. In silico protein modeling and drug screening was able to identify potential ligands against the ZnF domain of USP16.

  View details for DOI 10.1002/alz.093300

  View details for PubMedID 39750542

• Expression, purification, and characterization of diacylated Lipo-YcjN from Escherichia coli. The Journal of biological chemistry Trevino, M. A., Amankwah, K. A., Fernandez, D., Weston, S. A., Stewart, C. J., Gallardo, J. M., Shahgholi, M., Sharaf, N. G. 2024: 107853

  Abstract

  YcjN is a putative substrate binding protein expressed from a cluster of genes involved in carbohydrate import and metabolism in Escherichia coli. Here, we determine the crystal structure of YcjN to a resolution of 1.95 A, revealing that its three-dimensional structure is similar to substrate binding proteins in subcluster D-I, which includes the well-characterized maltose binding protein (MBP). Furthermore, we found that recombinant overexpression of YcjN results in the formation of a lipidated form of YcjN that is posttranslationally diacylated at cysteine 21. Comparisons of size-exclusion chromatography profiles and dynamic light scattering measurements of lipidated and non-lipidated YcjN proteins suggest that lipidated YcjN aggregates in solution via its lipid moiety. Additionally, bioinformatic analysis indicates that YcjN-like proteins may exist in both Bacteria and Archaea, potentially in both lipidated and non-lipidated forms. Together, our results provide a better understanding of the aggregation properties of recombinantly expressed bacterial lipoproteins in solution and establish a foundation for future studies that aim to elucidate the role of these proteins in bacterial physiology.

  View details for DOI 10.1016/j.jbc.2024.107853

  View details for PubMedID 39362470

• Expression, purification, and characterization of diacylated Lipo-YcjN fromEscherichia coli. bioRxiv : the preprint server for biology Trevino, M. A., Amankwah, K., Fernandez, D., Weston, S., Stewart, C. J., Gallardo, J. M., Shahgholi, M., Sharaf, N. G. 2024

  Abstract

  YcjN is a putative substrate-binding protein expressed from a cluster of genes involved in carbohydrate import and metabolism in Escherichia coli . Here, we determine the crystal structure of YcjN to a resolution of 1.95 A, revealing that its three-dimensional structure is similar to substrate binding proteins in subcluster D-I, which includes the well-characterized maltose binding protein (MBP). Furthermore, we found that recombinant overexpression of YcjN results in the formation of a lipidated form of YcjN that is posttranslationally diacylated at cysteine 21. Comparisons of size-exclusion chromatography profiles and dynamic light scattering measurements of lipidated and non-lipidated YcjN proteins suggest that lipidated YcjN aggregates in solution via its lipid moiety. Additionally, bioinformatic analysis indicates that YcjN-like proteins may exist in both Bacteria and Archaea, potentially in both lipidated and non-lipidated forms. Together, our results provide a better understanding of the aggregation properties of recombinantly expressed bacterial lipoproteins in solution and establish a foundation for future studies that aim to elucidate the role of these proteins in bacterial physiology.

  View details for DOI 10.1101/2024.09.05.611266

  View details for PubMedID 39282304

• Dual quorum-sensing control of purine biosynthesis drives pathogenic fitness of Enterococcus faecalis. bioRxiv : the preprint server for biology Zlitni, S., Bowden, S., Sberro, H., Torres, M. D., Vaughan, J. M., Pinto, A. F., Pinto, Y., Fernandez, D., Röst, H., Saghatelian, A., de la Fuente-Nunez, C., Bhatt, A. S. 2024

  Abstract

  Enterococcus faecalis is a resident of the human gut, though upon translocation to the blood or body tissues, it can be pathogenic. Here we discover and characterize two peptide-based quorum-sensing systems that transcriptionally modulate de novo purine biosynthesis in E. faecalis. Using a comparative genomic analysis, we find that most enterococcal species do not encode this system; E. moraviensis, E. haemoperoxidus and E. caccae, three species that are closely related to E. faecalis, encode one of the two systems, and only E. faecalis encodes both systems. We show that these systems are important for the intracellular survival of E. faecalis within macrophages and for the fitness of E. faecalis in a murine wound infection model. Taken together, we combine comparative genomics, microbiological, bacterial genetics, transcriptomics, targeted proteomics and animal model experiments to describe a paired quorum sensing mechanism that directly influences central metabolism and impacts the pathogenicity of E. faecalis.

  View details for DOI 10.1101/2024.08.13.607696

  View details for PubMedID 39185165

  View details for PubMedCentralID PMC11343167

• Structural insights reveal interplay between LAG-3 homodimerization, ligand binding, and function. Proceedings of the National Academy of Sciences of the United States of America Silberstein, J. L., Du, J., Chan, K. W., Frank, J. A., Mathews, I. I., Kim, Y. B., You, J., Lu, Q., Liu, J., Philips, E. A., Liu, P., Rao, E., Fernandez, D., Rodriguez, G. E., Kong, X. P., Wang, J., Cochran, J. R. 2024; 121 (12): e2310866121

  Abstract

  Lymphocyte activation gene-3 (LAG-3) is an inhibitory receptor expressed on activated T cells and an emerging immunotherapy target. Domain 1 (D1) of LAG-3, which has been purported to directly interact with major histocompatibility complex class II (MHCII) and fibrinogen-like protein 1 (FGL1), has been the major focus for the development of therapeutic antibodies that inhibit LAG-3 receptor-ligand interactions and restore T cell function. Here, we present a high-resolution structure of glycosylated mouse LAG-3 ectodomain, identifying that cis-homodimerization, mediated through a network of hydrophobic residues within domain 2 (D2), is critically required for LAG-3 function. Additionally, we found a previously unidentified key protein-glycan interaction in the dimer interface that affects the spatial orientation of the neighboring D1 domain. Mutation of LAG-3 D2 residues reduced dimer formation, dramatically abolished LAG-3 binding to both MHCII and FGL1 ligands, and consequentially inhibited the role of LAG-3 in suppressing T cell responses. Intriguingly, we showed that antibodies directed against D1, D2, and D3 domains are all capable of blocking LAG-3 dimer formation and MHCII and FGL-1 ligand binding, suggesting a potential allosteric model of LAG-3 function tightly regulated by dimerization. Furthermore, our work reveals unique epitopes, in addition to D1, that can be targeted for immunotherapy of cancer and other human diseases.

  View details for DOI 10.1073/pnas.2310866121

  View details for PubMedID 38483996

• An orally bioavailable SARS-CoV-2 main protease inhibitor exhibits improved affinity and reduced sensitivity to mutations. Science translational medicine Westberg, M., Su, Y., Zou, X., Huang, P., Rustagi, A., Garhyan, J., Patel, P. B., Fernandez, D., Wu, Y., Hao, C., Lo, C. W., Karim, M., Ning, L., Beck, A., Saenkham-Huntsinger, P., Tat, V., Drelich, A., Peng, B. H., Einav, S., Tseng, C. K., Blish, C., Lin, M. Z. 2024; 16 (738): eadi0979

  Abstract

  Inhibitors of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) such as nirmatrelvir (NTV) and ensitrelvir (ETV) have proven effective in reducing the severity of COVID-19, but the presence of resistance-conferring mutations in sequenced viral genomes raises concerns about future drug resistance. Second-generation oral drugs that retain function against these mutants are thus urgently needed. We hypothesized that the covalent hepatitis C virus protease inhibitor boceprevir (BPV) could serve as the basis for orally bioavailable drugs that inhibit SARS-CoV-2 Mpro more efficiently than existing drugs. Performing structure-guided modifications of BPV, we developed a picomolar-affinity inhibitor, ML2006a4, with antiviral activity, oral pharmacokinetics, and therapeutic efficacy similar or superior to those of NTV. A crucial feature of ML2006a4 is a derivatization of the ketoamide reactive group that improves cell permeability and oral bioavailability. Last, ML2006a4 was found to be less sensitive to several mutations that cause resistance to NTV or ETV and occur in the natural SARS-CoV-2 population. Thus, anticipatory design can preemptively address potential resistance mechanisms to expand future treatment options against coronavirus variants.

  View details for DOI 10.1126/scitranslmed.adi0979

  View details for PubMedID 38478629

• Targeting <i>Aspergillus</i> allergen oryzin with a chemical probe at atomic precision SCIENTIFIC REPORTS Pattelli, O. N., Diec, D., Guo, W., Russi, S., Fernandez, D. 2023; 13 (1): 17926

  Abstract

  We report the molecular basis of Aspergillus fumigatus oryzin, allergen Asp f 13, or alkaline proteinase ALP1, containing the sequence motif His-Asp-Ser of the subtilisin family, structure, and function at atomic detail. Given the resolution of the data (1.06 Å), we use fragment molecular replacement with ideal polyalanine α-helices to determine the first crystal structure of oryzin. We probe the catalytic serine through formation of an irreversible bond to a small molecule compound, specifically labeling it, describing the amino acid residues performing the catalytic function. Defined by a self-processed pro-peptide, the active site architecture shapes up pocket-like subsites that bind to and unveil the S1'-S4' substrate binding preferences. We use molecular modeling to dock a model of the pro-peptide in the S1-S4 region and to dock collagen along the active site cleft. Opposite to the face harboring the catalytic serine, the enzyme binds to a calcium ion in a binding site created by backbone flipping. We use thermal unfolding to show that this metal ion provides structural stability. With no known host inhibitor identified thus far, this structure may hasten the progress of developing new therapeutic agents for diseases caused by pathogenic fungi.

