Biology subjects

Sgourakis, N. G.

Publications and source records attributed to Sgourakis, N. G..

13 recordsLinked to original sources

bioRxiv2026

A generalizable system for antigenic peptide targeting across HLA-I allotypes

T cell receptors (TCRs) and TCR-mimicking antibodies recognize peptide antigens in the context of specific Human Leucocyte Antigen (HLA-I) allotypes, and the extreme polymorphism of the HLA locus limits the breadth of immunotherapy development. Key barriers include divergent molecular surfaces on HLA proteins and differences in the peptide structure. As a result, existing modalities cannot confer therapeutic coverage across patients of divergent genetic backgrounds. Here, we develop an approach which combines a peptide conformational prediction tool, PepPred, with a cross-HLA binding protein engineering system, TRACeR-I1, to outline a generalized framework for developing binders (xTRACeRs) with compatibility across HLA allotypes while maintaining high levels of specificity towards the peptide antigen. We use our system to develop and validate xTRACeRs against clinically relevant, established peptide antigens presented across common alleles within five HLA-A/B/C supertypes2. Cryo-EM structures of xTRACeR-pHLA complexes for an oncofetal antigen from PRAME and a neuroblastoma-specific peptide from PHOX2B reveal effective mechanisms to navigate polymorphic HLA surface residues, and extensive interactions with the peptide. We implemented these two xTRACeRs as Chimeric Antigen Receptor (CAR) T cells and demonstrated their potent killing efficacy and specificity. Overcoming restriction across HLA supertypes lifts a key barrier in HLA-targeted immunotherapy by expanding patient coverage.

synthetic biology↗

bioRxiv2024

Ready-to-load MHC-I Nanoparticles for High-throughput T cell Screening Studies

MHC-I proteins present epitopic peptides to CD8+ T cells to elicit multifaceted adaptive immune responses. The affinity and avidity of interactions between peptide-MHC molecules and T-cell receptors (TCR) are fundamental parameters that contribute to the induction of activated or anergic T cell states. Here, we present a loadable system, VLP-Open HLA, featuring a virus-like particle (VLP) that can accommodate up to 60 loadable HLA (HLA - human leukocyte antigen) molecules. HLA nanoparticles, pre-loaded with a placeholder ligand, allow efficient peptide exchange upon incubation with target peptides. We show that fluorescently tagged VLP-Open HLA particles can be used to stain antigen-specific CD8+ T cells, providing a screening tool for novel TCRs. Finally, we demonstrate that our system can induce activation of T cells in an antigen-specific manner. Our platform can be adapted to encompass multiple HLA allotypes and co-stimulatory molecules as mosaic nanoparticles, to enable a range of applications in experimental immunology.

immunology↗

bioRxiv2024

Regulating IL-2 immune signaling function via a core allosteric structural network

Human interleukin-2 (IL-2) is a crucial cytokine for T cell regulation, with therapeutic potential in cancer and autoimmune diseases. However, IL-2s pleiotropic effects across different immune cell types often lead to toxicity and limited efficacy. Previous efforts to enhance IL-2s therapeutic profile have focused on modifying its receptor binding sites. Yet, the underlying dynamics and intramolecular networks contributing to IL-2 receptor recognition remain unexplored. This study presents a detailed characterization of IL-2 dynamics compared to two engineered IL-2 mutants, "superkines" S15 and S1, which exhibit biased signaling towards effector T cells. Using NMR spectroscopy and molecular dynamics simulations, we demonstrate significant variations in core dynamic pathways and conformational exchange rates across these three IL-2 variants. We identify distinct allosteric networks and excited state conformations in the superkines, despite their structural similarity to wild-type IL-2. Furthermore, we rationally design a mutation (L56A) in the S1 superkines core network, which partially reverts its dynamics, receptor binding affinity, and T cell signaling behavior towards that of wild-type IL-2. Our results reveal that IL-2 superkine core dynamics play a critical role in their enhanced receptor binding and function, suggesting that modulating IL-2 dynamics and core allostery represents an untapped approach for designing immunotherapies with improved immune cell selectivity profiles. HighlightsO_LINMR and molecular dynamics simulations revealed distinct conformational dynamics and allosteric networks in computationally re-designed IL-2 superkines compared to wild-type IL-2, despite their similar crystal structures. C_LIO_LIThe superkines S1 and S15 exhibit altered sampling of excited state conformations at an intermediate timescale, with slower conformational exchange rates compared to wild-type IL-2. C_LIO_LIA rationally designed mutation (L56A) in the S1 superkines core allosteric network partially reverted its dynamics, receptor binding affinity, and T cell signaling behavior towards that of wild-type IL-2. C_LIO_LIOur study demonstrates that IL-2 core dynamics play a critical role in receptor binding and signaling function, providing a foundation for engineering more selective IL-2-based immunotherapies. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=152 HEIGHT=200 SRC="FIGDIR/small/617024v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1e298adorg.highwire.dtl.DTLVardef@195b602org.highwire.dtl.DTLVardef@1e06205org.highwire.dtl.DTLVardef@4351e5_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

