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

描述（由申请人提供）：T细胞受体（TCR）-CD 3复合物由不同的TCR？？与不变的CD 3二聚体CD 3？？非共价结合的异源二聚体，CD3？？，CD3？TCR介导肽-MHC（pMHC）识别，而相关的CD 3分子向T细胞传递活化信号。尽管人们对下游T细胞信号传导了解很多，但TCR与pMHC结合启动信号传导的机制仍然是一个谜。已经提出了三种基本机制：聚集、分离和/或构象变化。构象变化机制的一个主要吸引力在于，原则上，它可以解释在抗原呈递细胞上非常低密度的pMHC激动剂的触发。然而，X射线晶体学研究迄今未能确定明确和一致的构象变化的TCR C？？或C？可以明确地归因于抗原结合的结构域。一种可能性是，这种变化根本不会发生。另一个是，晶体堆积效应可能掩盖了C？？还是C第三种可能性是，相关的变化可能是蛋白质动力学的变化，这是一个X射线晶体学无法获得的参数。为了区分这些可能性，我们提出对处于未结合状态并与pMHC结合的全长TCR胞外域（V和C区）进行第一溶液NMR分析。作为我们的测试系统，我们将使用人类自身免疫TCR（MS 2 - 3C 8），其识别来自髓鞘碱性蛋白（MBP）的肽和MHC II类分子HLA-DR 4。我们最近确定的MS 2 - 3C 8-MBP-DR 4复合物的晶体结构揭示了TCR MS 2 - 3C 8以抗外源TCR的典型对接模式接合pMHC。此外，MS 2 - 3C 8与最强烈的抗外来TCR一样紧密地结合MBP-DR 4。我们的目标是：1. TCR的游离态和pMHC结合态的结构分析？？胞外域在溶液中。我们将确定游离形式和与MBP-DR 4结合的TCR MS 2 - 3C 8的NMR溶液结构。因为TCR是一种异源二聚体，然后呢？链，每条链的单独同位素标记将降低光谱复杂性并促进NMR分析。初步NMR光谱支持对于单个结构域和总体折叠分别实现<2.0 A和<3.0 A的骨架RSD的可行性，这将允许严格评估MS 2 - 3C 8中的任何配体诱导的构象变化。2. TCR的游离和pMHC结合状态的骨架动力学分析？？胞外域我们的目标是研究pMHC结合是否诱导TCR骨架动力学的变构变化。将检查宽时间尺度范围（皮秒至秒），以确保检测到主链动力学的任何变化。总的来说，这些研究应该解决TCR在溶液中是否经历构象和/或动力学的长距离变化，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连接导致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CD 3信号传导装置：聚集、分离和/或构象变化。在这里，我们解决的构象变化假说，提出进行有史以来第一次解决NMR分析的全长TCR胞外域（V和C区）在未结合状态，并结合其肽-MHC配体，允许详细的分析，以前不可能使用其他方法。

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.