Christopher Garcia, Postdoctoral Faculty Sponsor
Identification of orphan ligand-receptor relationships using a cell-based CRISPRa enrichment screening platform.
Secreted proteins, which include cytokines, hormones and growth factors, are extracellular ligands that control key signaling pathways mediating cell-cell communication within and between tissues and organs. Many drugs target secreted ligands and their cell-surface receptors. Still, there are hundreds of secreted human proteins that either have no identified receptors ('orphans') and are likely to act through cell surface receptors that have not yet been characterized. Discovery of secreted ligand-receptor interactions by high-throughput screening has been problematic, because the most commonly used high-throughput methods for protein-protein interaction (PPI) screening do not work well for extracellular interactions. Cell-based screening is a promising technology for definition of new ligand-receptor interactions, because multimerized ligands can enrich for cells expressing low affinity cell-surface receptors, and such methods do not require purification of receptor extracellular domains. Here, we present a proteo-genomic cell-based CRISPR activation (CRISPRa) enrichment screening platform employing customized pooled cell surface receptor sgRNA libraries in combination with a magnetic bead selection-based enrichment workflow for rapid, parallel ligand-receptor deorphanization. We curated 80 potentially high value orphan secreted proteins and ultimately screened 20 secreted ligands against two cell sgRNA libraries with targeted expression of all single-pass (TM1) or multi-pass (TM2+) receptors by CRISPRa. We identified previously unknown interactions in 12 of these screens, and validated several of them using surface plasmon resonance and/or cell binding. The newly deorphanized ligands include three receptor tyrosine phosphatase (RPTP) ligands and a chemokine like protein that binds to killer cell inhibitory receptors (KIR's). These new interactions provide a resource for future investigations of interactions between the human secreted and membrane proteomes.
View details for DOI 10.7554/eLife.81398
View details for PubMedID 36178190
Organizing Structural Principles of the Interleukin-17 Ligand-Receptor Axis.
The interleukin-17 (IL-17) family of cytokines and receptors play central roles in host defense against infection, and development of inflammatory diseases1. The compositions and structures of functional IL-17 family ligand-receptor signaling assemblies remain unclear. Interleukin-17E (IL-17E or IL-25) is a key regulator of Th2 immune responses and driver of inflammatory diseases such as allergic asthma and requires both IL-17 receptor A (IL-17RA) and IL-17RB to elicit functional responses2. Here, we studied IL-25-IL-17RB binary and IL-25-IL-17RB-IL-17RA ternary complexes using a combination of cryo-electron microscopy (cryo-EM), single-molecule imaging, and cell-based signaling approaches. The IL-25-IL-17RB-IL-17RA ternary signaling assembly is a c2-symmetric complex in which the IL-25-IL-17RB homodimer is flanked by two "wing-like" IL-17RA co-receptors through a "tip-to-tip" geometry that is the key receptor-receptor interaction required for initiation of signal transduction. IL-25 interacts solely with IL-17RB to allosterically promote the formation of the IL-17RB-IL-17RA tip-to-tip interface. The resulting large separation between the receptors at the membrane-proximal level may reflect proximity constraints by the intracellular domains for signaling. Cryo-EM structures of IL-17A-IL-17RA and IL-17A-IL-17RA-IL-17RC complexes reveal that this tip-to-tip architecture is a key organizing principle of the IL-17 receptor family, Furthermore, these studies reveal dual actions for IL-17RA sharing amongst IL-17 cytokine complexes, by either directly engaging IL-17 cytokines, or alternatively functioning as a co-receptor.
View details for DOI 10.1038/s41586-022-05116-y
View details for PubMedID 35863378
Structures of neurexophilin-neurexin complexes reveal a regulatory mechanism of alternative splicing.
The EMBO journal
Neurexins are presynaptic, cell-adhesion molecules that specify the functional properties of synapses via interactions with trans-synaptic ligands. Neurexins are extensively alternatively spliced at six canonical sites that regulate multifarious ligand interactions, but the structural mechanisms underlying alternative splicing-dependent neurexin regulation are largely unknown. Here, we determined high-resolution structures of the complex of neurexophilin-1 and the second laminin/neurexin/sex-hormone-binding globulin domain (LNS2) of neurexin-1 and examined how alternative splicing at splice site #2 (SS2) regulates the complex. Our data reveal a unique, extensive, neurexophilin-neurexin binding interface that extends the jelly-roll β-sandwich of LNS2 of neurexin-1 into neurexophilin-1. The SS2A insert of LNS2 augments this interface, increasing the binding affinity of LNS2 for neurexophilin-1. Taken together, our data reveal an unexpected architecture of neurexophilin-neurexin complexes that accounts for the modulation of binding by alternative splicing, which in turn regulates the competition of neurexophilin for neurexin binding with other ligands.