A Chicken Tapasin ortholog can chaperone empty HLA molecules independently of other peptide-loading components

Human Tapasin (hTapasin) is the main chaperone of MHC-I molecules, enabling peptide loading and antigen repertoire optimization across HLA allotypes. However, it is restricted to the endoplasmic reticulum (ER) lumen as part of the protein loading complex (PLC) and therefore is highly unstable when expressed in recombinant form. Additional stabilizing co-factors such as ERp57 are required to catalyze peptide exchange in vitro, limiting uses for the generation of pMHC-I molecules of desired antigen specificities. Here, we show that the chicken Tapasin (chTapasin) ortholog can be expressed recombinantly at high yields in stable form, independently of co-chaperones. chTapasin can bind the human HLA-B*37:01 with low micromolar-range affinity to form a stable tertiary complex. Biophysical characterization by methyl-based NMR methods reveals that chTapasin recognizes a conserved {beta}2m epitope on HLA-B*37:01, consistent with previously solved X-ray structures of hTapasin. Finally, we provide evidence that the B*37:01/chTapasin complex is peptide-receptive and can be dissociated upon binding of high-affinity peptides. Our results highlight the use of chTapasin as a stable scaffold for future protein engineering applications aiming to expand the ligand exchange function on human MHC-I and MHC-like molecules.

immunology↗

bioRxiv2023

Structural principles of peptide-centric Chimeric Antigen Receptor recognition guide therapeutic expansion

Peptide-Centric Chimeric Antigen Receptors (PC-CARs), which recognize oncoprotein epitopes displayed by human leukocyte antigens (HLAs) on the cell surface, offer a promising strategy for targeted cancer therapy1. We have previously developed a PC-CAR targeting a neuroblastoma- associated PHOX2B peptide, leading to robust tumor cell lysis restricted by two common HLA allotypes2. Here, we determine the 2.1 [A] structure of the PC-CAR:PHOX2B/HLA-A*24:02/{beta}2m complex, which reveals the basis for antigen-specific recognition through interactions with CAR complementarity-determining regions (CDRs). The PC-CAR adopts a diagonal docking mode, where interactions with both conserved and polymorphic HLA framework residues permit recognition of multiple HLA allotypes from the A9 serological cross-reactivity group, covering a combined American population frequency of up to 25.2%. Comprehensive characterization using biochemical binding assays, molecular dynamics simulations, and structural and functional analyses demonstrate that high-affinity PC-CAR recognition of cross-reactive pHLAs necessitates the presentation of a specific peptide backbone, where subtle structural adaptations of the peptide are critical for high-affinity complex formation and CAR-T cell killing. Our results provide a molecular blueprint for engineering CARs with optimal recognition of tumor-associated antigens in the context of different HLAs, while minimizing cross-reactivity with self-epitopes.

biochemistry↗

bioRxiv2023

HLA3DB: comprehensive annotation of peptide/HLA complexes enables blind structure prediction of T cell epitopes

