{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,4,18]],"date-time":"2026-04-18T17:44:49Z","timestamp":1776534289142,"version":"3.51.2"},"posted":{"date-parts":[[2026,4,15]]},"group-title":"Biochemistry","reference-count":42,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,4,15]],"date-time":"2026-04-15T00:00:00Z","timestamp":1776211200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,4,15]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Major histocompatibility complex class I\u2013related 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 (\u03b2\n                  <jats:sub>2<\/jats:sub>\n                  m) 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 \u03b1\n                  <jats:sub>1<\/jats:sub>\n                  \/\u03b1\n                  <jats:sub>2<\/jats:sub>\n                  ligand-binding platform fused to a helical stabilizing domain that functionally replaces the \u03b1\n                  <jats:sub>3<\/jats:sub>\n                  and \u03b2\n                  <jats:sub>2<\/jats:sub>\n                  m 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.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Significance Statement<\/jats:title>\n                  <jats:p>MR1 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\u2013ligand and MR1\u2013TCR interactions, this platform provides a versatile tool for exploring how antigens shape immune responses and for accelerating discovery of therapeutic strategies targeting MR1.<\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.04.13.718121","type":"posted-content","created":{"date-parts":[[2026,4,15]],"date-time":"2026-04-15T23:20:12Z","timestamp":1776295212000},"source":"Crossref","is-referenced-by-count":0,"title":["A miniaturized MR1 metabolite display system with native-like protein features"],"prefix":"10.64898","author":[{"given":"Photis","family":"Rotsides","sequence":"first","affiliation":[{"name":"Center for Computational and Genomic Medicine, Department of Pathology and Laboratory Medicine, The Children\u2019s Hospital of Philadelphia, Philadelphia, PA, 19104, USA"},{"name":"Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Omkar","family":"Shinde","sequence":"additional","affiliation":[{"name":"Center for Computational and Genomic Medicine, Department of Pathology and Laboratory Medicine, The Children\u2019s Hospital of Philadelphia, Philadelphia, PA, 19104, USA"},{"name":"Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Julia N.","family":"Danon","sequence":"additional","affiliation":[{"name":"Center for Computational and Genomic Medicine, Department of Pathology and Laboratory Medicine, The Children\u2019s Hospital of Philadelphia, Philadelphia, PA, 19104, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3655-3902","authenticated-orcid":false,"given":"Nikolaos G.","family":"Sgourakis","sequence":"additional","affiliation":[{"name":"Center for Computational and Genomic Medicine, Department of Pathology and Laboratory Medicine, The Children\u2019s Hospital of Philadelphia, Philadelphia, PA, 19104, USA"},{"name":"Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2026041810250526000_2026.04.13.718121v1.1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.immunol.16.1.323"},{"key":"2026041810250526000_2026.04.13.718121v1.2","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2020.01961"},{"key":"2026041810250526000_2026.04.13.718121v1.3","doi-asserted-by":"publisher","DOI":"10.1038\/ni.3416"},{"key":"2026041810250526000_2026.04.13.718121v1.4","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2011260117"},{"key":"2026041810250526000_2026.04.13.718121v1.5","doi-asserted-by":"crossref","first-page":"859","DOI":"10.1038\/s41589-022-01049-9","article-title":"TAPBPR employs a ligand-independent docking mechanism to chaperone MR1 molecules","volume":"18","year":"2022","journal-title":"Nature Chemical Biology"},{"key":"2026041810250526000_2026.04.13.718121v1.6","first-page":"1439","article-title":"The MHC-related protein 1 (MR1) is expressed by a