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

游离状态和肽 MHC 结合状态下 TCR 的溶液结构和动力学

基本信息

批准号：
8301177
负责人：
Roy A Mariuzza
金额：
$ 22.8万
依托单位：
UNIV OF MARYLAND, COLLEGE PARK
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-02-01 至 2014-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8301177
关键词：
Accounting Address Agonist Amides Anisotropy Antigen-Presenting Cells Autoantigens Autoimmune Process Binding Biological Assay Biological Testing CD3 Antigens Cell membrane Cells Chemicals Complement Complex Coupling DNA Sequence Rearrangement Data Detection Docking Failure Fluorescence Spectroscopy Goals HLA-DR4 Antigen Histocompatibility Antigens Class II Human Hydrogen Immunology Individual Isotope Labeling Label Length Ligands Ligation MHC binding peptide Masks Measurement Measures Mediating Methods Modeling Molecular Molecular Conformation Molecular Immunology Motion Multiple Sclerosis Mutation Myelin Basic Proteins Peptide/MHC Complex Peptides Process Protein Binding Protein Dynamics Relaxation Residual state Resolution Roentgen Rays Sampling Signal Transduction Solutions Structure System T-Cell Activation T-Cell Receptor T-Lymphocyte TNFRSF10A gene Testing Time Vertebral column X-Ray Crystallography antigen binding base density dimer electron density methyl group millisecond nanosecond research study restraint segregation

项目摘要

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.

描述（由申请人提供）：T细胞受体（TCR）-CD3复合物由多种TCR组成？异二聚体与不变的CD3二聚体CD3 ??，CD3 ??和CD3 ??无关。 TCR介导了肽-MHC（PMHC）识别，而相关的CD3分子将激活信号转换为T细胞。尽管对下游T细胞信号传导有很多了解，但PMHC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，隔离和/或构象变化。构象变化机制的主要吸引力是，它原则上可以解释在抗原呈递细胞上以非常低的PMHC激动剂触发。但是，到目前为止，X射线晶体学研究未能识别出TCR C中的清晰，一致的构象变化？或可以明确归因于抗原结合的C？域。一种可能性是根本不会发生这种变化。另一个是晶体包装效应可能掩盖了C中的构象变化？还是C？然而，第三个可能性是相关的变化可能是蛋白质动力学，这是X射线晶体学无法访问的参数。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外域（V和C区）进行第一个溶液NMR分析。作为我们的测试系统，我们将使用人类自身免疫性TCR（MS2-3C8），该TCR（MS2-3C8）识别髓磷脂碱性蛋白（MBP）和MHC II类分子HLA-DR4的肽。我们最近确定的MS2-3C8-MBP-DR4复合物的晶体结构表明，TCR MS2-3C8在反外国TCR的规范对接模式下与PMHC接合。此外，MS2-3C8的结合MBP-DR4与最狂热的反外国TCR一样紧密。我们的目标是：1。对TCR的自由和PMHC结合状态的结构分析？溶液中的胞外域。我们将以自由形式确定TCR MS2-3C8的NMR溶液结构，并与MBP-DR4结合。由于TCR是？ - 和？ - 链的异二聚体，因此每个链的单独同位素标记将降低光谱复杂性并促进NMR分析。初步的NMR光谱支持分别针对单个域和整体折叠的骨架RMSD <2.0 A和<3.0 A的可行性，这将允许对MS2-3C8中任何配体诱导的构象变化进行严格的评估。 2。对TCR的自由和PMHC结合状态的主干动力学分析？外生域。我们的目标是研究PMHC结合是否会引起TCR主链动力学的变构变化。将检查广泛的时间尺度范围（比秒至秒），以确保检测骨干动力学的任何变化。总的来说，这些研究应该解决TCR是否会在溶液中构象和/或动力学的远程变化，这可以用作T细胞触发的机制。公共卫生相关性：TCR连接导致T细胞触发的机制一直是免疫学上的基本谜团，已有20多年了。已经提出了三种基本机制来解释如何将TCR连接传达到CD3信号传导设备：聚集，隔离和/或构象变化。在这里，我们通过提议在未结合状态的全长TCR外域（V和C区）进行第一个溶液NMR分析来解决构象变化假设，并与其肽-MHC配体绑定，允许使用其他方法不可能使用其他方法。