View details for DOI 10.15252/embj.2019101603
View details for PubMedID 31566781
Production and Characterization of Synthetic Carboxysome Shells with Incorporated Luminal Proteins
2016; 170 (3): 1868-1877
Spatial segregation of metabolism, such as cellular-localized CO2 fixation in C4 plants or in the cyanobacterial carboxysome, enhances the activity of inefficient enzymes by selectively concentrating them with their substrates. The carboxysome and other bacterial microcompartments (BMCs) have drawn particular attention for bioengineering of nanoreactors because they are self-assembling proteinaceous organelles. All BMCs share an architecturally similar, selectively permeable shell that encapsulates enzymes. Fundamental to engineering carboxysomes and other BMCs for applications in plant synthetic biology and metabolic engineering is understanding the structural determinants of cargo packaging and shell permeability. Here we describe the expression of a synthetic operon in Escherichia coli that produces carboxysome shells. Protein domains native to the carboxysome core were used to encapsulate foreign cargo into the synthetic shells. These synthetic shells can be purified to homogeneity with or without luminal proteins. Our results not only further the understanding of protein-protein interactions governing carboxysome assembly, but also establish a platform to study shell permeability and the structural basis of the function of intact BMC shells both in vivo and in vitro. This system will be especially useful for developing synthetic carboxysomes for plant engineering.
View details for DOI 10.1104/pp.15.01822
View details for Web of Science ID 000375420300049
View details for PubMedID 26792123
View details for PubMedCentralID PMC4775138
Biogenesis of a Bacterial Organelle: The Carboxysome Assembly Pathway
2013; 155 (5): 1131-1140
The carboxysome is a protein-based organelle for carbon fixation in cyanobacteria, keystone organisms in the global carbon cycle. It is composed of thousands of subunits including hexameric and pentameric proteins that form a shell to encapsulate the enzymes ribulose 1,5-bisphosphate carboxylase/oxygenase and carbonic anhydrase. Here, we describe the stages of carboxysome assembly and the requisite gene products necessary for progression through each. Our results demonstrate that, unlike membrane-bound organelles of eukaryotes, in carboxysomes the interior of the compartment forms first, at a distinct site within the cell. Subsequently, shell proteins encapsulate this procarboxysome, inducing budding and distribution of functional organelles within the cell. We propose that the principles of carboxysome assembly that we have uncovered extend to diverse bacterial microcompartments.
View details for DOI 10.1016/j.cell.2013.10.044
View details for Web of Science ID 000327500600016
View details for PubMedID 24267892
Two new high-resolution crystal structures of carboxysome pentamer proteins reveal high structural conservation of CcmL orthologs among distantly related cyanobacterial species
2013; 118 (1-2): 9-16
Cyanobacteria have evolved a unique carbon fixation organelle known as the carboxysome that compartmentalizes the enzymes RuBisCO and carbonic anhydrase. This effectively increases the local CO2 concentration at the active site of RuBisCO and decreases its relatively unproductive side reaction with oxygen. Carboxysomes consist of a protein shell composed of hexameric and pentameric proteins arranged in icosahedral symmetry. Facets composed of hexameric proteins are connected at the vertices by pentameric proteins. Structurally homologous pentamers and hexamers are also found in heterotrophic bacteria where they form architecturally related microcompartments such as the Eut and Pdu organelles for the metabolism of ethanolamine and propanediol, respectively. Here we describe two new high-resolution structures of the pentameric shell protein CcmL from the cyanobacteria Thermosynechococcus elongatus and Gloeobacter violaceus and provide detailed analysis of their characteristics and comparison with related shell proteins.
View details for DOI 10.1007/s11120-013-9909-z
View details for Web of Science ID 000327065500002
View details for PubMedID 23949415