subpopulation of CD38+, IgA+ cells in the human intestinal mucosa","volume":"24","year":"2009","journal-title":"Histol Histopathol"},{"key":"2026041810250526000_2026.04.13.718121v1.7","doi-asserted-by":"publisher","DOI":"10.1084\/jem.20072579"},{"key":"2026041810250526000_2026.04.13.718121v1.8","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1005524"},{"key":"2026041810250526000_2026.04.13.718121v1.9","doi-asserted-by":"crossref","first-page":"4797","DOI":"10.1038\/s41598-019-41402-y","article-title":"MR1 recycling and blockade of endosomal trafficking reveal distinguishable antigen presentation pathways between Mycobacterium tuberculosis infection and exogenously delivered antigens","volume":"9","year":"2019","journal-title":"Scientific Reports"},{"key":"2026041810250526000_2026.04.13.718121v1.10","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2003136117"},{"key":"2026041810250526000_2026.04.13.718121v1.11","doi-asserted-by":"crossref","first-page":"228","DOI":"10.1038\/s42003-024-05912-4","article-title":"Delivery of loaded MR1 monomer results in efficient ligand exchange to host MR1 and subsequent MR1T cell activation","volume":"7","year":"2024","journal-title":"Communications Biology"},{"key":"2026041810250526000_2026.04.13.718121v1.12","doi-asserted-by":"crossref","unstructured":"H. J. Lim et al., A specialized tyrosine-based endocytosis signal in MR1 controls antigen presentation to MAIT cells. Journal of Cell Biology 221 (2022).","DOI":"10.1083\/jcb.202110125"},{"key":"2026041810250526000_2026.04.13.718121v1.13","doi-asserted-by":"crossref","unstructured":"C. L. Ashley et al., Suppression of MR1 by human cytomegalovirus inhibits MAIT cell activation. Frontiers in Immunology Volume 14 - 2023 (2023).","DOI":"10.3389\/fimmu.2023.1107497"},{"key":"2026041810250526000_2026.04.13.718121v1.14","doi-asserted-by":"publisher","DOI":"10.1038\/s41577-023-00934-1"},{"key":"2026041810250526000_2026.04.13.718121v1.15","doi-asserted-by":"crossref","first-page":"eaao2556","DOI":"10.1126\/sciimmunol.aao2556","article-title":"MR1 displays the microbial metabolome driving selective MR1-restricted T cell receptor usage","volume":"3","year":"2018","journal-title":"Science Immunology"},{"key":"2026041810250526000_2026.04.13.718121v1.16","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.R115.685990"},{"key":"2026041810250526000_2026.04.13.718121v1.17","doi-asserted-by":"publisher","DOI":"10.1038\/nature13160"},{"key":"2026041810250526000_2026.04.13.718121v1.18","doi-asserted-by":"publisher","DOI":"10.1038\/nature11605"},{"key":"2026041810250526000_2026.04.13.718121v1.19","doi-asserted-by":"publisher","DOI":"10.1084\/jem.20140484"},{"key":"2026041810250526000_2026.04.13.718121v1.20","doi-asserted-by":"publisher","DOI":"10.1038\/s41590-020-0616-6"},{"key":"2026041810250526000_2026.04.13.718121v1.21","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M501087200"},{"key":"2026041810250526000_2026.04.13.718121v1.22","doi-asserted-by":"crossref","first-page":"933","DOI":"10.4049\/jimmunol.2300609","article-title":"Multiple Isomers of Photolumazine V Bind MR1 and Differentially Activate MAIT Cells","volume":"212","year":"2024","journal-title":"The Journal of Immunology"},{"key":"2026041810250526000_2026.04.13.718121v1.23","doi-asserted-by":"crossref","first-page":"2142","DOI":"10.1038\/ncomms3142","article-title":"Recognition of vitamin B metabolites by mucosal-associated invariant T cells","volume":"4","year":"2013","journal-title":"Nature Communications"},{"key":"2026041810250526000_2026.04.13.718121v1.24","doi-asserted-by":"crossref","unstructured":"M. R. Abdelaal et al., The antigen-presenting molecule MR1 binds host-generated riboflavin catabolites. J Exp Med 223 (2026).","DOI":"10.1084\/jem.20250711"},{"key":"2026041810250526000_2026.04.13.718121v1.25","doi-asserted-by":"crossref","first-page":"191","DOI":"10.1007\/978-1-0716-0207-2_13","article-title":"Production of MR1 Tetramers Loaded with Microbial Ligands","volume":"2098","year":"2020","journal-title":"Methods Mol