Structural Analysis of the TCR-CD3 Complex and TCR Signaling

TCR-CD3 复合物和 TCR 信号传导的结构分析

• 批准号: 9251684
• 负责人: Roy A Mariuzza
• 金额: $ 70.04万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9251684
• 关键词: Address; Affinity; Antigens; Antiviral Agents; Architecture; Autoimmune Process; Binding; Binding Proteins; Biological; Biological Assay; CD3 Antigens; CRISPR/Cas technology; Cell Line; Cell physiology; Cell surface; Cells; Chemicals; Complex; Coupled; Crystallization; Data; Development; Directed Molecular Evolution; Docking; Elements; Epitopes; Event; Flow Cytometry; Goals; HLA-A2 Antigen; HLA-DR4 Antigen; Health; Heteronuclear NMR; Human T-lymphotropic virus 1; Immune response; Immunology; In Vitro; Individual; Length; Libraries; Ligands; Ligation; Link; MHC Class I Genes; MHC Class II Genes; MHC binding peptide; Malignant Neoplasms; Maps; Mature T-Lymphocyte; Measurable; Mediating; Microbe; Molecular; Molecular Conformation; Molecular Immunology; Multiprotein Complexes; Mus; Mutagenesis; Mutation; Myelin Basic Proteins; NMR Spectroscopy; Peptide/MHC Complex; Peptides; Physiological; Play; Process; Receptor Signaling; Relaxation; Research Personnel; Resolution; Retroviral Vector; Roentgen Rays; Role; Signal Pathway; Signal Transduction; Site; Structure; Surface; System; T-Cell Activation; T-Cell Development; T-Cell Receptor; T-Lymphocyte; Taxes; Technology; Testing; Time; Validation; Variant; Viral Antigens; X-Ray Crystallography; Yeasts; adaptive immunity; base; design; dimer; extracellular; genome editing; mutant; novel strategies; receptor; reconstitution

 DESCRIPTION (provided by applicant): The T cell receptor (TCR)-CD3 complex is composed of a diverse TCRαβ heterodimer noncovalently associated with the invariant CD3 dimers CD3εγ, CD3εδ, and CD3ζζ. The TCR mediates peptide-MHC (pMHC) recognition, while the CD3 molecules transduce activation signals to the T cell. Whereas much is known about downstream T cell signaling pathways, the mechanism whereby TCR engagement by pMHC initiates signaling remains a mystery. Our goal is to understand the early events of TCR signaling by obtaining information on: 1) the spatial organization of the TCR-CD3 complex; and 2) possible allosteric changes in TCR conformation and/or dynamics upon binding pMHC that are relayed to CD3. These studies will be undertaken through a highly integrated and comprehensive project that combines multiple approaches, technologies, and investigators. To assure the generality of our findings, we will study both an MHC class I-restricted TCR (A6) and an MHC class II-restricted TCR (MS2-3C8). A6 recognizes a viral antigen from HTLV-1 bound to HLA-A2; MS2-3C8 recognizes a self-peptide from myelin basic protein bound to HLA-DR4. Our Specific Aims are: 1. Structure determination of the wild-type TCR-CD3 complex by NMR. Although the extracellular regions of CD3 are known to interact with the extracellular regions of the TCR, all attempts to crystallize TCR-CD3 complexes have been thwarted by the very low affinity of TCR-CD3 interactions in solution. We will employ NMR chemical shift perturbation and PRE to determine binding epitopes between CD3 and TCR. These data will be used to determine a structure for the wild-type TCR-CD3 complex. We will address whether pMHC binding alters TCR-CD3 interactions, possibly triggering T cell signaling. 2. Structural analysis of affinity- matured TCR-CD3 complexes by X-ray crystallography. We will attempt to overcome the weak association between TCR and CD3 ectodomains by in vitro directed evolution using yeast surface display to stabilize TCR-CD3 complexes for crystallization. Information on binding epitopes from NMR will be used to design TCR mutant libraries by focusing mutagenesis on those regions that contact CD3 (or vice versa). 3. Biological validation of the TCR-CD3 structure. We will validate TCR-CD3 interfaces identified by NMR or X-ray crystallography by evaluating the effects of structure-based mutations in TCR-CD3 interfaces on complex assembly, cell surface expression, signaling, and T cell function and development. The TCR-CD3 complex will be reconstituted using 2A-linked retroviral vectors to generate T cells or retrogenic mice expressing defined combinations of TCR and CD3 components. 4. Structural and dynamics analysis of free and pMHC-bound states of TCRαβ ectodomains. We will address the allosteric change hypothesis by carrying out solution NMR analysis of full-length TCR ectodomains in free form and bound to pMHC, for both TCRs A6 and MS2- 3C8. We will investigate whether pMHC ligation induces changes in TCR conformation and/or dynamics and, if so, relate these changes to a possible mechanism for TCR triggering involving interactions with CD3.