The class I proteins of the major histocompatibility complex (MHC-I) display epitopic peptides derived from endogenous proteins on the cell surface for immune surveillance. Accurate modeling of peptide/HLA (pHLA, the human MHC) structures has been mired by conformational diversity of the central peptide residues, which are critical for recognition by T cell receptors. Here, analysis of X-ray crystal structures within a curated database (HLA3DB) shows that pHLA complexes encompassing multiple HLA allotypes present a discrete set of peptide backbone conformations. Leveraging these representative backbones, we employ a regression model trained on terms of a physically relevant energy function to develop a comparative modeling approach for nonamer peptide/HLA structures named RepPred. Our method outperforms the top pHLA modeling approach by up to 19% in terms of structural accuracy, and consistently predicts blind targets not included in our training set. Insights from our work provide a framework for linking conformational diversity with antigen immunogenicity and receptor cross-reactivity.

bioinformatics↗

bioRxiv2023

Universal open MHC-I molecules for rapid peptide loading and enhanced complex stability across HLA allotypes

The polymorphic nature and intrinsic instability of class I major histocompatibility complex (MHC-I) and MHC-like molecules loaded with suboptimal peptides, metabolites, or glycolipids presents a fundamental challenge for identifying disease-relevant antigens and antigen-specific T cell receptors (TCRs), hindering the development of autologous therapeutics. Here, we leverage the positive allosteric coupling between the peptide and light chain ({beta}2 microglobulin, {beta}2m) subunits for binding to the MHC-I heavy chain (HC) through an engineered disulfide bond bridging conserved epitopes across the HC/{beta}2m interface, to generate conformationally stable, open MHC-I molecules. Biophysical characterization shows that open MHC-I molecules are properly folded protein complexes of enhanced thermal stability compared to the wild type, when loaded with low- to intermediate-affinity peptides. Using solution NMR, we characterize the effects of the disulfide bond on the conformation and dynamics of the MHC-I structure, ranging from local changes in {beta}2m interacting sites of the peptide binding groove to long-range effects on the 2-1 helix and 3 domain. The interchain disulfide bond stabilizes empty MHC-I molecules in a peptide-receptive, open conformation to promote peptide exchange across multiple human leucocyte antigen (HLA) allotypes, covering representatives from five HLA-A, six HLA-B supertypes, and oligomorphic HLA-Ib molecules. Our structural design, combined with conditional {beta}-peptide ligands, provides a universal platform for generating ready-to-load MHC-I systems of enhanced stability, enabling a range of approaches to screen antigenic epitope libraries and probe polyclonal TCR repertoires in the context of highly polymorphic HLA-I allotypes, as well as oligomorphic nonclassical molecules. Significance StatementWe outline a structure-guided approach for generating conformationally stable, open MHC-I molecules with enhanced ligand exchange kinetics spanning five HLA-A, all HLA-B supertypes, and oligomorphic HLA-Ib allotypes. We present direct evidence of positive allosteric cooperativity between peptide binding and {beta}2m association with the heavy chain by solution NMR and HDX-MS spectroscopy. We demonstrate that covalently linked {beta}2m serves as a conformational chaperone to stabilize empty MHC-I molecules in a peptide-receptive state, by inducing an open conformation and preventing intrinsically unstable heterodimers from irreversible aggregation. Our study provides structural and biophysical insights into the conformational properties of MHC-I ternary complexes, which can be further applied to improve the design of ultra-stable, universal ligand exchange systems in a pan-HLA allelic setting.

