Structural Immunology & Functional Annotation

For the past two decades, the major scientific focus of my laboratory has been high-throughput structure discovery and functional annotation. My laboratory has been directly involved in a number of large-scale programs involved in technology development and high-throughput applications. We have contributed to efforts focused on the large scale annotation of enzyme function, including the discovery of novel antivirals and new targets for controlling inflammatory processes.

My laboratory has made extensive contributions to the structural, functional and mechanistic analysis of the cell surface and secreted proteins that modulate adaptive and innate immunity, and we have developed a series of platforms for the high-throughput evaluation of protein interactions and their functional implications. Finally, we have a number of programs that depend on the design of new selectivities and affinities for the realization of novel biologics that will greatly benefit the proposed work.

1. Strategies for Functional Annotation and Metabolism Discovery. The number of newly reported protein sequences inferred from genome sequencing continues to grow at a rate that severely outpaces the assignment of function through comparative genomics or direct biochemical analysis. This situation results in a large proportion of unannotated and misannotated protein sequences precluding the discovery of novel enzymes, activities, and metabolic pathways important to (1) understanding the contributions of the gut microbiome to human health, (2) the realization of new chemical processes for industry, and (3) our understanding of critical environmental issues, including global nutrient cycles and the evolution of complex microbial communities. To address these challenges our laboratory is devising experimental strategies based on the solute binding protein (SBP) components of small molecule transport systems, since the first step in a catabolic pathway is frequently the passage of a metabolite across the cellular membrane by SBP-dependent transport machinery. The ability to identify the initial reactant (or a closely related molecule) for a catabolic pathway provides an immediate toe-hold by placing significant constraints on the regions of chemical space that need to be considered and, in conjunction with knowledge of colocalized and coregulated genes, begins to define details of the in vivo biochemical transformations operating within the metabolic pathway. Using our high-throughput infrastructure we produced and screened 158 TRAP SBPs against a small molecule library by differential scanning fluorimetry (DSF). These efforts led to the identification of 40 new TRAP SBP ligands, the generation of experiment-based annotations for 2084 individual SBPs in 71 isofunctional clusters, and the definition of numerous metabolic pathways, including novel catabolic pathways for the utilization of ethanolamine as sole nitrogen source and the use of D-Ala-D-Ala as sole carbon source1. Other comparable large scale functional annotation studies were performed for the Isoprenoid Synthase2 and Haloacid Dehalogenase3 Superfamilies.
   1. Vetting MW, Al-Obaidi N, Zhao S, San Francisco B, Kim J, Wichelecki DJ, Bouvier JT, Solbiati JO, Vu H, Zhang X, Rodionov DA, Love JD, Hillerich BS, Seidel RD, Quinn RJ, Osterman AL, Cronan JE, Jacobson MP, Gerlt JA, Almo SC (2015) “Experimental strategies for functional annotation and metabolism discovery: targeted screening of solute binding proteins and unbiased panning of metabolomes.” Biochemistry 54, 909-931.
   2. Wallrapp FH, Pan JJ, Ramamoorthy G, Almonacid DE, Hillerich BS, Seidel R, Patskovsky Y, Babbitt PC, Almo SC, Jacobson MP, Poulter CD (2013) “Prediction of function for the polyprenyl transferase subgroup in the isoprenoid synthase superfamily.” Proc Natl Acad Sci U S A 110, 196-202.
   3. Huang H, Pandya C, Liu C, Al-Obaidi NF, Wang M, Zheng L, Toews Keating S, Aono M, Love JD, Evans B, Seidel RD, Hillerich BS, Garforth SJ, Almo SC, Mariano PS, Dunaway-Mariano D, Allen KN, Farelli JD. (2015) “Panoramic view of a superfamily of phosphatases through substrate profiling.” Proc Natl Acad Sci U S A 112, 74-83.
   4. Gizzi AS, Grove TL, Arnold JJ, Jose J, Jangra RK, Garforth SJ, Du Q, Cahill SM, Dulyaninova NG, Love JD, Chandran K, Bresnick AR, Cameron CE, Almo SC (2018) “A naturally occurring antiviral ribonucleotide encoded by the human genome.” Nature 558, 610-614.
   5. Funabashi M, Grove TL, Wang M, Varma Y, McFadden ME, Brown LC, Guo C, Higginbottom S, Almo SC, Fischbach MA (2020) “A metabolic pathway for bile acid dehydroxylation by the gut microbiome.” Nature 582, 566–570.