  View details for DOI 10.1038/s41598-023-45028-z

  View details for Web of Science ID 001087596300059

  View details for PubMedID 37864071

  View details for PubMedCentralID PMC10589352

• Structure-guided stabilization improves the ability of the HIV-1 gp41 hydrophobic pocket to elicit neutralizing antibodies. The Journal of biological chemistry Bruun, T. J., Tang, S., Erwin, G., Deis, L., Fernandez, D., Kim, P. 2023: 103062

  Abstract

  The hydrophobic pocket found in the N-heptad repeat (NHR) region of HIV-1 gp41 is a highly conserved epitope that is the target of various HIV-1 neutralizing monoclonal antibodies. Although the high conservation of the pocket makes it an attractive vaccine candidate, it has been challenging to elicit potent anti-NHR antibodies via immunization. Here, we solved a high-resolution structure of the NHR mimetic IQN17, and, consistent with previous ligand-bound gp41 pocket structures, we observed remarkable conformational plasticity of the pocket. The high malleability of this pocket led us to test whether we could improve the immunogenicity of the gp41 pocket by stabilizing its conformation. We show that the addition of five amino acids at the C-terminus of IQN17, to generate IQN22, introduces a stabilizing salt bridge at the base of the peptide that rigidifies the pocket. Mice immunized with IQN22 elicited higher avidity antibodies against the gp41 pocket and a more potent, albeit still weak, neutralizing response against HIV-1 compared to IQN17. Stabilized epitope-focused immunogens could serve as the basis for future HIV-1 fusion-inhibiting vaccines.

  View details for DOI 10.1016/j.jbc.2023.103062

  View details for PubMedID 36841484

• Human neutralizing antibodies to cold linear epitopes and subdomain 1 of the SARS-CoV-2 spike glycoprotein. Science immunology Bianchini, F., Crivelli, V., Abernathy, M. E., Guerra, C., Palus, M., Muri, J., Marcotte, H., Piralla, A., Pedotti, M., De Gasparo, R., Simonelli, L., Matkovic, M., Toscano, C., Biggiogero, M., Calvaruso, V., Svoboda, P., Cervantes Rincon, T., Fava, T., Podesvova, L., Shanbhag, A. A., Celoria, A., Sgrignani, J., Stefanik, M., Honig, V., Pranclova, V., Michalcikova, T., Prochazka, J., Guerrini, G., Mehn, D., Ciabattini, A., Abolhassani, H., Jarrossay, D., Uguccioni, M., Medaglini, D., Pan-Hammarstrom, Q., Calzolai, L., Fernandez, D., Baldanti, F., Franzetti-Pellanda, A., Garzoni, C., Sedlacek, R., Ruzek, D., Varani, L., Cavalli, A., Barnes, C. O., Robbiani, D. F. 2023: eade0958

  Abstract

  Emergence of SARS-CoV-2 variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike glycoprotein that are both functionally relevant and averse to change, we identified human neutralizing antibodies to highly conserved viral epitopes. Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four genera, including the nine human coronaviruses, through recognition of a conserved motif that includes the S2 site of proteolytic cleavage. Antibody hr2.016 targets the stem helix and neutralizes SARS-CoV-2 variants. Antibody sd1.040 binds to subdomain 1, synergizes with antibody rbd.042 for neutralization and, like fp.006 and hr2.016, protects mice expressing human ACE2 against infection when present as bispecific antibody. Thus, coldspot-guided antibody discovery reveals donor-derived neutralizing antibodies that are cross-reactive with Orthocoronavirinae, including SARS-CoV-2 variants.

  View details for DOI 10.1126/sciimmunol.ade0958

  View details for PubMedID 36701425

• Human neutralizing antibodies to cold linear epitopes and to subdomain 1 of SARS-CoV-2. bioRxiv : the preprint server for biology Bianchini, F., Crivelli, V., Abernathy, M. E., Guerra, C., Palus, M., Muri, J., Marcotte, H., Piralla, A., Pedotti, M., De Gasparo, R., Simonelli, L., Matkovic, M., Toscano, C., Biggiogero, M., Calvaruso, V., Svoboda, P., Rincón, T. C., Fava, T., Podešvová, L., Shanbhag, A. A., Celoria, A., Sgrignani, J., Stefanik, M., Hönig, V., Pranclova, V., Michalcikova, T., Prochazka, J., Guerrini, G., Mehn, D., Ciabattini, A., Abolhassani, H., Jarrossay, D., Uguccioni, M., Medaglini, D., Pan-Hammarström, Q., Calzolai, L., Fernandez, D., Baldanti, F., Franzetti-Pellanda, A., Garzoni, C., Sedlacek, R., Ruzek, D., Varani, L., Cavalli, A., Barnes, C. O., Robbiani, D. F. 2022

  Abstract

  Emergence of SARS-CoV-2 variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike that are both functionally relevant and averse to change, we identified human neutralizing antibodies to highly conserved viral epitopes. Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four genera , including the nine human coronaviruses, through recognition of a conserved motif that includes the S2' site of proteolytic cleavage. Antibody hr2.016 targets the stem helix and neutralizes SARS-CoV-2 variants. Antibody sd1.040 binds to subdomain 1, synergizes with antibody rbd.042 for neutralization and, like fp.006 and hr2.016, protects mice when present as bispecific antibody. Thus, coldspot-guided antibody discovery reveals donor-derived neutralizing antibodies that are cross-reactive with Orthocoronavirinae , including SARS-CoV-2 variants.Broadly cross-reactive antibodies that protect from SARS-CoV-2 variants are revealed by virus coldspot-driven discovery.

  View details for DOI 10.1101/2022.11.24.515932

  View details for PubMedID 36482967

  View details for PubMedCentralID PMC9727766

• De Novo Design of a Highly Stable Ovoid TIM Barrel: Unlocking Pocket Shape towards Functional Design. Biodesign research Chu, A. E., Fernandez, D., Liu, J., Eguchi, R. R., Huang, P. S. 2022; 2022: 9842315

  Abstract

  The ability to finely control the structure of protein folds is an important prerequisite to functional protein design. The TIM barrel fold is an important target for these efforts as it is highly enriched for diverse functions in nature. Although a TIM barrel protein has been designed de novo, the ability to finely alter the curvature of the central beta barrel and the overall architecture of the fold remains elusive, limiting its utility for functional design. Here, we report the de novo design of a TIM barrel with ovoid (twofold) symmetry, drawing inspiration from natural beta and TIM barrels with ovoid curvature. We use an autoregressive backbone sampling strategy to implement our hypothesis for elongated barrel curvature, followed by an iterative enrichment sequence design protocol to obtain sequences which yield a high proportion of successfully folding designs. Designed sequences are highly stable and fold to the designed barrel curvature as determined by a 2.1 Å resolution crystal structure. The designs show robustness to drastic mutations, retaining high melting temperatures even when multiple charged residues are buried in the hydrophobic core or when the hydrophobic core is ablated to alanine. As a scaffold with a greater capacity for hosting diverse hydrogen bonding networks and installation of binding pockets or active sites, the ovoid TIM barrel represents a major step towards the de novo design of functional TIM barrels.

  View details for DOI 10.34133/2022/9842315

  View details for PubMedID 37850141

  View details for PubMedCentralID PMC10521652

• ENPP1's regulation of extracellular cGAMP is a ubiquitous mechanism of attenuating STING signaling. Proceedings of the National Academy of Sciences of the United States of America Carozza, J. A., Cordova, A. F., Brown, J. A., AlSaif, Y., Bohnert, V., Cao, X., Mardjuki, R. E., Skariah, G., Fernandez, D., Li, L. 2022; 119 (21): e2119189119

  Abstract

  SignificanceThe immune system strikes a careful balance between launching a robust response to threats and avoiding overactivation. The molecule cGAMP is an immunotransmitter that activates innate immunity and signals extracellularly, where it is subject to degradation by the enzyme ENPP1. Here, we engineer ENPP1 to lose activity toward cGAMP but not other substrates, thus creating a biochemically precise tool to understand how ENPP1 regulates extracellular cGAMP and thus innate immunity. We uncover that ENPP1's degradation of extracellular cGAMP has a long evolutionary history, and that this mechanism is critical for controlling diverse immune threats, including viral infection and inflammation.

  View details for DOI 10.1073/pnas.2119189119

  View details for PubMedID 35588451

• Structure-guided mutagenesis of a mucin-selective metalloprotease from Akkermansia muciniphila alters substrate preferences. The Journal of biological chemistry Shon, D. J., Fernandez, D., Riley, N. M., Ferracane, M. J., Bertozzi, C. R. 2022: 101917

  Abstract

  Akkermansia muciniphila, a mucin-degrading microbe found in the human gut, is often associated with positive health outcomes. The abundance of Akkermansia muciniphila is modulated by the presence and accessibility of nutrients, which can be derived from diet or host glycoproteins. In particular, the ability to degrade host mucins, a class of proteins carrying densely O-glycosylated domains, provides a competitive advantage in the sustained colonization of niche mucosal environments. Although Akkermansia muciniphila is known to rely on mucins as a carbon and nitrogen source, the enzymatic machinery used by this microbe to process mucins in the gut is not yet fully characterized. Here, we focus on the mucin-selective metalloprotease, Amuc_0627 (AM0627), which is known to cleave between adjacent residues carrying truncated core 1 O-glycans. We showed that this enzyme is capable of degrading purified mucin 2 (MUC2), the major protein component of mucus in the gut. An X-ray crystal structure of AM0627 (1.9 A resolution) revealed O-glycan binding residues that are conserved between structurally characterized enzymes from the same family. We further rationalized the substrate cleavage motif using molecular modeling to identify nonconserved glycan-interacting residues. We conclude that mutagenesis of these residues resulted in altered substrate preferences down to the glycan level, providing insight into the structural determinants of O-glycan recognition.

