Solution structure and dynamics of TCR in free and peptide-MHC-bound states

描述(申请人提供)：T细胞受体(TCR)-CD3复合体由不同的TCR？？组成。异源二聚体与不变的CD3二聚体CD3？、CD3？和CD3？？非共价相关。TCR介导肽-MHC(PMHC)识别，而相关的CD3分子将激活信号转导到T细胞。尽管对下游T细胞信号转导有很多了解，但pMHC参与TCR启动信号转导的机制仍然是个谜。已经提出了三种基本机制：聚集、分离和/或构象变化。构象改变机制的一个主要吸引力是，原则上，它可以解释在非常低密度的pMHC激动剂在抗原提呈细胞上触发的原因。然而，到目前为止，X射线结晶学研究还没有确定TCRC？？的明显和一致的构象变化。或可明确归因于抗原结合的C？？结构域。一种可能性是，这样的变化根本不会发生。另一种是晶体堆积效应可能掩盖了C？？的构象变化。还是C？然而，第三种可能性是，相关的变化可能发生在蛋白质动力学中，这是X射线结晶学无法获得的参数。为了区分这些可能性，我们建议对未结合状态并与pMHC结合的全长TCR胞外结构域(V和C区)进行第一次溶液核磁共振分析。作为我们的测试系统，我们将使用人类自身免疫TCR(MS2-3C8)，它可以识别髓鞘碱性蛋白(MBP)和MHC II类分子HLA-DR4中的一种肽。我们最近测定的MS2-3C8-MBP-DR4络合物的晶体结构表明，TCRMS2-3C8以抗外源TCRs的正则对接模式与pMHC对接。此外，MS2-3C8与MBP-DR4的结合与最狂热的反外来TCR一样紧密。我们的目标是：1.对TCR的自由态和pMHC束缚态进行结构分析。溶液中的胞外区域。我们将确定TCR MS2-3C8的自由形式并与MBP-DR4结合的核磁共振溶液结构。由于TCR是？-链和？-链的杂二聚体，所以对每条链进行单独的同位素标记将降低光谱的复杂性并便于核磁共振分析。初步的核磁共振谱支持分别为单个结构域和整个折叠实现&lt；2.0 A和&lt；3.0 A主链RMSD的可行性，这将允许对配体诱导的MS2-3C8中的任何构象变化进行严格评估。2.TCR？？自由态和pMHC束缚态的主干动力学分析外域。我们的目标是研究pMHC结合是否导致TCR骨架动力学的变构变化。将检查大范围的时间范围(皮秒到秒)，以确保检测到主干动态中的任何变化。总而言之，这些研究应该确定TCR在溶液中是否经历了构象和/或动力学的长期变化，这可以作为T细胞触发的机制。 公共卫生相关性：20多年来，TCR结扎导致T细胞触发的机制一直是免疫学中的一个基本谜团。已经提出了三种基本机制来解释TCR连接如何传递到CD3信号装置：聚集、分离和/或构象变化。在这里，我们通过首次对未结合状态并与其肽-MHC配体结合的全长TCR胞外结构域(V和C区)进行溶液核磁共振分析来解决构象变化假说，从而进行以前使用其他方法无法进行的详细分析。

DESCRIPTION (provided by applicant): The T cell receptor (TCR)-CD3 complex is composed of a diverse TCR?? heterodimer noncovalently associated with the invariant CD3 dimers CD3??, CD3??, and CD3??. The TCR mediates peptide-MHC (pMHC) recognition, while the associated CD3 molecules transduce activation signals to the T cell. Whereas much is known about downstream T cell signaling, the mechanisms whereby TCR engagement by pMHC initiates signaling remain a mystery. Three basic mechanisms have been proposed: aggregation, segregation, and/or conformational change. A major attraction of the conformational change mechanism is that it can, in principle, account for triggering at very low densities of pMHC agonists on antigen-presenting cells. However, X-ray crystallographic studies have so far failed to identify clear and consistent conformational changes in the TCR C?? or C??domains that could be unambiguously attributed to antigen binding. One possibility is that such changes simply do not occur. Another is that crystal packing effects may have masked conformational changes in C?? or C? Yet a third possibility is that the relevant changes may be in protein dynamics, a parameter which cannot be accessed by X-ray crystallography. To distinguish among these possibilities, we propose to carry out the first solution NMR analysis of a full- length TCR ectodomain (V and C regions) in the unbound state and bound to pMHC. As our test system, we will use a human autoimmune TCR (MS2-3C8) that recognizes a peptide from myelin basic protein (MBP) and the MHC class II molecule HLA-DR4. The crystal structure of the MS2-3C8-MBP-DR4 complex, which we recently determined, revealed that TCR MS2-3C8 engages pMHC in the canonical docking mode of anti-foreign TCRs. Moreover, MS2-3C8 binds MBP-DR4 as tightly as the most avid anti- foreign TCRs. Our objectives are: 1. Structural analysis of free and pMHC-bound states of the TCR?? ectodomain in solution. We will determine the NMR solution structure of TCR MS2-3C8 in free form and bound to MBP-DR4. Because TCR is a heterodimer of ?- and ?-chains, separate isotope labeling of each chain will reduce spectral complexity and facilitate NMR analysis. Preliminary NMR spectra support the feasibility of achieving backbone RMSDs of <2.0 A and <3.0 A for individual domains and overall folds, respectively, which will permit a rigorous assessment of any ligand-induced conformational changes in MS2-3C8. 2. Backbone dynamics analysis of free and pMHC-bound states of the TCR?? ectodomain. Our goal here is to investigate whether pMHC-binding induces allosteric changes in TCR backbone dynamics. A wide time scale range will be examined (picoseconds to seconds) to assure detection of any alterations in backbone dynamics. Collectively, these studies should resolve whether the TCR undergoes long-range changes in conformation and/or dynamics in solution that can serve as a mechanism for T cell triggering. PUBLIC HEALTH RELEVANCE: The mechanism by which TCR ligation leads to T cell triggering has remained a fundamental mystery in immunology for over 20 years. Three basic mechanisms have been suggested to explain how TCR ligation is communicated to the CD3 signaling apparatus: aggregation, segregation, and/or conformational change. Here, we address the conformational change hypothesis by proposing to carry out the first ever solution NMR analysis of a full-length TCR ectodomain (V and C regions) in the unbound state and bound to its peptide-MHC ligand, permitting detailed analyses that were not previously possible using other methods.