biochemistry↗

bioRxiv2020

TAPBPR Promotes Antigen Loading on MHC-I Molecules Using a Peptide Trap

Chaperones tapasin and TAP-binding protein related (TAPBPR) perform the important functions of stabilizing nascent MHC-I molecules (chaperoning) and selecting high affinity peptides in the MHC-I groove (editing). While X-ray and cryo-EM snapshots of MHC-I in complex with TAPBPR and tapasin, respectively, have provided important insights into the peptide-deficient MHC-I groove structure, the molecular mechanism through which these chaperones influence the selection of specific amino acid sequences remains incompletely characterized. Of particular importance is a 16 residue loop in TAPBPR (corresponding to 11 residues in tapasin), which has been proposed to actively compete with incoming peptides by forming direct contacts in the F-pocket of the empty MHC-I groove. Using a deep mutational scanning functional analysis of TAPBPR, we find that important residues for the chaperoning activity are located on the major interaction surfaces with nascent MHC-I molecules, excluding the loop. However, interactions with properly conformed molecules toward peptide editing are influenced by loop mutations, in an MHC-I allele- and peptide-dependent manner. Detailed biophysical characterization by ITC, FP and NMR reveals that the loop does not interact with the peptide-deficient MHC-I groove to compete with incoming peptides, but instead promotes peptide loading by acting as a kinetic trap. Our results suggest that the longer loop of TAPBPR lowers the affinity threshold for peptide selection, to promote loading within subcellular compartments of reduced peptide concentration and to prevent disassembly of high affinity peptide-MHC-I complexes that are transiently interrogated by TAPBPR during editing.

immunology↗

bioRxiv2020

Structure-based modeling of SARS-CoV-2 peptide/HLA-A02 antigens

As a first step toward the development of diagnostic and therapeutic tools to fight the Coronavirus disease (COVID-19), it is important to characterize CD8+ T cell epitopes in the SARS-CoV-2 peptidome that can trigger adaptive immune responses. Here, we use RosettaMHC, a comparative modeling approach which leverages existing high-resolution X-ray structures from peptide/MHC complexes available in the Protein Data Bank, to derive physically realistic 3D models for high-affinity SARS-CoV-2 epitopes. We outline an application of our method to model 439 9mer and 279 10mer predicted epitopes displayed by the common allele HLA-A*02:01, and we make our models publicly available through an online database (https://rosettamhc.chemistry.ucsc.edu). As more detailed studies on antigen-specific T cell recognition become available, RosettaMHC models of antigens from different strains and HLA alleles can be used as a basis to understand the link between peptide/HLA complex structure and surface chemistry with immunogenicity, in the context of SARS-CoV-2 infection.

immunology↗

bioRxiv2019

Interleukin-2 druggability is modulated by global conformational transitions controlled by a helical capping switch

Interleukin-2 (IL-2) is a small -helical cytokine that regulates immune cell homeostasis through its recruitment to a high-affinity heterotrimeric receptor complex (IL-2R/IL-2R{beta}/{gamma}c). IL-2 has been shown to have therapeutic efficacy for immune diseases by preferentially expanding distinct T-cell compartments, and several Treg-biasing anti-IL-2 antibodies have been developed for combination therapies. The conformational plasticity of IL-2 plays an important role in its biological actions by modulating the strength of receptor and drug interactions. Through a NMR analysis of milliseconds-timescale dynamics of free mIL-2 we identify a global transition to an auto-inhibitory conformation controlled by an -helical capping \"switch\". Binding to either an anti-mouse IL-2 mAb or a small molecule inhibitor near the AB loop induces changes in dynamics at the core of the structure, and perturbations of the capping hydrogen-bond network abrogate Ab binding by destabilizing the auto-inhibitory conformation. Our results highlight a paradigm for designing precision therapeutics targeting a continuum of IL-2 conformational states.

biochemistry↗

bioRxiv2019

Molecular determinants of chaperone interactions on MHC-I for folding and antigen repertoire selection