  View details for DOI 10.1016/j.jbc.2022.101917

  View details for PubMedID 35405095

• Targeting colorectal cancer with small-molecule inhibitors of ALDH1B1 Nature Chemical Biology Feng, Z., Hom, M. E., Bearrood, T. E., Rosenthal, Z. C., Fernández, D., Ondrus, A. E., Gu, Y., McCormick, A. K., Tomaske, M. G., Marshall, C. R., Chen, C., Mochly-Rosen, D., Kuo, C. J., Chen, J. K. 2022

  View details for DOI 10.1038/s41589-022-01048-w

• 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

  Abstract

  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

• Inter-cellular CRISPR screens reveal regulators of cancer cell phagocytosis. Nature Kamber, R. A., Nishiga, Y., Morton, B., Banuelos, A. M., Barkal, A. A., Vences-Catalan, F., Gu, M., Fernandez, D., Seoane, J. A., Yao, D., Liu, K., Lin, S., Spees, K., Curtis, C., Jerby-Arnon, L., Weissman, I. L., Sage, J., Bassik, M. C. 2021

  Abstract

  Monoclonal antibody therapies targeting tumour antigens drive cancer cell elimination in large part by triggering macrophage phagocytosis of cancer cells1-7. However, cancer cells evade phagocytosis using mechanisms that are incompletely understood. Here we develop a platform for unbiased identification of factors that impede antibody-dependent cellular phagocytosis (ADCP) using complementary genome-wide CRISPR knockout and overexpression screens in both cancer cells and macrophages. In cancer cells, beyond known factors such as CD47, we identify many regulators of susceptibility to ADCP, including the poorly characterized enzyme adipocyte plasma membrane-associated protein (APMAP). We find that loss of APMAP synergizes with tumour antigen-targeting monoclonal antibodies and/or CD47-blocking monoclonal antibodies to drive markedly increased phagocytosis across a wide range of cancer cell types, including those that are otherwise resistant to ADCP. Additionally, we show that APMAP loss synergizes with several different tumour-targeting monoclonal antibodies to inhibit tumour growth in mice. Using genome-wide counterscreens in macrophages, we find that the G-protein-coupled receptor GPR84 mediates enhanced phagocytosis of APMAP-deficient cancer cells. This work reveals a cancer-intrinsic regulator of susceptibility to antibody-driven phagocytosis and, more broadly, expands our knowledge of the mechanisms governing cancer resistance to macrophage phagocytosis.

  View details for DOI 10.1038/s41586-021-03879-4

  View details for PubMedID 34497417

• Dissecting O-GalNAc glycosylation by glycosyltransferase engineering Tastan, O. Y., Debets, M. F., Malaker, S. A., Wisnovsky, S. P., Angelis, N., Wagner, L. J. S., Choi, J., Browne, W. M., Bineva-Todd, G., Cioce, A., Agbay, A. J., Li, Z., Briggs, D. C., Flynn, H., Roustan, C., Fernandez, D., Douglas, H. L., Kjaer, S., Snijders, A. P., Li, V. S. W., Bertozzi, C. R., Schumann, B. OXFORD UNIV PRESS INC. 2020: 1028

  View details for Web of Science ID 000606577100022

• Structure-Aided Development of Small-Molecule Inhibitors of ENPP1, the Extracellular Phosphodiesterase of the Immunotransmitter cGAMP. Cell chemical biology Carozza, J. A., Brown, J. A., Bohnert, V., Fernandez, D., AlSaif, Y., Mardjuki, R. E., Smith, M., Li, L. 2020

  Abstract

  Cancer cells initiate an innate immune response by synthesizing and exporting the small-molecule immunotransmitter cGAMP, which activates the anti-cancer Stimulator of Interferon Genes (STING) pathway in the host. An extracellular enzyme, ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1), hydrolyzes cGAMP and negatively regulates this anti-cancer immune response. Small-molecule ENPP1 inhibitors are much needed as tools to study the basic biology of extracellular cGAMP and as investigational cancer immunotherapy drugs. Here, we surveyed structure-activity relationships around a series of cell-impermeable and thus extracellular-targeting phosphonate inhibitors of ENPP1. In addition, we solved the crystal structure of an exemplary phosphonate inhibitor to elucidate the interactions that drive potency. This study yielded several best-in-class inhibitors with Ki< 2nM and excellent physicochemical and pharmacokinetic properties. Finally, we demonstrate that an ENPP1 inhibitor delays tumor growth in a breast cancer mouse model. Together, we have developed ENPP1 inhibitors that are excellent tool compounds and potential therapeutics.

  View details for DOI 10.1016/j.chembiol.2020.07.007

  View details for PubMedID 32726585

• A Plasma Protein Network Regulates PM20D1 and N-Acyl Amino Acid Bioactivity. Cell chemical biology Kim, J. T., Jedrychowski, M. P., Wei, W., Fernandez, D., Fischer, C. R., Banik, S. M., Spiegelman, B. M., Long, J. Z. 2020

  Abstract

  N-acyl amino acids are a family of cold-inducible circulating lipids that stimulate thermogenesis. Their biosynthesis is mediated by a secreted enzyme called PM20D1. The extracellular mechanisms that regulate PM20D1 or N-acyl amino acid activity in the complex environment of blood plasma remains unknown. Using quantitative proteomics, here we show that PM20D1 circulates in tight association with both low- and high-density lipoproteins. Lipoprotein particles are powerful co-activators of PM20D1 activity invitro and N-acyl amino acid biosynthesis invivo. We also identify serum albumin as a physiologic N-acyl amino acid carrier, which spatially segregates N-acyl amino acids away from their sites of production, confers resistance to hydrolytic degradation, and establishes an equilibrium between thermogenic "free" versus inactive "bound" fractions. These data establish lipoprotein particles as principal extracellular sites of N-acyl amino acid biosynthesis and identify a lipoprotein-albumin network that regulates the activity of a circulating thermogenic lipid family.

  View details for DOI 10.1016/j.chembiol.2020.04.009

  View details for PubMedID 32402239

• Bump-and-Hole Engineering Identifies Specific Substrates of Glycosyltransferases in Living Cells. Molecular cell Schumann, B. n., Malaker, S. A., Wisnovsky, S. P., Debets, M. F., Agbay, A. J., Fernandez, D. n., Wagner, L. J., Lin, L. n., Li, Z. n., Choi, J. n., Fox, D. M., Peh, J. n., Gray, M. A., Pedram, K. n., Kohler, J. J., Mrksich, M. n., Bertozzi, C. R. 2020

  Abstract

  Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein, and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context, and glycan fine structure. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide N-acetylgalactosaminyl transferases. This approach bestows a gain-of-chemical-functionality modification on cells, where the products of individual glycosyltransferases can be selectively characterized or manipulated to understand glycan contribution to major physiological processes.

  View details for DOI 10.1016/j.molcel.2020.03.030

  View details for PubMedID 32325029

• Protein structural changes on a CubeSat under rocket acceleration profile. NPJ microgravity Luna, A. n., Meisel, J. n., Hsu, K. n., Russi, S. n., Fernandez, D. n. 2020; 6 (1): 12

  Abstract

  Catalyzing life-sustaining reactions, proteins are composed by 20 different amino acids that fold into a compact yet flexible three-dimensional architecture, which dictates what their function(s) might be. Determining the spatial arrangement of the atoms, the protein's 3D structure, enables key advances in fundamental and applied research. Protein crystallization is a powerful technique to achieve this. Unlike Earth's crystallization experiments, biomolecular crystallization in space in the absence of gravitational force is actively sought to improve crystal growth techniques. However, the effects of changing gravitational vectors on a protein solution reaching supersaturation remain largely unknown. Here, we have developed a low-cost crystallization cell within a CubeSat payload module to exploit the unique experimental conditions set aboard a sounding rocket. We designed a biaxial gimbal to house the crystallization experiments and take measurements on the protein solution in-flight with a spectrophotometry system. After flight, we used X-ray diffraction analysis to determine that flown protein has a structural rearrangement marked by loss of the protein's water and sodium in a manner that differs from crystals grown on the ground. We finally show that our gimbal payload module design is a portable experimental setup to take laboratory research investigations into exploratory space flights.

  View details for DOI 10.1038/s41526-020-0102-3

  View details for PubMedID 33580006

• STING Polymer Structure Reveals Mechanisms for Activation, Hyperactivation, and Inhibition. Cell Ergun, S. L., Fernandez, D., Weiss, T. M., Li, L. 2019

  Abstract

  How the central innate immune protein, STING, is activated by its ligands remains unknown. Here, using structural biology and biochemistry, we report that the metazoan second messenger 2'3'-cGAMP induces closing of the human STING homodimer and release of the STING C-terminal tail, which exposes a polymerization interface on the STING dimer and leads to the formation of disulfide-linked polymers via cysteine residue 148. Disease-causing hyperactive STING mutations either flank C148 and depend on disulfide formation or reside in the C-terminal tail binding site and cause constitutive C-terminal tail release and polymerization. Finally, bacterial cyclic-di-GMP induces an alternative active STING conformation, activates STING in a cooperative manner, and acts as a partial antagonist of 2'3'-cGAMP signaling. Our insights explain the tight control of STING signaling given varying background activation signals and provide a therapeutic hypothesis for autoimmune syndrome treatment.

