Molecular mechanisms of HLA-DM mediated peptide exchange

描述（由申请人提供）：主要组织相容性复合体（MHC）是许多常见人类自身免疫性疾病最重要的易感位点，包括1型糖尿病、类风湿关节炎和多发性硬化症，表明抗原呈递到CD4 T细胞是其发病机制的重要一步。HLA-DM （DM）在MHCII类（MHCII）抗原递呈途径中发挥核心作用：它诱导不变链衍生的CLIP肽解离并编辑肽库，有利于高亲和力肽的递呈。该项目的中心目标是在结构水平上定义DM的作用机制，并促进我们对自身免疫性疾病中自身抗原呈递的理解。初步研究表明，DM与HLA-DR （DR）分子的相互作用受DR肽结合槽的占据状态控制。我们发现，当肽结合槽被肽完全占据时，DM不能与DR蛋白结合。相反，DM结合到DR蛋白的不稳定过渡状态，其中肽n端部分与凹槽分离。我们利用这一见解来开发DM结合DR1复合体的结晶策略。自提交原始提案以来，我们已经能够以2.6Å的分辨率确定DR1-DM复合物的晶体结构。该结构提示了一种快速选择高亲和力肽（也称为“编辑”）的新机制。在与dm结合的状态下，三个D残基已经移动到凹槽的关键部分（P1口袋和P2位点），使肽最初无法进入。多肽需要竞争进入这些位点，这种能量屏障驱动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肽与空DR1-DM复合物的快速结合。这项技术现在将用于定义DM编辑肽库的关键机制步骤。目标2是基于对DR1-DM结构的新观察。HLA-DQ8 （DQ8）赋予1型糖尿病易感性，而HLA-DQ6 （DQ6）诱导显性保护。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的肽结合特性存在显著差异。我们将使用一系列功能方法来剖析这些DQ多态性对DQ8和DQ6肽呈现的影响。此外，使DR1-DM复合物结晶的新策略将用于DQ-DM复合物的结晶试验。我们预计这些研究将对我们对自身免疫性疾病中DM功能和MHCII抗原呈递的理解产生重大影响。

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.