The antigen-presenting molecule MR1 presents riboflavin-based metabolites to Mucosal-Associated Invariant T (MAIT) cells. While MR1 egress to the cell surface is ligand-dependent, the ability of small-molecule ligands to impact on MR1 cellular trafficking remains unknown. Arising from an in silico screen of the MR1 ligand-binding pocket, we identify one ligand, 3-([2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]formamido)propanoic acid, DB28, as well as an analog, methyl 3-([2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]formamido)propanoate, NV18.1, that down-regulate MR1 from the cell surface and retain MR1 molecules in the endoplasmic reticulum (ER) in an immature form. DB28 and NV18.1 compete with the known MR1 ligands, 5-OP-RU and acetyl-6-FP, for MR1 binding and inhibit MR1-dependent MAIT cell activation. Crystal structures of the MAIT T cell receptor (TCR) complexed with MR1-DB28 and MR1-NV18.1, show that these two ligands reside within the A′pocket of MR1. Neither ligand forms a Schiff base with MR1 molecules; both are nevertheless sequestered by a network of hydrophobic and polar contacts. Accordingly, we define a class of compounds that inhibits MR1 cellular trafficking.
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ACKNOWLEDGMENTS. We thank the late V.C. as an inspirational colleague, mentor, and pioneer in the field of iNKT/CD1/MAIT cell biology, who passed away during the completion of the revision of this work. We thank Dr. Ted Hansen for the provision of 26.5 and 8F2F9 antibodies and Prof. A. Sewell for the provision of THP1-MR1 KO cells. We thank Dr. B. Park for help with the statistical analysis. We acknowledge the contribution of the WIMM Flow cytometry facility for cell sorting of MAIT cells and of the Weatherall Institute of Molecular Medicine (WIMM) Wolfson Imaging Facility for help with confocal microscopy, and Dr. Giorgio Napolitani for critical reading of the manuscript. This work was supported by Cancer Research UK (Programme Grant C399/A2291 to V.C.), the Medical Research Council (MRC Human Immunology Unit Core funding), Veteran Affairs (VA) Merit Award I01BX000533 (to D.M.L.), and NIH Grant R01AI134790. J.R. is an Australian Research Council (ARC) Laureate Fellow. We thank the staff at the Monash Macromolecular crystallization facility. This research was undertaken in part using the MX2 beamline at the Australian Synchrotron, part of Australian Nuclear Science and Technology Organisation (ANSTO), and made use of the Australian Cancer Research Foundation detector. G.S.B. acknowledges support from the UK Medical Research Council (MR/S000542/1) and The Wellcome Trust (081569/Z/06/Z). The contents do not represent the views of the US Department of Veterans Affairs or the US government.
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