  View details for DOI 10.1016/j.cell.2019.05.036

  View details for PubMedID 31230712

• Discovery of Mechanism-Based Inactivators for Human Pancreatic Carboxypeptidase A from a Focused Synthetic Library ACS MEDICINAL CHEMISTRY LETTERS Testero, S. A., Granados, C., Fernandez, D., Gallego, P., Covaleda, G., Reverter, D., Vendrell, J., Aviles, F. X., Pallares, I., Mobashery, S. 2017; 8 (10): 1122-1127

  Abstract

  Metallocarboxypeptidases (MCPs) are involved in many biological processes such as fibrinolysis or inflammation, development, Alzheimer's disease, and various types of cancer. We describe the synthesis and kinetic characterization of a focused library of 22 thiirane- and oxirane-based potential mechanism-based inhibitors, which led to discovery of an inhibitor for the human pro-carboxypeptidase A1. Our structural analyses show that the thiirane-based small-molecule inhibitor penetrates the barrier of the pro-domain to bind within the active site. This binding leads to a chemical reaction that covalently modifies the catalytic Glu270. These results highlight the importance of combined structural, biophysical, and biochemical evaluation of inhibitors in design strategies for the development of spectroscopically nonsilent probes as effective beacons for in vitro, in cellulo, and/or in vivo localization in clinical and industrial applications.

  View details for DOI 10.1021/acsmedchemlett.7b00346

  View details for Web of Science ID 000413135800024

  View details for PubMedID 29057062

  View details for PubMedCentralID PMC5641956

• Calcium-dependent oligomerization of CAR proteins at cell membrane modulates ABA signaling PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Diaz, M., Jose Sanchez-Barrena, M., Maria Gonzalez-Rubio, J., Rodriguez, L., Fernandez, D., Antoni, R., Yunta, C., Belda-Palazon, B., Gonzalez-Guzman, M., Peirats-Llobet, M., Menendez, M., Boskovic, J., Marquez, J. A., Rodriguez, P. L., Albert, A. 2016; 113 (3): E396-E405

  Abstract

  Regulation of ion transport in plants is essential for cell function. Abiotic stress unbalances cell ion homeostasis, and plants tend to readjust it, regulating membrane transporters and channels. The plant hormone abscisic acid (ABA) and the second messenger Ca(2+) are central in such processes, as they are involved in the regulation of protein kinases and phosphatases that control ion transport activity in response to environmental stimuli. The identification and characterization of the molecular mechanisms underlying the effect of ABA and Ca(2+) signaling pathways on membrane function are central and could provide opportunities for crop improvement. The C2-domain ABA-related (CAR) family of small proteins is involved in the Ca(2+)-dependent recruitment of the pyrabactin resistance 1/PYR1-like (PYR/PYL) ABA receptors to the membrane. However, to fully understand CAR function, it is necessary to define a molecular mechanism that integrates Ca(2+) sensing, membrane interaction, and the recognition of the PYR/PYL interacting partners. We present structural and biochemical data showing that CARs are peripheral membrane proteins that functionally cluster on the membrane and generate strong positive membrane curvature in a Ca(2+)-dependent manner. These features represent a mechanism for the generation, stabilization, and/or specific recognition of membrane discontinuities. Such structures may act as signaling platforms involved in the recruitment of PYR/PYL receptors and other signaling components involved in cell responses to stress.

  View details for DOI 10.1073/pnas.1512779113

  View details for Web of Science ID 000368458800019

  View details for PubMedID 26719420

  View details for PubMedCentralID PMC4725540

• C2-Domain Abscisic Acid-Related Proteins Mediate the Interaction of PYR/PYL/RCAR Abscisic Acid Receptors with the Plasma Membrane and Regulate Abscisic Acid Sensitivity in <i>Arabidopsis</i> (Publication with Expression of Concern. See vol. 37, 2025) PLANT CELL Rodriguez, L., Gonzalez-Guzman, M., Diaz, M., Rodrigues, A., Izquierdo-Garcia, A. C., Peirats-Llobet, M., Fernandez, M. A., Antoni, R., Fernandez, D., Marquez, J. A., Mulet, J. M., Albert, A., Rodriguez, P. L. 2014; 26 (12): 4802-4820

  Abstract

  Membrane-delimited abscisic acid (ABA) signal transduction plays a critical role in early ABA signaling, but the molecular mechanisms linking core signaling components to the plasma membrane are unclear. We show that transient calcium-dependent interactions of PYR/PYL ABA receptors with membranes are mediated through a 10-member family of C2-domain ABA-related (CAR) proteins in Arabidopsis thaliana. Specifically, we found that PYL4 interacted in an ABA-independent manner with CAR1 in both the plasma membrane and nucleus of plant cells. CAR1 belongs to a plant-specific gene family encoding CAR1 to CAR10 proteins, and bimolecular fluorescence complementation and coimmunoprecipitation assays showed that PYL4-CAR1 as well as other PYR/PYL-CAR pairs interacted in plant cells. The crystal structure of CAR4 was solved, which revealed that, in addition to a classical calcium-dependent lipid binding C2 domain, a specific CAR signature is likely responsible for the interaction with PYR/PYL receptors and their recruitment to phospholipid vesicles. This interaction is relevant for PYR/PYL function and ABA signaling, since different car triple mutants affected in CAR1, CAR4, CAR5, and CAR9 genes showed reduced sensitivity to ABA in seedling establishment and root growth assays. In summary, we identified PYR/PYL-interacting partners that mediate a transient Ca(2+)-dependent interaction with phospholipid vesicles, which affects PYR/PYL subcellular localization and positively regulates ABA signaling.

  View details for DOI 10.1105/tpc.114.129973

  View details for Web of Science ID 000348646000020

  View details for PubMedID 25465408

  View details for PubMedCentralID PMC4311195

• A functional and structural study of the major metalloprotease secreted by the pathogenic fungus <i>Aspergillus fumigatus</i> ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY Fernandez, D., Russi, S., Vendrell, J., Monod, M., Pallares, I. 2013; 69: 1946-1957

  Abstract

  Fungalysins are secreted fungal peptidases with the ability to degrade the extracellular matrix proteins elastin and collagen and are thought to act as virulence factors in diseases caused by fungi. Fungalysins constitute a unique family among zinc-dependent peptidases that bears low sequence similarity to known bacterial peptidases of the thermolysin family. The crystal structure of the archetype of the fungalysin family, Aspergillus fumigatus metalloprotease (AfuMep), has been obtained for the first time. The 1.8 Å resolution structure of AfuMep corresponds to that of an autoproteolyzed proenzyme with separate polypeptide chains corresponding to the N-terminal prodomain in a binary complex with the C-terminal zinc-bound catalytic domain. The prodomain consists of a tandem of cystatin-like folds whose C-terminal end is buried into the active-site cleft of the catalytic domain. The catalytic domain harbouring the key catalytic zinc ion and its ligands, two histidines and one glutamic acid, undergoes a conspicuous rearrangement of its N-terminal end during maturation. One key positively charged amino-acid residue and the C-terminal disulfide bridge appear to contribute to its structural-functional properties. Thus, structural, biophysical and biochemical analysis were combined to provide a deeper comprehension of the underlying properties of A. fumigatus fungalysin, serving as a framework for the as yet poorly known metallopeptidases from pathogenic fungi.

  View details for DOI 10.1107/S0907444913017642

  View details for Web of Science ID 000325403900009

  View details for PubMedID 24100314

• Metallocarboxypeptidases and their Inhibitors: Recent Developments in Biomedically Relevant Protein and Organic Ligands CURRENT MEDICINAL CHEMISTRY Fernandez, D., Pallares, I., Covaleda, G., Aviles, F. X., Vendrell, J. 2013; 20 (12): 1595-1608

  Abstract

  Metallocarboxypeptidases (MCPs) are zinc-dependent exoproteases that have been for long considered benchmark enzymes, perform a wide range of physiological roles and have been regarded as interesting drug targets. Several crystal structures of MCPs in complex with protein and small molecular weight inhibitors have recently been obtained providing a framework for understanding the binding properties of these ligands. Much of the latest research focused on carboxypeptidase U or thrombin-activable fibrinolysis inhibitor (CPU/TAFI) which has fueled new designs in the field of cardiovascular drugs. Further, new details on the catalytic mechanism of MCPs have emerged from recent crystal structures of covalently modified forms and the pace of investigations on inhibitors has been steadily fastening in the last years. This paper will focus on the latest research carried on metallocarboxypeptidase small molecular weight inhibitors as drug candidates and will give an update of protein inhibitors to emphasize the growing interest for products isolated from natural sources.