The interplay between a highly polymorphic set of MHC-I alleles and molecular chaperones shapes the repertoire of peptide antigens displayed on the cell surface for T cell surveillance. Here, we demonstrate that the molecular chaperone TAPBPR associates with a broad range of partially folded MHC-I species inside the cell. Bimolecular fluorescence complementation and deep mutational scanning reveal that TAPBPR recognition is polarized towards one side of the peptide-binding groove, and depends on the formation of a conserved MHC-I disulfide epitope in the 2 domain. Conversely, thermodynamic measurements of TAPBPR binding for a representative set of properly conformed, peptide-loaded molecules suggest a narrower MHC-I specificity range. Using solution NMR, we find that the extent of dynamics at \"hotspot\" surfaces confers TAPBPR recognition of a sparsely populated MHC-I state attained through a global conformational change. Consistently, restriction of MHC-I groove plasticity through the introduction of a disulfide bond between the 1/2 helices abrogates TAPBPR binding, both in solution and on a cellular membrane, while intracellular binding is tolerant of many destabilizing MHC-I substitutions. Our data support parallel TAPBPR functions of i) chaperoning unstable MHC-I molecules at early stages of their folding process, akin to a holdase with broad allele-specificity, and ii) editing the peptide cargo of properly conformed MHC-I molecules en route to the surface, which demonstrates a narrower specificity. Our results suggest that TAPBPR exploits localized structural adaptations, both near and distant to the peptide-binding groove, to selectively recognize discrete conformational states sampled by MHC-I alleles, towards editing Sithe repertoire of displayed antigens.\n\nSignificance StatementThe human population contains thousands of MHC-I alleles, showing a range of dependencies on molecular chaperones for loading of their peptide cargo, which are then displayed on the cell surface for T cell surveillance. Using the chaperone TAPBPR as a model, we combine deep mutagenesis with functional and biophysical data, especially solution NMR, to provide a complete view of the molecular determinants of chaperone recognition. Our data provide significant evidence that localized protein motions define the intrinsic ability of MHC-I molecules to interact with chaperones. The importance of MHC-I dynamics unifies all our findings, with broad recognition of conformationally unstable, nascent MHC-I molecules becoming restricted to a smaller set of MHC-I alleles that retain relevant dynamic motions in their folded state.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC=\"FIGDIR/small/779777v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (66K):\norg.highwire.dtl.DTLVardef@19da2fforg.highwire.dtl.DTLVardef@383607org.highwire.dtl.DTLVardef@63171eorg.highwire.dtl.DTLVardef@182bf14_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDeep mutagenesis identifies a conformational disulfide-linked epitope as the main requirement for association of nascent MHC-I molecules with the TAPBPR chaperone\nC_LIO_LIAnalysis of s-ms timescale conformational dynamics by methyl NMR reveals allele-specific profiles at the TAPBPR interaction surfaces of peptide-loaded MHC-I molecules\nC_LIO_LIs-ms dynamics dictate the specificity of TAPBPR interactions for different MHC-I alleles through the sampling of a minor, \"excited state\" conformation\nC_LIO_LIRestriction of dynamics though an engineered disulfide bond abrogates interactions with TAPBPR, both in solution and on a cellular membrane\nC_LI

immunology↗

bioRxiv2019

High Throughput pMHC-I Tetramer Library Production Using Chaperone Mediated Peptide Exchange

Peptide exchange technologies are essential for the generation of pMHC-multimer libraries, used to probe highly diverse, polyclonal TCR repertoires. Using the molecular chaperone TAPBPR, we present a robust method for the capture of stable, empty MHC-I molecules which can be readily tetramerized and loaded with peptides of choice in a high-throughput manner. Combined with tetramer barcoding using multi-modal cellular indexing technology (ECCITE-seq), our approach allows a combined analysis of TCR repertoires and other T-cell transcription profiles together with their cognate pMHC-I specificities in a single experiment.

immunology↗

bioRxiv2019

De novo design of proteins with two isoenergetic but structurally divergent ground states

The plasticity of naturally occurring protein structures, which can change shape considerably in response to changes in environmental conditions, is critical to biological function. While computational methods have been used to de novo design proteins that fold to a single state with a deep free energy minima (Huang et al., 2016), and to reengineer natural proteins to alter their dynamics (Davey et al., 2017) or fold (Alexander et al., 2009), the de novo design of closely related sequences which adopt well-defined, but structurally divergent structures remains an outstanding challenge. Here, we design closely related sequences (over 94% identity) that can adopt two very different homotrimeric helical bundle conformations -- one short ([~]66 [A] height) and the other long ([~]100 [A] height) -- reminiscent of the conformational transition of viral fusion proteins (Ivanovic et al., 2013; Podbilewicz, 2014; Skehel and Wiley, 2000). Crystallographic and NMR spectroscopic characterization show that both the short and long state sequences fold as designed. We sought to design bistable sequences for which both states are accessible, and obtained a single designed protein sequence that populates either the short state or the long state depending on the measurement conditions. The design of sequences which are poised to adopt two very different conformations sets the stage for creating large scale conformational switches between structurally divergent forms.

biochemistry↗