The Major Histocompatibility Complex class I (MHC-I) related protein 1 (MR1) presents small molecule metabolites, drugs, and drug-like molecules that are recognized by MR1-reactive T cells. While we have an understanding of how antigens bind to MR1 and upregulate MR1 cell surface expression, a quantitative, cell-free, assessment of MR1-ligand binding affinity was lacking. Here, we developed a fluorescence polarization (FP)-based assay in which fluorescent MR1 ligand was loaded into MR1 protein in vitro, and competitively displaced by candidate ligands over a range of concentrations. Using this assay, ligand affinity for MR1 could be differentiated as strong (IC₅₀ < 1 μM), moderate (1 μM < IC₅₀ < 100 μM), and weak (IC₅₀ > 100 μM). We demonstrated a clear correlation between ligand binding affinity for MR1, the presence of a covalent bond between MR1 and ligand, and the number of salt bridge and hydrogen bonds formed between MR1 and ligand. Using this newly developed FP-based assay to screen for candidate ligands, we identified the dietary molecules vanillin and ethylvanillin as weak bona fide MR1 ligands. Both upregulated MR1 on the surface of C1R.MR1 cells and the crystal structure of a MAIT cell T cell receptor (TCR)-MR1-ethylvanillin complex revealed that ethylvanillin formed a Schiff base with K43 of MR1 and was buried within the Aʹ-pocket. Collectively, we developed and validated a method to quantitate the binding affinities of ligands for MR1 that will enable an efficient and rapid screening of candidate MR1 ligands.

主要组织相容性复合体 I 类 (MHC-I) 相关蛋白 1 (MR1) 呈现小分子代谢物、药物和药物样分子，可被 MR1 反应性 T 细胞识别。虽然我们了解抗原如何与 MR1 结合并上调 MR1 细胞表面表达，但缺乏对 MR1 配体结合亲和力的定量、无细胞评估。在这里，我们开发了一种基于荧光偏振 (FP) 的测定法，其中荧光 MR1 配体在体外加载到 MR1 蛋白中，并在一定浓度范围内被候选配体竞争性取代。使用该测定法，配体对 MR1 的亲和力可分为强 (IC ₅₀ < 1 μM)、中等 (1 μM < IC ₅₀ < 100 μM) 和弱 (IC ₅₀> 100 微米）。我们证明了配体对 MR1 的结合亲和力、MR1 和配体之间共价键的存在以及 MR1 和配体之间形成的盐桥和氢键的数量之间存在明显的相关性。使用这种新开发的基于 FP 的检测方法来筛选候选配体，我们将饮食分子香兰素和乙基香兰素鉴定为弱的真正MR1 配体。C1R.MR1 细胞表面上调的 MR1 和 MAIT 细胞 T 细胞受体 (TCR)-MR1-乙基香兰素复合物的晶体结构表明，乙基香兰素与 MR1 的 K43 形成席夫碱，并被埋在 A'-pocket 中。总的来说，我们开发并验证了一种方法来定量配体对 MR1 的结合亲和力，这将能够高效快速地筛选候选 MR1 配体。