  View details for DOI 10.2174/0929867311320120009

  View details for Web of Science ID 000316617600009

  View details for PubMedID 23432588

• Structure of a novel M32 peptidase from disease-causing <i>Trypanosoma cruzi</i> Pallares, I., Fabrega, M., Llonch, S., Reverter, D., Vendrell, J., Fernandez, D. WILEY. 2012: 456-457

  View details for Web of Science ID 000308128602491

• Structural and Functional Analysis of the Complex between Citrate and the Zinc Peptidase Carboxypeptidase A. Enzyme research Fernandez, D., Boix, E., Pallares, I., Aviles, F. X., Vendrell, J. 2011; 2011: 128676

  Abstract

  A high-resolution carboxypeptidase-Zn(2+)-citrate complex was studied by X-ray diffraction and enzyme kinetics for the first time. The citrate molecule acts as a competitive inhibitor of this benchmark zinc-dependent peptidase, chelating the catalytic zinc ion in the active site of the enzyme and inducing a conformational change such that carboxypeptidase adopts the conformation expected to occur by substrate binding. Citrate adopts an extended conformation with half of the molecule facing the zinc ion, while the other half is docked in the S1' hydrophobic specificity pocket of the enzyme, in contrast with the binding mode expected for a substrate like phenylalanine or a peptidomimetic inhibitor like benzylsuccinic acid. Combined structural and enzymatic analysis describes the characteristics of the binding of this ligand that, acting against physiologically relevant zinc-dependent proteases, may serve as a general model in the design of new drug-protecting molecules for the oral delivery of drugs of peptide origin.

  View details for DOI 10.4061/2011/128676

  View details for PubMedID 21804935

• Progress in metallocarboxypeptidases and their small molecular weight inhibitors BIOCHIMIE Fernandez, D., Pallares, I., Vendrell, J., Aviles, F. X. 2010; 92 (11): 1484-1500

  Abstract

  In what corresponds to a life span, metallocarboxypeptidases (MCPs) have jumped from being mere contaminants in animal pancreas powders (in depression year 1929) to be key players in cellular and molecular processes (in yet-another-depression years 2009-2010). MCPs are unique zinc-dependent enzymes that catalyze the breakdown of the amide bond at the C-terminus of peptide and protein substrates and participate in the recovery of dietary amino acids, tissue organogenesis, neurohormone and cytokine maturation and other important physiological processes. More than 26 genes code for MCPs in the human genome, many of them still waiting to be fully understood in terms of physiological function. A variety of MCPs have been linked to diseases in man: acute pancreatitis and pancreas cancer, type 2 diabetes, Alzheimer's Disease, various types of cancer, and fibrinolysis and inflammation. Many of these discoveries have been made possible thanks to recent advances, as exemplified by plasma carboxypeptidases N and B, known for fifty and twenty years, respectively, which have had their structures released only very recently. Plasma carboxypeptidase B is a biological target for therapy because of its involvement in the coagulation/fibrinolysis processes. Besides, the widespread use of carboxypeptidase A as a benchmark metalloprotease since the early days of Biochemistry has allowed the identification and design of an increasingly vast repertory of small molecular weight inhibitors. With these two examples we wish to emphasize that MCPs have become part of the drug discovery portfolio of pharmaceutical companies and academic research laboratories. This paper will review key developments in the discovery and design of MCP small molecular weight inhibitors, with an emphasis on the discovery of chemically diverse entities. Although encouraging advances have been achieved in the last few years, the specificity and oral bioavailability of the new chemotherapeutic agents seem to pose a challenge to medicinal chemists.

  View details for DOI 10.1016/j.biochi.2010.05.002

  View details for Web of Science ID 000284717300003

  View details for PubMedID 20466032

• Analysis of a New Crystal Form of Procarboxypeptidase B: Further Insights into the Catalytic Mechanism BIOPOLYMERS Fernandez, D., Boix, E., Pallares, I., Aviles, F. X., Vendrell, J. 2010; 93 (2): 178-185

  Abstract

  A new triclinic crystal structure form of porcine pancreatic procarboxypeptidase B (PCPB) was obtained at higher resolution than the previously known tetragonal crystal structure. This new crystal polymorph has allowed for a corrected, accurate assignment of residues along the polypeptide chain based on the currently available gene sequence information and crystallographic data. The present structure shows unbound PCPB in a distinct molecular packing as compared to the previous benzamidine complexed form. Its catalytically important Tyr248 residue is oriented and hydrogen-bonded to solvent water molecules, and locates the furthest away from the catalytic zinc ion as compared to previous structures. A relatively long stretch of residues flanking Tyr248 and guarding the access to the catalytic zinc ion was found to be sequentially unique to the M14 family of peptidases. Predictions from a normal mode analysis indicated that this stretch of residues belongs to a rigid subdomain in the protein structure. The specific presence of a tyrosyl residue at the most exposed position in this region would allow for a delicate balance between extreme hydrophobicity and hydrophilicity, and affect substrate binding and the kinetic efficiency of the enzyme.

  View details for DOI 10.1002/bip.21320

  View details for Web of Science ID 000272809100007

  View details for PubMedID 19802820

• The X-Ray Structure of Carboxypeptidase A Inhibited by a Thiirane Mechanism-Based Inhibitor CHEMICAL BIOLOGY & DRUG DESIGN Fernandez, D., Testero, S., Vendrell, J., Aviles, F. X., Mobashery, S. 2010; 75 (1): 29-34

  Abstract

  The three-dimensional X-ray crystal structure of carboxypeptidase A, a zinc-dependent hydrolase, covalently modified by a mechanism-based thiirane inactivator, 2-benzyl-3,4-epithiobutanoic acid, has been solved to 1.38 A resolution. The interaction of the thiirane moiety of the inhibitor with the active site zinc ion promotes its covalent modification of Glu-270 with the attendant opening of the thiirane ring. The crystal structure determination at high resolution allowed for the clear visualization of the covalent ester bond to the glutamate side chain. The newly generated thiol from the inhibitor binds to the catalytic zinc ion in a monodentate manner, inducing a change in the zinc ion geometry and coordination, while its benzyl group fits into the S1' specificity pocket of the enzyme. The inhibitor molecule is distorted at the position of the carbon atom that is involved in the ester bond linkage on one side and the zinc coordination on the other. This particular type of thiirane-based metalloprotease inhibitor is for the first time analyzed in complex to the target protease at high resolution and may be used as a general model for zinc-dependent proteases.

  View details for DOI 10.1111/j.1747-0285.2009.00907.x

  View details for Web of Science ID 000272306500004

  View details for PubMedID 19895506

  View details for PubMedCentralID PMC2908478

• A new type of five-membered heterocyclic inhibitors of basic metallocarboxypeptidases EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY Fernandez, D., Aviles, F. X., Vendrell, J. 2009; 44 (8): 3266-3271

  Abstract

  A structure-based virtual screening survey was used to identify potential inhibitors of the human M14 family of metallocarboxypeptidases. A good correlation between docking energy scores and measured K(i) values was observed, indicating an efficient performance of the screening procedure. Among various compounds displaying K(i) values in the low micromolar range, N-(3-chlorophenyl)-4-((5-(3-methoxybenzylthio)-1,3,4-oxadiazol-2-yl)methyl)thiazol-2-amine emerged as the most powerful inhibitor for human carboxypeptidase B (CPB). According to molecular docking, this compound fits into CPB active site cleft through coordination of the catalytic zinc ion with the 1,3,4-oxadiazole moiety. This represents a novel five-membered heterocyclic type of inhibitor for disease-linked metallocarboxypeptidases and an interesting lead for further development.

  View details for DOI 10.1016/j.ejmech.2009.03.034

  View details for Web of Science ID 000266709700019

  View details for PubMedID 19386397

• Cyclobutane-containing peptides: Evaluation as novel metallocarboxypeptidase inhibitors and modelling of their mode of action BIOORGANIC & MEDICINAL CHEMISTRY Fernandez, D., Torres, E., Aviles, F. X., Ortuno, R. M., Vendrell, J. 2009; 17 (11): 3824-3828

  Abstract

  Different types of cyclobutane-containing peptides (CBPs) were screened for the first time as ligands of metallocarboxypeptidases (MCPs). CBPs are conformationally constrained, low molecular-weight compounds which showed moderate yet selective inhibitory activity against mammalian MCPs. The most potent compound was a carboxypeptidase B inhibitor. Docked protein-ligand complexes indicated that CBPs may bind to the target proteases via electrostatic interactions and aromatic stacking to catalytically crucial residues and that the placement of functional groups seems to be assisted by the rigid CBP backbone. The easily obtainable CBPs may offer a valuable alternative in the design of novel inhibitors to disease-linked metallocarboxypeptidases like human plasma carboxypeptidase B.

  View details for DOI 10.1016/j.bmc.2009.04.035

  View details for Web of Science ID 000266117400011

  View details for PubMedID 19414265

• Aromatic Organic Compounds as Scaffolds for Metallocarboxypeptidase Inhibitor Design CHEMICAL BIOLOGY & DRUG DESIGN Fernandez, D., AvilEs, F. X., Vendrell, J. 2009; 73 (1): 75-82

  Abstract

  We have identified and characterized a set of quinoline, naphthalene and quinazoline derivatives as inhibitors of metallocarboxypeptidases, a class of metal-dependent proteolytic enzymes. The aromatic organic compounds were selected from a high-throughput screening survey and, with some exceptions, showed a good correlation between inhibitory potency and docking energy value. The in vitro inhibition tests gave K(i) values in the lower micromolar range for metallocarboxypeptidases with different specificities, and a tendency to behave as more powerful inhibitors of CPB was observed for most of the compounds tested. The kinetic results were further analyzed by structural analysis via molecular docking. The most potent aromatic organic inhibitor docks to human CPB mostly through burial of its hydrophobic moiety deep into the enzyme's active site cleft and by interacting with the catalytic zinc ion. The significance of our results in designing inhibitors against disease-related CPs from the identified ligands is examined herein.

