Human leukocyte antigen-DM (HLA-DM) is an integral component of the major histocompatibility complex class II (MHCII) antigen-processing and -presentation pathway. HLA-DM shapes the immune system by differentially catalyzing peptide exchange on MHCII molecules, thereby editing the peptide-MHCII (pMHCII) repertoire by imposing a bias on the foreign and self-derived peptide cargos that are presented on the cell surface for immune surveillance and tolerance induction by CD4+ T cells. To better understand DM selectivity, here we developed a real-time fluorescence anisotropy assay to delineate the pMHCII intrinsic stability, DM-binding affinity, and catalytic turnover, independent kinetic parameters of HLA-DM enzymatic activity. We analyzed prominent pMHCII contacts by differentiating the kinetic parameters in pMHCII homologs, observing that peptide interactions throughout the MHCII binding cleft influence both the rate of peptide dissociation from the DM-pMHCII catalytic complex and the binding affinity of HLA-DM for a pMHCII. We show that the intrinsic stability of a pMHCII linearly correlates with DM catalytic turnover, but is non-linearly correlated with its binding affinity. Surprisingly, interactions at the peptides N-terminus up to and including the MHCII position one (P1) anchor affected the catalytic turnover, suggesting that the active DM-pMHCII catalytic complex operates on pMHCII complexes with full peptide occupancy. Furthermore, interactions at the peptide C-terminus modulated DM-binding affinity, suggesting distal communication between peptide interactions with the MHCII and the DM-pMHCII binding interface. Our results imply an intimate linkage between the DM-pMHCII interface and peptide-MHCII interactions throughout the peptide-binding cleft.

中文翻译：

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HLA-DM通过感测整个肽结合裂口中的II类MHC相互作用，催化增强肽的解离。

人白细胞抗原-DM（HLA-DM）是主要组织相容性复合物II类（MHCII）抗原加工和呈递途径的组成部分。HLA-DM通过差异催化MHCII分子上的肽交换来塑造免疫系统，从而通过对存在于细胞表面的外源和自衍生肽货物施加偏见来编辑肽-MHCII（pMHCII）谱，以进行免疫监控和CD4 + T细胞诱导耐受性。为了更好地理解DM的选择性，在这里我们开发了实时荧光各向异性测定法来描述pMHCII固有稳定性，DM结合亲和力和催化周转率，HLA-DM酶活性的独立动力学参数。我们通过区分pMHCII同源物中的动力学参数来分析重要的pMHCII接触，观察到贯穿整个MHCII结合裂隙的肽相互作用既影响肽从DM-pMHCII催化复合物中解离的速率，也影响HLA-DM对pMHCII的结合亲和力。我们表明，pMHCII的固有稳定性与DM催化转换线性相关，但与其结合亲和力非线性相关。出人意料的是，直至和包括MHCII位置一（P1）锚的肽N末端的相互作用影响了催化转换，表明活性DM-pMHCII催化复合物在具有完整肽占用的pMHCII复合物上起作用。此外，肽C末端的相互作用调节了DM结合亲和力，表明肽与MHCII和DM-pMHCII结合界面之间的远距离通讯。