Biol"},{"key":"2026041810250526000_2026.04.13.718121v1.26","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2304055120"},{"key":"2026041810250526000_2026.04.13.718121v1.27","doi-asserted-by":"crossref","first-page":"e2505932123","DOI":"10.1073\/pnas.2505932123","article-title":"Design of solubly expressed miniaturized SMART MHCs","volume":"123","year":"2026","journal-title":"Proceedings of the National Academy of Sciences"},{"key":"2026041810250526000_2026.04.13.718121v1.28","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.89.8.3429"},{"key":"2026041810250526000_2026.04.13.718121v1.29","doi-asserted-by":"crossref","unstructured":"A. Chancellor et al., Promiscuous recognition of MR1 drives self-reactive mucosal-associated invariant T cell responses. Journal of Experimental Medicine 220 (2023).","DOI":"10.1084\/jem.20221939"},{"key":"2026041810250526000_2026.04.13.718121v1.30","doi-asserted-by":"crossref","first-page":"eadn0126","DOI":"10.1126\/sciimmunol.adn0126","article-title":"Nucleobase adducts bind MR1 and stimulate MR1-restricted T cells","volume":"9","year":"2024","journal-title":"Science Immunology"},{"key":"2026041810250526000_2026.04.13.718121v1.31","doi-asserted-by":"crossref","first-page":"e2418525122","DOI":"10.1073\/pnas.2418525122","article-title":"Mitochondria regulate MR1 protein expression and produce self-metabolites that activate MR1-restricted T cells","volume":"122","year":"2025","journal-title":"Proceedings of the National Academy of Sciences"},{"key":"2026041810250526000_2026.04.13.718121v1.32","doi-asserted-by":"crossref","unstructured":"T. Schmidlin et al., MR1-ligand cross-linking identifies vitamin B6 metabolites as TCR-reactive antigens. Cell Reports Methods 5 (2025).","DOI":"10.1016\/j.crmeth.2025.101120"},{"key":"2026041810250526000_2026.04.13.718121v1.33","doi-asserted-by":"publisher","DOI":"10.1038\/s41590-019-0578-8"},{"key":"2026041810250526000_2026.04.13.718121v1.34","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1012632"},{"key":"2026041810250526000_2026.04.13.718121v1.35","doi-asserted-by":"crossref","first-page":"22539","DOI":"10.1038\/s41598-022-26259-y","article-title":"Deaza-modification of MR1 ligands modulates recognition by MR1-restricted T cells","volume":"12","year":"2022","journal-title":"Scientific Reports"},{"key":"2026041810250526000_2026.04.13.718121v1.36","doi-asserted-by":"crossref","first-page":"e2506016122","DOI":"10.1073\/pnas.2506016122","article-title":"Solution mapping of MHC-I:TCR interactions using a minimalistic protein system","volume":"122","year":"2025","journal-title":"Proceedings of the National Academy of Sciences"},{"key":"2026041810250526000_2026.04.13.718121v1.37","doi-asserted-by":"publisher","DOI":"10.1111\/tan.14390"},{"key":"2026041810250526000_2026.04.13.718121v1.38","doi-asserted-by":"publisher","DOI":"10.1016\/j.pep.2005.01.016"},{"key":"2026041810250526000_2026.04.13.718121v1.39","doi-asserted-by":"publisher","DOI":"10.1006\/jmbi.1998.2091"},{"key":"2026041810250526000_2026.04.13.718121v1.40","doi-asserted-by":"publisher","DOI":"10.1007\/BF00197809"},{"key":"2026041810250526000_2026.04.13.718121v1.41","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btab180"},{"key":"2026041810250526000_2026.04.13.718121v1.42","doi-asserted-by":"crossref","first-page":"102714","DOI":"10.1016\/j.jbc.2022.102714","article-title":"Quantitative affinity measurement of small molecule ligand binding to major histocompatibility complex class-I\u2013related protein 1 MR1","volume":"298","year":"2022","journal-title":"Journal of Biological Chemistry"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.04.13.718121","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,4,18]],"date-time":"2026-04-18T17:25:14Z","timestamp":1776533114000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.04.13.718121"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4,15]]},"references-count":42,"URL":"https:\/\/doi.org\/10.64898\/2026.04.13.718121","relation":{},"subject":[],"published":{"date-parts":[[2026,4,15]]},"subtype":"preprint"}}