  View details for DOI 10.1111/j.1747-0285.2008.00752.x

  View details for Web of Science ID 000261837700010

  View details for PubMedID 19152637

• The molecular analysis of <i>Trypanosoma cruzi</i> metallocarboxypeptidase 1 provides insight into fold and substrate specificity MOLECULAR MICROBIOLOGY Niemirowicz, G., Fernandez, D., Sola, M., Cazzulo, J. J., Aviles, F. X., Xavier Gomis-Ruth, F. 2008; 70 (4): 853-866

  Abstract

  Trypanosoma cruzi is the aetiological agent of Chagas' disease, a chronic infection that affects millions in Central and South America. Proteolytic enzymes are involved in the development and progression of this disease and two metallocarboxypeptidases, isolated from T. cruzi CL Brener clone, have recently been characterized: TcMCP-1 and TcMCP-2. Although both are cytosolic and closely related in sequence, they display different temporary expression patterns and substrate preferences. TcMCP-1 removes basic C-terminal residues, whereas TcMCP-2 prefers hydrophobic/aromatic residues. Here we report the three-dimensional structure of TcMCP-1. It resembles an elongated cowry, with a long, deep, narrow active-site cleft mimicking the aperture. It has an N-terminal dimerization subdomain, involved in a homodimeric catalytically active quaternary structure arrangement, and a proteolytic subdomain partitioned by the cleft into an upper and a lower moiety. The cleft accommodates a catalytic metal ion, most likely a cobalt, which is co-ordinated by residues included in a characteristic zinc-binding sequence, HEXXH and a downstream glutamate. The structure of TcMCP-1 shows strong topological similarity with archaeal, bacterial and mammalian metallopeptidases including angiotensin-converting enzyme, neurolysin and thimet oligopeptidase. A crucial residue for shaping the S(1') pocket in TcMCP-1, Met-304, was mutated to the respective residue in TcMCP-2, an arginine, leading to a TcMCP-1 variant with TcMCP-2 specificity. The present studies pave the way for a better understanding of a potential target in Chagas' disease at the molecular level and provide a template for the design of novel therapeutic approaches.

  View details for DOI 10.1111/j.1365-2958.2008.06444.x

  View details for Web of Science ID 000260194300009

  View details for PubMedID 18793339

• Direct interaction between a human digestive protease and the mucoadhesive poly(acrylic acid) ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY Pallares, I., Fernandez, D., Comellas-Bigler, M., Fernandez-Recio, J., Ventura, S., Aviles, F. X., Bode, W., Vendrell, J. 2008; 64: 784-791

  Abstract

  Carboxypeptidase A1 has been the subject of extensive research in the last 30 y and is one of the most widely studied zinc metalloenzymes. However, the three-dimensional structure of the human form of the enzyme is not yet available. This report describes the three-dimensional structure of human carboxypeptidase A1 (hCPA1) derived from crystals that belong to the tetragonal space group P4(3)2(1)2 and diffract to 1.6 angstroms resolution. A description of the ternary complex hCPA1-Zn2+-poly(acrylic acid) is included as a model of the interaction of mucoadhesive polymers with proteases in the gastrointestinal tract. The direct mode of interaction between poly(acrylic acid) and the active site of the target protease was confirmed by in vitro inhibition assays. The structure was further analyzed in silico through the optimal docking-area method. The characterization of binding sites on the surface of hCPA1 and a comparison with other available carboxypeptidase structures provided further insights into the formation of multiprotein complexes and the activation mechanisms of carboxypeptidase zymogens. The high-resolution structure of hCPA1 provides an excellent template for the modelling of physiologically relevant carboxypeptidases and could also contribute to the design of specific agents for biomedical purposes.

  View details for DOI 10.1107/S0907444908013474

  View details for Web of Science ID 000256856100008

  View details for PubMedID 18566513

• Recent structural and computational insights into conformational diseases CURRENT MEDICINAL CHEMISTRY Fernandez-Busquets, X., de Groot, N. S., Fernandez, D., Ventura, S. 2008; 15 (13): 1336-1349

  Abstract

  Protein aggregation correlates with the development of several deleterious human disorders such as Alzheimer's disease, Parkinson's disease, prion-associated transmissible spongiform encephalopathies and type II diabetes. The polypeptides involved in these disorders may be globular proteins with a defined 3D-structure or natively unfolded proteins in their soluble conformations. In either case, proteins associated with these pathogeneses all aggregate into amyloid fibrils sharing a common structure, in which beta-strands of polypeptide chains are perpendicular to the fibril axis. Because of the prominence of amyloid deposits in many of these diseases, much effort has gone into elucidating the structural basis of protein aggregation. A number of recent experimental and theoretical studies have significantly increased our understanding of the process. On the one hand, solid-state NMR, X-ray crystallography and single molecule methods have provided us with the first high-resolution 3D structures of amyloids, showing that they exhibit conformational plasticity and are able to adopt different stable tertiary folds. On the other hand, several computational approaches have identified regions prone to aggregation in disease-linked polypeptides, predicted the differential aggregation propensities of their genetic variants and simulated the early, crucial steps in protein self-assembly. This review summarizes these findings and their therapeutic relevance, as by uncovering specific structural or sequential targets they may provide us with a means to tackle the debilitating diseases linked to protein aggregation.

  View details for DOI 10.2174/092986708784534938

  View details for Web of Science ID 000256616700010

  View details for PubMedID 18537613

• Thioxophosphoranyl aryl- and heteroaryloxiranes as the representants of a new class of metallocarboxypeptidase inhibitors BIOORGANIC & MEDICINAL CHEMISTRY Fernandez, D., Illa, O., Aviles, F. X., Branchadell, V., Vendrell, J., Ortuno, R. M. 2008; 16 (9): 4823-4828

  Abstract

  A novel and potent family of metallocarboxypeptidase inhibitors based on thioxophosphoranyl oxiranes is presented. These compounds bear aryl or heteroaryl substituents with trans-stereochemistry with respect to the phosphorylated group and they have been synthesized by the addition of [bis(diisopropylamino)phosphino](trimethylsilyl)carbene to the corresponding aldehydes and the subsequent thiolation of the phosphine. These oxiranes contain a tetrahedral P atom harboring shielded N,N-groups. The screening of their biological activity as metallocarboxypeptidase inhibitors and some structural studies, as well as full experimental details for the new compounds, is disclosed. Thus, from the analysis of their activity against the prototypical metallocarboxypeptidases A and B (CPA and CPB), we have observed that hydrophobic phosphorylated oxiranes perform better as CPB inhibitors, reaching K(i) values comparable to classical synthetic carboxypeptidase inhibitors. X-ray diffraction analysis revealed that the packing in the structure of one phosphorylated oxirane is mediated mainly by hydrophobic contacts and that the N,N-groups are highly flexible. Consequently, phosphorylated oxiranes might constitute an attractive material for subsequent improvements in the design of novel inhibitors against human proteolytic enzymes with enhanced oral availability.

  View details for DOI 10.1016/j.bmc.2008.03.047

  View details for Web of Science ID 000255605600004

  View details for PubMedID 18396051

• Structural and functional characterization of binding sites in metallocarboxypeptidases based on optimal docking area analysis PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS Fernandez, D., Vendrell, J., Aviles, F. X., Fernandez-Recio, J. 2007; 68 (1): 131-144

  Abstract

  The metallocarboxypeptidases (MCPs) belonging to the clan MC were studied by the Optimal Docking Area (ODA) method to evaluate protein-protein binding sites and to provide a basis for the identification of binding partners for this class of enzymes. The ODA method identifies surface patches with optimal desolvation energy based on the selection of low-energy docking regions, generated from a set of surface points around the protein. With few exceptions, the ODA method identified surface patches with a significant low-energy docking surface for all the MCPs with known three-dimensional structure. Overall, in 14 out of 24 cases, the detected ODA patches were correctly located (i.e. more than 50% of the predicted residues were in known protein-protein binding sites), yielding a global success rate of 58%. More specifically, the success rate increased up to 80% on the ODA patches detected for the catalytic domains of the M14A subfamily, independently on the partner. Interestingly, the ODA residues on the catalytic domain were correctly located in the interface with the N-terminal pro domain in all MCPs. The spatial distribution of the ODA patches for the different members of the family is in relation to the origin and function of the particular MCP, which allowed distinguishing between them. In good agreement with the experimentally characterized protein interfaces, the total average surface area of the theoretically derived ODA patches for the catalytic domain of MCPs is around 1700 A2 and their content in hydrophobic residues is about 40%. As a particular case, the average surface area of the ODA patches in MCPs of crop insect pests is about twice that of the MCPs of vertebrates, which might be related to their particular function. We recognized two binding regions for the catalytic domain of the MCPs, one of them accounting for nearly all the known intermolecular interactions made up by the enzymes. Protein inhibitors seem to have evolved to dock on this subset of ODA patches, evoking the binding mode of the N-terminal pro domains. The second binding region detected, for which no ligands have been identified so far, seems to be related to the acquisition/maintenance of the native structure of the peptidase. Overall, the ODA method has been successful in identifying low-energy docking areas in a set of structurally and functionally related proteins, suggesting that it can be easily extended to other families in the search for protein-protein binding sites and for their functional significance.