2. High-throughput Protein Production Infrastructure. Despite a multitude of recent technical breakthroughs speeding high-resolution structural and functional analysis of biological macromolecules, production of sufficient quantities of well-behaved, active protein continues to represent the rate-limiting step in many structure discovery and functional annotation efforts. These challenges are amplified when considered in the context of ongoing large scale efforts to systematically define structure, function and mechanism of a wide range of macromolecules including multi-domain eukaryotic proteins, secreted proteins, and ever larger macromolecular assemblies. As part of our programs at Einstein, we have established robust bacterial expression platforms for the high-throughput discovery of new metabolism. Unique to the Almo group is the world’s first integrated system for high-throughput functional and structural biology of oxygen sensitive proteins. This resource has allowed for the recapitulation of the entire high-throughput protein production and crystallization pipeline within an oxygen-free environment (see http://www.nysgrc.org/psi3/anaerobic.html). We have also established high-throughput eukaryotic expression platforms, including insect and mammalian-based systems, which represents a unique resource in academics. We have extensively described the capabilities of our protein production platforms in the literature4. These capabilities are being leveraged to realize a wide range of cutting-edge platform technologies, including receptor-ligand deorphaning, epitope discovery, the generation of novel biologics and the development of new clonal-specific T cell strategies for the treatment of malignancies and autoimmunity⁵.
   1. Almo SC, Garforth SJ, Hillerich BS, Love JD, Seidel RD, Burley SK (2013) “Protein production from the structural genomics perspective: achievements and future needs.” Curr Opin Struct Biol 23, 335-44.
   2. Samanta D, Mukherjee G, Ramagopal UA, Chaparro RJ, Nathenson SG, DiLorenzo TP, Almo SC (2011) “Structural and functional characterization of a single-chain peptide-MHC molecule that modulates both naive and activated CD8+ T cells.” Proc Natl Acad Sci U S A 108, 13682-13687.
3. Structural, functional and mechanistic analysis of the cell surface and secreted proteins that modulate adaptive and innate immunity. Cell surface receptors and adhesion molecules are the gatekeepers of cellular function, and are responsible for the detection of signals arising from developmental, morphogenetic and environmental cues central to normal physiology and pathology. Notably, these receptors and ligands are not only therapeutic targets, but soluble versions of these molecules are themselves widely exploited therapeutics for the treatment of autoimmune diseases, infectious diseases and malignancies. High resolution structural characterization and biochemical analyses of these complexes are mechanistically invaluable as they define the chemical and physical determinants underlying receptor:ligand specificity, affinity, oligomeric state, and valency. We have made significant contributions in these areas, including the structures of complexes of CTLA-4:B7-2⁶, PD-1:PD-L2⁷, DcR3:TL1A⁸, DcR3:LIGHT, DcR3:FasL and HVEM:LIGHT, as well as B7-H3, B7-H4⁹, TIM-3, NTB-A, CD84, GITRL, TIGIT, CRTAM, nectins and CD160, all of which are potential/proven targets for immunotherapy. These structures defined the determinants responsible for receptor:ligand recognition, which are being leveraged to generate a wide range of variants with altered biochemical properties (e.g., affinities, selectivities) to probe mechanism and provide new functional/therapeutic insights. A major challenge in these efforts is the fact that many, if not most, receptor:ligand pairs remain undefined and thus cannot be structurally characterized or exploited for immunotherapy. To address this bottleneck, we are developing experimental platform technologies for the rapid, systematic and affordable identification of cell surface protein-protein interacting partners and the mapping of protein interaction interfaces. This same platform provides powerful approaches to generate costimulatory receptors and ligands with a wide range of affinities and selectivities, which can be leveraged for the design of “tunable” immune modulators.
   1. Schwartz J-C, Zhang X, Fedorov AA, Nathenson SG, Almo SC (2001) “Structural Basis for Costimulation by the Human CTLA-4/B7-2 Complex.” Nature 410, 604-608.
   2. Lázár-Molnár E, Yan Q, Cao E, Ramagopal U, Nathenson SG, Almo SC (2008) “Crystal structure of the complex between programmed death-1 (PD-1) and its ligand PD-L2.” Proc Natl Acad Sci U S A. 105, 10483-10488.
   3. Zhan C, Patskovsky Y, Yan Q, Li Z, Ramagopal U, Cheng H, Brenowitz M, Hui X, Nathenson SG, Almo SC (2011) “Decoy Strategies: The Structure of TL1A-DcR3 Complex.” Structure 19, 162-171.
   4. Jeon H, Vigdorovich V, Garrett-Thomson SC, Janakiram M, Ramagopal UA, Abadi YM, Lee JS, Scandiuzzi L, Ohaegbulam KC, Chinai JM, Zhao R, Yao Y, Mao Y, Sparano JA, Almo SC, Zang X (2014) “Structure and cancer immunotherapy of the B7 family member B7x.” Cell Rep 9, 1089-1098.
   5. Liu W, Garrett SC, Fedorov EV, Ramagopal UA, Garforth SJ, Bonanno JB, Almo SC (2019) “Structural Basis of CD160:HVEM Recognition.” Structure 27, 1286-1295.
   6. Ramagopal UA, Liu W, Garrett-Thomson SC, Bonanno JB, Yan Q, Srinivasan M, Wong SC, Bell A, Mankikar S, Rangan VS, Deshpande S, Korman AJ, Almo SC (2019) “Structural basis for cancer immunotherapy by the first-in-class checkpoint inhibitor ipilimumab.” Proc Natl Acad Sci USA 114, E4223-E4232.
   7. Quayle SN, Girgis N, Thapa DR, Merazga Z, Kemp MM, Histed A, Zhao F, Moreta M, Ruthardt P, Hulot S, Nelson A, Kraemer LD, Beal DR, Witt L, Ryabin J, Soriano J, Haydock M, Spaulding E, Ross JF, Kiener PA, Almo SC, Chaparro R, Seidel R, Suri A, Cemerski S, Pienta KJ, Simcox ME. (2020) “CUE-101, a Novel E7-pHLA-IL2-Fc Fusion Protein, Enhances Tumor Antigen-Specific T-Cell Activation for the Treatment of HPV16-Driven Malignancies” Clin Cancer Res 26, 1953-1964.