 描述（由适用提供）：T细胞受体（TCR）-CD3复合物由与不变的CD3 DimersCD3εγ，CD3εδ和CD33εδ和CD33ζ无共价相关的发散TCRαβ异二聚体组成。 TCR介导了肽-MHC（PMHC）识别，而CD3分子将激活信号转换为T细胞。尽管对下游T细胞信号通路有很多了解，但PMHC启动信号传导的机制仍然是一个谜。我们的目标是通过获取以下信息来了解TCR信号的早期事件：1）TCR-CD3复合物的空间组织； 2）在结合PMHC时可能会发生变构变化，并传递到CD3。这些研究将通过一个高度综合和全面的项目进行，该项目结合了多种方法，技术和研究人员。为了确保我们的发现的普遍性，我们将同时研究MHC I类限制的TCR（A6）和MHC II级限制性TCR（MS2-3C8）。 A6识别从HTLV-1结合到HLA-A2的病毒抗原； MS2-3C8识别与HLA-DR4结合的髓磷脂碱性蛋白的一种自肽。我们的具体目的是：1。通过NMR对野生型TCR-CD3复合物的结构测定。尽管已知CD3的细胞外区域与TCR的细胞外区域相互作用，但所有试图使TCR-CD3复合物结晶的尝试都受到溶液中TCR-CD3相互作用的非常低的亲和力的阻碍。我们将采用NMR化学位移扰动和预先确定CD3和TCR之间的结合表位。这些数据将用于确定野生型TCR-CD3复合物的结构。我们将解决PMHC结合是否改变了TCR-CD3相互作用，可能触发T细胞信号传导。 2。通过X射线晶体学对亲和力 - 到期TCR-CD3复合物的结构分析。我们将尝试通过使用酵母表面显示出来稳定TCR-CD3复合物进行结晶的体外定向进化来克服TCR和CD3外生域之间的弱关联。 NMR的结合表位的信息将用于通过将诱变聚焦在与CD3（或VICE反之亦然）的区域上，以设计TCR突变库。 3。TCR-CD3结构的生物学验证。我们将通过评估TCR-CD3接口中基于结构的突变对复杂组装，细胞表面表达，信号传导以及T细胞功能和开发的影响，来验证通过NMR或X射线晶体学鉴定的TCR-CD3接口。 TCR-CD3复合物将使用2A连接的逆转录病毒载体重组，以产生TCR和CD3成分的定义组合的T细胞或后源性小鼠。 4。tcrαβ胞外域的自由和PMHC结合状态的结构和动力学分析。对于TCRS A6和MS2- 3C8，我们将通过对全长TCR外生求解并与PMHC结合的全长TCR外生域的解决方案NMR分析来解决变构的变化假设。我们将研究PMHC连接是否诱导TCR构象和/或动力学的变化，如果是的，则将这些更改与TCR触发与CD3相互作用的可能机制有关。