  View details for DOI 10.1002/prot.21390

  View details for Web of Science ID 000246894800015

  View details for PubMedID 17407161

• Caught after the act:: A human a-type metallocarboxypeptidase in a product complex with a cleaved hexapeptide BIOCHEMISTRY Bayes, A., Fernandez, D., Sola, M., Marrero, A., Garcia-Pique, S., Aviles, F. X., Vendrell, J., Gomis-Ruth, F. 2007; 46 (23): 6921-6930

  Abstract

  A/B-type metallocarboxypeptidases (MCPs) are among the most thoroughly studied proteolytic enzymes, and their catalytic mechanisms have been considered as prototypes even for several unrelated metalloprote(in)ase families. It has long been postulated that the nature of the side chains of at least five substrate residues, i.e., P4-P1', influence Km and kcat and that once the peptide or protein substrate is cleaved, both products remain in the first instance bound to the active-site cleft of the enzyme in a double-product complex. Structural details of binding of substrate to the nonprimed side of the cleft have largely relied on complexes with protein inhibitors and peptidomimetic small-molecule inhibitors that do not span the entire groove. In the former, the presence of N-terminal globular protein domains participating in large-scale interactions with the surface of the cognate catalytic domain outside the active-site cleft mostly conditions the way their C-terminal tails bind to the cleft. Accordingly, they may not be accurate models for a product complex. We hereby provide the structural details of a true cleaved double-product complex with a hexapeptide of an MCP engaged in prostate cancer, human carboxypeptidase A4, employing diffraction data to 1.6 A resolution (Rcryst and Rfree = 0.159 and 0.176, respectively). These studies provide detailed information about subsites S5-S1' and contribute to our knowledge of the cleavage mechanism, which is revisited in light of these new structural insights.

  View details for DOI 10.1021/bi700480b

  View details for Web of Science ID 000247141700024

  View details for PubMedID 17506531

• Polymorphism on leflunomide:: Stability and crystal structures JOURNAL OF PHARMACEUTICAL SCIENCES Vega, D., Petragalli, A., Fernández, D., Ellena, J. A. 2006; 95 (5): 1075-1083

  Abstract

  Two polymorphs of Leflunomide were found and studied (form I and II). Both of them were characterized by X-ray powder diffraction and thermal analysis. Single crystals were obtained and both structures were solved. Forms I and II crystallize in the space group P2(1)/c with two and one independent molecules per asymmetric unit, respectively. Thermodynamic stability of the two forms is assessed by differential scanning calorimetry. The cohesion in the crystal of form I (the more stable) is provided by both by H bonding as well as pi...pi interactions, while in form II it is given only by the former. The independent molecules in form I adopt different conformations thus allowing for a larger number of intermolecular interactions.

  View details for DOI 10.1002/jps.20382

  View details for Web of Science ID 000237236800012

  View details for PubMedID 16570307

• Thermal behaviour and stability in Olanzapine INTERNATIONAL JOURNAL OF PHARMACEUTICS Polla, G. I., Vega, D. R., Lanza, H., Tombari, D. G., Baggio, R., Ayala, A. P., Mendes, J., Fernández, D., Leyva, G., Dartayet, G. 2005; 301 (1-2): 33-40

  Abstract

  The stability and thermal behaviour of two anhydrate phases and a new mixed water:DMSO solvate of Olanzapine (2-methyl-4-(4-methyl-1-piperazinyl)-10H-thieno-[2,3-b][1,5]benzodiazepine) are studied by different methods: differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD) and Raman scattering (RS). Single crystal structural data for the latter phase are presented, confirming the presence of the (Olanzapine)(2) dimer as the structural building unit of all known phases of the drug, either anhydrate or solvated. An apparent interconversion between both solid state forms is shown to be an artifact and explained in terms of a melting-recrystallization process.

  View details for DOI 10.1016/j.ijpharm.2005.05.035

  View details for Web of Science ID 000232238900005

  View details for PubMedID 16040214

• Candesartan cilexetil, an anti hypertensive agent containing an extended double ester chain ACTA CRYSTALLOGRAPHICA SECTION E-CRYSTALLOGRAPHIC COMMUNICATIONS Fernández, D., Vega, D., Ellena, J. A. 2005; 61: O309-O312

  View details for DOI 10.1107/S1600536805000097

  View details for Web of Science ID 000226692400126

• The purine transferase from <i>Trypanosoma cruzi</i> as a potential target for bisphosphonate-based chemotherapeutic compounds BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Fernández, D., Wenck, M. A., Craig, S. P., Delfino, J. M. 2004; 14 (17): 4501-4504

  Abstract

  We identified and tested bisphosphonates as inhibitors of a protozoan molecular target. Computational modeling studies demonstrated that these compounds are mimics of the natural substrate of the enzyme. The most potent bisphosphonates in vitro are pamidronate and risedronate, which inhibit the purine transferase from Trypanosoma cruzi in the micromolar range.

  View details for DOI 10.1016/j.bmcl.2004.06.042

  View details for Web of Science ID 000223382000026

  View details for PubMedID 15357980

• The Zn<SUP>2+</SUP> salt of pamidronate:: a role for water in the metal-cation binding properties of bisphosphonates ACTA CRYSTALLOGRAPHICA SECTION C-CRYSTAL STRUCTURE COMMUNICATIONS Fernández, D., Polla, G., Vega, D., Ellena, J. A. 2004; 60: M73-M75

  Abstract

  Pamidronate (3-ammonium-1-hydroxypropylidene-1,1-bisphosphonate) is used clinically in the treatment of diseases affecting bone tissue. In the salt zinc pamidronate dihydrate, Zn(2+).2C(3)H(10)NO(7)P(2)(-).2H(2)O, pamidronate is a zwitterion with an overall charge of -1. The carbon chain adopts a trans conformation, separating maximally the positively charged N atom from the negative phosphonate groups. The Zn(2+) ion lies on an inversion center and is surrounded by a sixfold coordination sphere provided by two bidentate chelating zwitterions and two water molecules. The bidentate O.Zn.O bond angle is 92.70 (7) degrees, while the O.O bite distance is 3.018 (3) A.

  View details for DOI 10.1107/S0108270104000216

  View details for Web of Science ID 000188555300016

  View details for PubMedID 14767118

• Alendronate zwitterions bind to calcium cations arranged in columns ACTA CRYSTALLOGRAPHICA SECTION C-CRYSTAL STRUCTURE COMMUNICATIONS Fernández, D., Vega, D., Goeta, A. 2003; 59: M543-M545

  Abstract

  Alendronate is used clinically in the treatment of skeletal disorders, the mode of action depending on the adsorption to calcium hydroxyapatite crystals (bone). In the title compound, calcium 4-ammonium-1-hydroxybutylidene-1,1-bisphosphonate, Ca(2+).2C(4)H(12)NO(7)P(2)(-), alendronate is a zwitterion, possessing one negative charge on each PO(3) group and a protonated N atom. The zwitterion is disposed with its negative end facing the Ca(2+) ion, while its positive end is stretched in the opposite direction. The geometry of the carbon chain is all-trans, while the hydroxy group is approximately gauche. The Ca(2+) ion lies on a twofold axis parallel to b. The coordination sphere around the metal cation is octahedral and is determined by monodentate- and bidentate-coordinated alendronate zwitterions. The O.O bite distance is 3.080 (2) A. Coordinated Ca(2+) metal cations are arranged at the centre of a column running along c.

  View details for DOI 10.1107/S0108270103025599

  View details for Web of Science ID 000187793800022

  View details for PubMedID 14671354

• Head-to-head dimers in the zwitterion of 1-hydroxy-1-phosphono-3-(1-piperidino)propylidene-1-phosphonate (PHPBP) ACTA CRYSTALLOGRAPHICA SECTION C-CRYSTAL STRUCTURE COMMUNICATIONS Fernández, D., Vega, D. 2003; 59: O661-O663

  Abstract

  The title compound, C(8)H(19)NO(7)P(2), is a member of the bisphosphonate family of therapeutic compounds. PHPBP has inner-salt character, consisting of a negatively charged PO(3) group and a positively charged N atom. The six-membered piperidine ring adopts an almost-perfect chair conformation. The hydroxyl group and the N atom have gauche and trans conformations in relation to the O-C-C-C-N backbone, respectively. Hydrogen bonding is the main contributor to the packing in the crystal, which consists of head-to-head dimers formed through phosphonyl-phosphonyl hydrogen bonds, while O-H.O and N-H.O interactions join the dimers into a plane parallel to crystallographic b and c axes.

  View details for DOI 10.1107/S0108270103023710

  View details for Web of Science ID 000187793800030

  View details for PubMedID 14671362

• Two members of the bisphosphonate class of drugs:: a zwitterion and a molecular compound ACTA CRYSTALLOGRAPHICA SECTION C-CRYSTAL STRUCTURE COMMUNICATIONS Fernández, D., Vega, D., Ellena, J. A. 2003; 59: O289-O292

  Abstract

  The compounds studied in this paper, viz. (1-ammonio-1-phosphonopropyl)phosphonate, C(3)H(11)NO(6)P(2), (I), and 1-(acetylamino)propylidene-1,1-bisphosphonic acid dihydrate, C(5)H(13)NO(7)P(2).2H(2)O, (II), are members of a commonly used family of therapeutic agents. Compound (I) is an inner salt with separated negative (on the ionized PO(3) group) and positive (on the tetrahedral N atom) charges, while (II) possesses neutral phosphonyl groups and one amide N atom. Both structures have a C-C-C-N backbone, which has comparable geometric parameters in (I) and (II); the main difference was found in one of the N-C-P bond angles, which is lengthened in (II) because of an intramolecular O(PO(3))-H.O(C=O) interaction. The hydrogen-bonding scheme in the crystal of (I) includes all possible donor atoms, namely all the H atoms of the ammonium group and the phosphonic acid functions. As a result of these interactions, the zwitterions are organized into a plane running along the crystallographic x axis. In (II), the intermolecular interactions include all possible donor atoms, except for the N atom; the packing differs from that of (I) in that the molecules are arranged in a chain running parallel to the x axis. In the chains, the molecules form head-to-head dimers, while the crystallization water molecules contribute to the intra- and interchain cohesion.

