Biology subjects

Sgourakis, N.

Publications and source records attributed to Sgourakis, N..

6 recordsLinked to original sources

bioRxiv2026

A miniaturized MR1 metabolite display system with native-like protein features

Major histocompatibility complex class I-related protein 1 (MR1) presents metabolite-derived antigens to mucosal-associated invariant T (MAIT) cells and other MR1-restricted T cells, playing a critical role in immune surveillance during infection and disease. Biochemical and structural studies of MR1 have been limited by the intrinsic instability of the molecule, which requires both ligand binding and association with beta-2-microglobulin ({beta}2m) for proper folding and stability. Here, we adapt MR1 to the SMART protein platform to generate a minimalistic system for studying MR1 ligand presentation and T cell receptor (TCR) recognition. SMART-MR1 consists of the MR1 1/2 ligand-binding platform fused to a helical stabilizing domain that functionally replaces the 3 and {beta}2m domains, resulting in a truncated protein that preserves the architecture of the antigen-binding groove. We show that SMART-MR1 can be efficiently produced recombinantly and retains the ability to bind chemically diverse classes of MR1 ligands. The reduced size of SMART-MR1 enables amide-based solution NMR experiments, and its simplified structure allows for ligand screening using fluorescence polarization. Importantly, SMART-MR1 maintains binding to the MAIT-derived A-F7 TCR, as confirmed by isothermal titration calorimetry. Finally, cryo-EM structural analysis of SMART-MR1/5-OP-RU bound to A-F7 revealed that ligand presentation and TCR recognition are nearly identical to those observed in native MR1. Together, these results establish SMART-MR1 as a minimal yet native-like system, expanding the experimental toolkit available for studying MR1 interactions and facilitating future efforts aimed at targeting MR1 pathways. Significance StatementMR1 is a highly conserved antigen-presenting molecule that enables T cells to detect metabolite signals from microbial infections and host metabolism. Despite its importance in immunity, mechanistic studies of MR1 have been limited by the instability of the native protein. We developed a simplified and stabilized version of MR1 that preserves the ligand-binding platform while eliminating structural elements that complicate biochemical analysis. This minimal system retains native-like antigen presentation and T cell receptor (TCR) recognition, while enabling experimental approaches that are difficult with full-length MR1. By lowering technical barriers to studying MR1-ligand and MR1-TCR interactions, this platform provides a versatile tool for exploring how antigens shape immune responses and for accelerating discovery of therapeutic strategies targeting MR1.

biochemistry↗

bioRxiv2025

Design of solubly expressed miniaturized SMART MHCs

The precise recognition of specific peptide-MHC (pMHC) complexes by T-cell receptors (TCRs) plays a key role in infectious disease, cancer and autoimmunity. A critical step in many immunobiological studies is the identification of T-cells expressing TCRs specific to a given pMHC antigen. However, the intrinsic instability of empty class-I MHCs limits their soluble expression in Escherichia coli (E. coli) and makes it very difficult to characterize even a small fraction of possible pMHC/TCR interactions. To overcome this limitation, we designed small proteins which buttress the peptide binding groove of class I MHCs, replacing {beta}2-microglobulin ({beta}2m) and the heavy chain 3 domain, and enable soluble expression of both H-2Db and A*02:01 in E. coli. We demonstrate that these soluble, monomeric, antigen-receptive, truncated (SMART) MHCs retain both peptide- and TCR-binding specificity, and that peptide-bound structures of both allomorphs are similar to their full-length, native counterparts. With extension to the majority of HLA alleles, SMART MHCs should be broadly useful for probing the T-cell repertoire in approaches ranging from yeast display to T-cell staining. SignificanceDespite the critical role that TCR/pMHC interactions play in human health, it has remained difficult to produce reagents necessary to study them. Requirements for refolding or sequence optimization limit immunologists and biochemists ability to characterize diverse pMHC/TCR interactions. Here, we develop a de-novo designed protein domain that stabilizes the H-2Db and A*02:01 class I MHC allomorphs, allowing soluble expression in E. coli without the need for a stabilizing peptide, and improving display on the yeast surface, while maintaining peptide and TCR binding interactions. These features facilitate a wide range of experiments to more fully understand the nature of pMHC/TCR interactions, and pave the way for the development of stabilizing domains for all MHC allomorphs.

biochemistry↗

bioRxiv2024

CryoEM structure of an MHC-I/TAPBPR peptide bound intermediate reveals the mechanism of antigen proofreading

