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bioRxiv · 10.64898/2026.04.13.718121

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

Rotsides, P.·Shinde, O.·Danon, J. N.·Sgourakis, N.
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Abstract

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.

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Rotsides, P., Shinde, O., Danon, J. N., Sgourakis, N.. 2026-04-15. A miniaturized MR1 metabolite display system with native-like protein features. https://doi.org/10.64898/2026.04.13.718121

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