  View details for DOI 10.1107/S0108270103007649

  View details for Web of Science ID 000183345100028

  View details for PubMedID 12794342

• Head-to-head dimers in the zwitterion of 1-hydroxy-3-(pyrrolidin-1-yl)propylidene-1,1-bisphosphonic acid (EB 1053) ACTA CRYSTALLOGRAPHICA SECTION C-CRYSTAL STRUCTURE COMMUNICATIONS Fernández, D., Vega, D. 2003; 59: O228-O230

  Abstract

  The title compound, 1-hydroxy-1-phosphono-3-(1-pyrrolidinio)propylidene-1-phosphonate, C(7)H(17)NO(7)P(2), is a member of the bisphosphonate class of drugs. As a zwitterion, it possesses a negative charge on one of the PO(3) groups and a positive charge on the pyrrolidine N atom. A zwitterion makes a contact with a neighbouring ion through the hydroxyl O atom and two phosphonyl O atoms, one each from two different PO(3) groups. Hydrogen bonding involves O-H.O and N-H.O interactions; the former are involved in the formation of head-to-head dimers, while the latter join the dimers into a chain running along the crystallographic b axis.

  View details for DOI 10.1107/S0108270103006085

  View details for Web of Science ID 000182656300020

  View details for PubMedID 12743399

• 17α,21-dihydroxy-16β-methyl-pregna-1,4-diene-3,11,20-trione (meprednisone) ACTA CRYSTALLOGRAPHICA SECTION C-CRYSTAL STRUCTURE COMMUNICATIONS Fernández, D., Vega, D., Ellena, J. A. 2003; 59: O187-O189

  Abstract

  The title compound, C(22)H(28)O(5), is a commercial therapeutic agent of the steroid class. Both independent molecules in the asymmetric unit have six-membered A rings that are planar, while the B and C rings adopt normal chair conformations. The five-membered D ring is in a 13beta,14alpha-half-chair conformation, and the B/C and C/D ring junctions are in trans positions. Cohesion in the crystal is provided by O-H.O hydrogen bonds, which generate chains of molecules that are organized in a plane that lies along the crystallographic b axis.

  View details for DOI 10.1107/S0108270103003184

  View details for Web of Science ID 000182560600022

  View details for PubMedID 12682403

• The calcium-binding properties of pamidronate, a bone-resorption inhibitor ACTA CRYSTALLOGRAPHICA SECTION C-CRYSTAL STRUCTURE COMMUNICATIONS Fernández, D., Vega, D., Goeta, A. 2002; 58: M494-M497

  Abstract

  The title compound, calcium bis(3-ammonio-1-hydroxypropylidene-1,1-bisphosphonate) dihydrate, Ca(2+).2C(3)H(10)NO(7)P(2)(-).2H(2)O, consists of calcium octahedra arranged in columns along the c axis and coordinated by hydrogen-bonded molecular anions. The Ca(2+) cation lies on a twofold axis. Pamidronate adopts a twisted conformation of the hydroxyalkylamine backbone that enables the formation of an intramolecular N-H...O hydrogen bond. The molecular anion is chelating monodentate as well as bidentate, with an O...O bite distance of 3.0647 (15) A.

  View details for DOI 10.1107/S010827010201497X

  View details for Web of Science ID 000178399800007

  View details for PubMedID 12359924

• Losartan potassium, a non-peptide agent for the treatment of arterial hypertension ACTA CRYSTALLOGRAPHICA SECTION C-STRUCTURAL CHEMISTRY Fernández, D., Vega, D., Ellena, J. A., Echeverría, G. 2002; 58: m418-m420

  Abstract

  In the title compound, potassium 2-butyl-4-chloro-1-[[2'-(5-tetrazolido)biphenyl-4-yl]methyl]-1H-imidazol-5-ylmethanol, K(+) x C(22)H(22)ClN(6)O(-), the imidazole and tetrazole rings are at angles of 85.0 (2) and 51.8 (1) degrees, respectively, to the phenyl rings to which they are attached, while the dihedral angle between the latter two rings is 46.7 (1) degrees. The coordination sphere of the metal cation consists of six tetrazoyl N atoms, the methanol O atom and the pi cloud of one of the phenyl rings. These interactions determine the formation of columns of molecular anions that lie parallel to the b axis, while hydrogen bonding contributes to intercolumnar cohesion. Far from the centre of the columns, the hydrocarbon chain is immersed in a hydrophobic environment.

  View details for DOI 10.1107/S0108270102009745

  View details for Web of Science ID 000176567300025

  View details for PubMedID 12094046

• Disodium pamidronate ACTA CRYSTALLOGRAPHICA SECTION C-CRYSTAL STRUCTURE COMMUNICATIONS Vega, D., Fernéndez, D., Ellena, J. A. 2002; 58: M77-M80

  Abstract

  The title compound, disodium 3-ammonium-1-hydroxypropylidene-1,1-bisphosphonate pentahydrate, 2Na(+).C(3)H(9)NO(7)P(2)(2-).5H(2)O, is used for the diagnosis and treatment of a number of bone disorders. In the solid state, disodium pamidronate shows zwitterionic character and has four different modes of chelation to sodium. The metal is octahedrally coordinated by zwitterion and water O atoms. Both coordination to sodium and hydrogen bonding determine the packing in the crystal, which comprises columns lying parallel to the crystallographic a axis.

  View details for DOI 10.1107/S0108270101019655

  View details for Web of Science ID 000173796300007

  View details for PubMedID 11828086

• 1-(2-{4-[6-Hydroxy-2-(4-hydroxyphenyl)benzo[<i>b</i>]thiol-3-ylcarbonyl]-phenoxy}ethyl)piperidinium chloride ACTA CRYSTALLOGRAPHICA SECTION C-STRUCTURAL CHEMISTRY Vega, D., Fernández, D., Ellena, J. A. 2001; 57: 1092-1094

  Abstract

  The title compound, raloxifene hydrochloride, C(28)H(28)NO(4)S(+).-Cl(-), belongs to the benzothiophene class of antiosteoporotic drugs. In the molecular cation, the 2-phenol ring sustains a dihedral angle of 45.3 (1) degrees relative to the benzo[b]thiophene system. The benzo[b]thiophene and phenyl ring planes are twisted with respect to the carbonyl plane, with the smallest twist component occurring between the phenyl and carbonyl planes. The N atom bears the positive charge in the molecular cation and the piperidine ring adopts an almost perfect chair conformation. The Cl(-) anion is involved in the formation of N-H...Cl and O-H...Cl intermolecular hydrogen bonds, which lead to the formation of a layer of molecular cations.

  View details for DOI 10.1107/S0108270101009763

  View details for Web of Science ID 000171202500034

  View details for PubMedID 11588381

• Ketazolam ACTA CRYSTALLOGRAPHICA SECTION C-STRUCTURAL CHEMISTRY Vega, D., Fernández, D., Echeverría, G. 2001; 57: 848-850

  Abstract

  The title compound, 11-chloro-8,12b-dihydro-2,8-dimethyl-12b-phenyl-4H-[1,3]oxazino[3,2-d][1,4]benzodiazepine-4,7(6H)-dione, C20H17ClN2O3, is a benzodiazepine with an additional d-face-fused heterocyclic ring. In the molecule, a dihedral angle of 86.2 (1) degrees is formed by the planes of the phenyl and benzo rings and the former is axially oriented from the core, i.e. the fused 6,7,6-tricyclic system. Both heterocycles in the core suffer significant deviations from planarity. The central diazepine ring is a twist-boat and the oxazine ring exhibits a conformation intermediate between half-chair and sofa.

  View details for DOI 10.1107/S0108270101006175

  View details for Web of Science ID 000169706700027

  View details for PubMedID 11443263

• 1-[2-(1-Hydroxycyclohexyl)-2-(4-methoxyphenyl)ethyl]dimethylammonium chloride (venlafaxine hydrochloride) ACTA CRYSTALLOGRAPHICA SECTION C-STRUCTURAL CHEMISTRY Vega, D., Fernández, D., Echeverría, G. 2000; 56: 1009-1010

  Abstract

  The crystal structure of racemic Venlafaxine hydrochloride, C(17)H(28)NO(2)(+).Cl(-), consists of two types of parallel chains formed by translated Venlafaxine(+) cations, hydrogen bonded by Cl(-) anions, and characterized by the opposite chirality of their constituent molecules. These chains organize in two different types of broad layers of opposite handedness, related by a glide plane.

  View details for DOI 10.1107/S0108270100007009

  View details for Web of Science ID 000088694400038

  View details for PubMedID 10944307