Molecular mechanisms of HLA-DM mediated peptide exchange

描述(申请人提供)：主要组织相容性复合体(MHC)代表了许多常见人类自身免疫性疾病的最重要的易感基因，包括1型糖尿病、类风湿性关节炎和多发性硬化症，这表明抗原呈递到CD4T细胞是其发病机制中的重要一步。HL A-DM(DM)在MHC-II类(MHCII)抗原提呈途径中起核心作用：它诱导不变链源性片段多肽的解离，并编辑多肽库，有利于高亲和力多肽的提呈。这个项目的中心目标是在结构水平上确定DM的作用机制，并促进我们对自身免疫性疾病中自身抗原提呈的理解。初步研究表明，DM与人类白细胞抗原DR(DR)分子的相互作用受DR多肽结合槽的占据状态控制。我们发现，当肽结合槽被多肽完全占据时，DM不能与DR蛋白结合。相反，DM与DR蛋白的一种不稳定的过渡态结合，在这种过渡态中，多肽N端已部分从凹槽中解离。我们利用这一洞察力开发了一种与DR1结合的DM络合物的结晶策略。目标1自提交原始提案以来，我们已经能够确定DR1-DM络合物的晶体结构，分辨率为2.6？这种结构暗示了一种快速选择高亲和力多肽(也称为编辑)的新机制。在DM结合状态下，三个D残基已经移动到凹槽的关键部分(P1口袋和P2位置)，使其最初无法被肽访问。多肽需要竞争进入这些位置，而这种能量障碍推动了选择具有最高亲和力的多肽。我们最近还开发了一种新的方法来测量多肽与空的DR1-DM复合体的快速结合。这项技术现在将被用来定义DM编辑多肽库中的关键机械步骤。AIM 2是基于对DR1-DM结构的新观察。人类白细胞抗原-DQ8(DQ8)对1型糖尿病具有易感性，而人类白细胞抗原-DQ6(DQ6)对1型糖尿病具有显性保护作用。DR1-DM结构表明，位于DR1-DM界面的大量残基在DQ8和DQ6中是多态的。此外，初步数据显示，DQ8和DQ6的多肽结合特性有很大差异。我们将使用一系列功能方法来剖析这些DQ多态对DQ8和DQ6呈现多肽的影响。此外，实现DR1-DM络合物结晶的新策略将用于DQ-DM络合物的结晶试验。我们预计，这些研究将对我们理解自身免疫性疾病中DM的功能和MHCII抗原提呈产生重大影响。

DESCRIPTION (provided by applicant): The Major Histocompatibility Complex (MHC) represents the most important susceptibility locus for many common human autoimmune diseases, including type 1 diabetes, rheumatoid arthritis and multiple sclerosis, indicating that antigen presentation to CD4 T cells represents an important step in their pathogenesis. HLA-DM (DM) plays a central role in the MHC class II (MHCII) antigen presentation pathway: it induces dissociation of the invariant chain-derived CLIP peptide and edits the peptide repertoire, favoring presentation of high-affinity peptides. The central goals of this project are to define th mechanism of DM action at a structural level and to advance our understanding of self-antigen presentation in autoimmune diseases. Preliminary studies have shown that the interaction of DM and HLA-DR (DR) molecules is controlled by the occupancy state of the DR peptide binding groove. We found that DM cannot bind to DR proteins when the peptide binding groove is fully occupied by peptides. Rather, DM binds to an unstable transition state of DR proteins in which the peptide N-terminus has partially dissociated from the groove. We utilized this insight to develop a strategy for crystallization of the complex of DM bound to DR1. Aim 1 Since submission of the original proposal, we have been able to determine the crystal structure of the DR1-DM complex at a resolution of 2.6Å. The structure suggests a novel mechanism for rapid selection of high-affinity peptides (also referred to as 'editing'). In the DM-bound state, three D residues have moved into a critical part of the groove (P1 pocket and P2 site), rendering it initially inaccessible to peptides. Peptides need to compete for access to these sites, and this energetic barrier drives selection of peptides with the highest affinities. We have also recently developed a novel approach to measure rapid binding of peptides to empty DR1-DM complexes. This technique will now be used to define critical mechanistic steps in editing of the peptide repertoire by DM. Aim 2 is based on novel observations from the DR1-DM structure. HLA-DQ8 (DQ8) confers susceptibility to type 1 diabetes while HLA-DQ6 (DQ6) induces dominant protection. The DR1-DM structure shows that a substantial number of residues located at the DR1-DM interface are polymorphic in DQ8 and DQ6. Furthermore, preliminary data show substantial differences in the peptide binding properties of DQ8 and DQ6. We will use a series of functional approaches to dissect the impact of these DQ polymorphisms on peptide presentation by DQ8 and DQ6. Also, the novel strategy that enabled crystallization of the DR1-DM complex will be used for crystallization trials of a DQ-DM complex. We anticipate that these studies will have a significant impact on our understanding of DM function and MHCII antigen presentation in autoimmune diseases.