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（由申请方提供）：T细胞受体（TCR）-CD 3复合物由与不变的CD 3二聚体CD 3 εγ、CD 3 εδ和CD 3 β非共价结合的不同TCRαβ异源二聚体组成。TCR介导肽-MHC（pMHC）识别，而CD 3分子向T细胞传递活化信号。尽管对下游T细胞信号传导途径了解很多，但TCR与pMHC结合启动信号传导的机制仍然是一个谜。我们的目标是通过获得以下信息来了解TCR信号传导的早期事件：1）TCR-CD 3复合物的空间组织;和2）在结合pMHC时TCR构象和/或动力学中可能的变构变化，所述变构变化被传递到CD 3。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，技术和调查人员。为了确保我们研究结果的普遍性，我们将研究MHC I类限制性TCR（A6）和MHC II类限制性TCR（MS 2 - 3C 8）。A6识别来自与HLA-A2结合的HTLV-1的病毒抗原; MS 2 - 3C 8识别来自与HLA-DR 4结合的髓鞘碱性蛋白的自身肽。我们的具体目标是：1。通过NMR确定野生型TCR-⑶ 3复合物的结构。尽管已知CD 3的胞外区与TCR的胞外区相互作用，但所有使TCR-CD 3复合物结晶的尝试都因溶液中TCR-CD 3相互作用的非常低的亲和力而受阻。我们将采用NMR化学位移扰动和PRE来确定CD 3和TCR之间的结合表位。这些数据将用于确定野生型TCR-CD 3复合物的结构。我们将讨论pMHC结合是否改变TCR-CD 3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成熟的TCR-⑶ 3复合物的结构分析。我们将尝试克服TCR和CD 3胞外域之间的弱关联，通过体外定向进化，使用酵母表面展示来稳定TCR-CD 3复合物以用于结晶。来自NMR的结合表位的信息将用于通过将诱变集中在接触CD 3的那些区域（或反之亦然）来设计TCR突变体文库。3. TCR-CD 3结构的生物学验证。我们将通过评估TCR-CD 3界面中基于结构的突变对复合物组装、细胞表面表达、信号传导和T细胞功能和发育的影响，验证通过NMR或X射线晶体学鉴定的TCR-CD 3界面。将使用2A连接的逆转录病毒载体重建TCR-CD 3复合物，以产生表达TCR和CD 3组分的确定组合的T细胞或逆转录小鼠。4. TCRαβ胞外域的游离和pMHC结合状态的结构和动力学分析。我们将通过对TCR A6和MS 2 - 3C 8的游离形式和与pMHC结合的全长TCR胞外域进行溶液NMR分析来解决变构变化假说。我们将调查是否pMHC连接诱导TCR构象和/或动力学的变化，如果是这样，这些变化涉及到一个可能的机制TCR触发与CD 3的相互作用。