Class I major histocompatibility complex (MHC-I) proteins play a pivotal role in adaptive immunity by displaying epitopic peptides to CD8+ T cells. The chaperones tapasin and TAPBPR promote the selection of immunogenic antigens from a large pool of intracellular peptides. Interactions of chaperoned MHC-I molecules with incoming peptides are transient in nature, and as a result, the precise antigen proofreading mechanism remains elusive. Here, we leverage a high-fidelity TAPBPR variant and conformationally stabilized MHC-I, to determine the solution structure of the human antigen editing complex bound to a peptide decoy by cryogenic electron microscopy (cryo-EM) at an average resolution of 3.0 [A]. Antigen proofreading is mediated by transient interactions formed between the nascent peptide binding groove with the P2/P3 peptide anchors, where conserved MHC-I residues stabilize incoming peptides through backbone-focused contacts. Finally, using our high-fidelity chaperone, we demonstrate robust peptide exchange on the cell surface across multiple clinically relevant human MHC-I allomorphs. Our work has important ramifications for understanding the selection of immunogenic epitopes for T cell screening and vaccine design applications.

immunology↗

bioRxiv2024

A structure-guided approach to predict MHC-I restriction of T cell receptors for public antigens

Peptides presented by class I major histocompatibility complex (MHC-I) proteins provide biomarkers for therapeutic targeting using T cell receptors (TCRs), TCR-mimicking antibodies (TMAs), or other engineered protein binders. Despite the extreme sequence diversity of the Human Leucocyte Antigen (HLA, the human MHC), a given TCR or TMA is restricted to recognize epitopic peptides in the context of a limited set of different HLA allotypes. Here, guided by our analysis of 96 TCR:pHLA complex structures in the Protein Data Bank (PDB), we identify TCR contact residues and classify 148 common HLA allotypes into T-cell cross-reactivity groups (T-CREGs) on the basis of their interaction surface features. Insights from our work have actionable value for resolving MHC-I restriction of TCRs, guiding therapeutic expansion of existing therapies, and informing the selection of peptide targets for forthcoming immunotherapy modalities.

immunology↗

bioRxiv2021

Global Dynamics as Communication Sensors in Peptide Synthetase Cyclization Domains.

Structural biology is the foundation for deriving molecular mechanisms, where snapshots of macromolecules and binding partners inform on mutations that test or modify function. However, frequently, the impact of mutations violates the underpinnings of structural models, and mechanisms become cryptic. This conundrum applies to multidomain enzymatic systems called nonribosomal peptide synthetases (NRPSs), which assemble simple substrates into complex metabolites often with pharmaceutical properties. Engineering NRPSs can generate new pharmaceuticals1-3 but a dynamic domain organization challenges rational design.4-8 Using nuclear magnetic resonance (NMR), we determined the solution structure of a 52 kDa cyclization domain and demonstrate that global intra-domain dynamics enable sensing of substrates tethered to partner domains and draw an allosteric response encompassing the enzymes buried active site and two binding sites 40 [A] apart. We show that a point-site mutation that impedes the domain dynamics globally hampers the allosteric response. We demonstrate this mechanism through NMR experiments that provide atomic-level read-outs of allosteric responses during biochemical transformations in situ. Our results establish global structural dynamics as sensors of molecular events that can remodel domain interactions and illustrate the need for integrating structural dynamics explicitly when deriving molecular mechanisms through mutagenesis and structural biology.

biophysics↗

bioRxiv2020

A slow-exchange conformational switch regulates off-target cleavage by high-fidelity Cas9

The Cas9 endonuclease is broadly used for genome engineering applications by programming its single-guide RNA, where high specificity is required. Although mechanistic understanding of DNA cleavage in the CRISPR-Cas9 system has been guided by crystallographic structures and single-molecule FRET experiments, the role of conformational plasticity within its DNA recognition lobe (REC) for target accuracy remains elusive. Through NMR analysis of milliseconds-timescale dynamics, we show that the REC3 domain exchanges between a major, closed and a minor, open conformation. We find that a single mutation in the HiFi Cas9 variant (R691A) increases conformational dynamics in REC3, eliciting a global transition to the open conformation which is regulated by an intramolecular salt-bridge network. These observations suggest a mechanism for reduced off-target recognition by switching on a global conformational exchange process to allow REC3 scanning for proper base pairing. Our data establish a framework for rational engineering Cas9 variants with improved target discrimination.

biochemistry↗