Understanding the structural basis of T cell receptor (TCR) and preTCR mechanosensing: single molecule, NMR and molecular dynamics studies

了解 T 细胞受体 (TCR) 和 preTCR 机械传感的结构基础：单分子、NMR 和分子动力学研究

• 批准号: 10153682
• 负责人: MATTHEW J LANG
• 金额: $ 75.53万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-22 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10153682
• 关键词: Adaptive Immune System; Adhesions; Affinity; Antigen-Presenting Cells; Antigens; Binding; Biological; Biological Assay; CD8B1 gene; Cell Culture Techniques; Cell Death; Cell Line; Cell physiology; Cell surface; Cells; Clone Cells; Communicable Diseases; Complex; Computer Simulation; Critical Pathways; Data; Development; Discrimination; Dissociation; Elements; Engineering; Environment; Event; Exhibits; Fetal Liver; Fostering; Future; Hematopoietic stem cells; Immune; Immunologic Surveillance; Lead; Ligand Binding; Ligands; Location; Lymphoid; Major Histocompatibility Complex; Mature T-Lymphocyte; Measurement; Measures; Mechanics; Mediating; Medical; Methods; Molecular; Molecular Conformation; Mutation; Mutation Analysis; Pathway interactions; Peptide/MHC Complex; Peptides; Performance; Peripheral; Process; Production; Reading; Recombinant Proteins; Scanning; Science; Sea; Sensitivity and Specificity; Series; Signal Transduction; Specificity; Stromal Cells; Structure; System; T-Cell Activation; T-Cell Immunologic Specificity; T-Cell Receptor; T-Lymphocyte; T-cell receptor repertoire; TCR Activation; Thymus Gland; Variant; Work; adaptive immune response; antigen binding; autoreactivity; base; conformational conversion; cytokine; design; immune activation; in silico; interfacial; mechanotransduction; migration; molecular dynamics; mutant; neglect; optical traps; pathogen; protein expression; screening; shear stress; simulation; single molecule; thymocyte; tumor

ABSTRACT The mammalian adaptive immune system protects its host against infectious diseases as well as tumors in a highly specific manner. At the core of ab T lymphocyte recognition is self- vs. non-self-discrimination, a functionality endowed by clonal cell-surface T cell receptors (TCRs). The millions of distinct TCRs expressed in the mammalian thymus create a repertoire that is refined to eliminate unwanted autoreactive specificities prior to export into the peripheral lymphoid compartment. Once there, mature abT cells scan their environment during immune surveillance, generating tensile and shear stresses over a wide range of pN-nN forces. Direct evidence that the TCR acts as a mechanosensor has been provided, explaining its exquisite specificity and sensitivity yet low affinity for ligand in the absence of physical load. Recently, we showed that force-based abTCR discrimination extended to its developmental precursor, the preTCR, a pTa-b heterodimer. Moreover, reversible structural rearrangements necessary for strengthened ligand binding under force were observed in both TCR and preTCR. In this proposal, we shall combine single molecule (SM) and single cell (SMSC) methods using optical traps, structure-function mutational analyses, recombinant protein expression and molecular dynamic simulation to probe TCR and preTCR complexes with pMHC under load to provide a clear understanding for the structural basis of mechanosensing. It is our hypothesis that binding is "gated" for unloaded TCRs but that TCRs enter a "binding reading state" when force loaded, extend and either stabilize the bond with lifetime lengthening to facilitate signaling or, alternatively, quickly release from irrelevant ligands. In Aim 1, we will elucidate the critical TCR a and b subunit variable (V) and constant (C) domain structural elements including the Cb FG loop involved in mechanically modulating the strength of loaded TCR-pMHC interactions. Topologically stabilized structures as well as de-stabilizing mutations will be assessed for their ability to alter pMHC bond lifetime and conformational change as well as to impact ab T cell activation as measured by cytokine production. We will leverage newly developed single molecule and single tether assays for direct comparison of strength of loaded TCRs on isolated TCRab-pMHC complexes and ab T cell lines. Aim 2 will examine mechanical tuning of the preTCR and how preTCR pTa-b structures differ from those of TCRab. In both Aims 1 and 2, we will identify conformational transitions leading to bond strengthening and release pathways critical to T cell activation and development. The effects of preTCR mutations on thymocyte developmental progression will be determined experimentally using the thymic stromal cell line OP9-DL4 and fetal liver hematopoietic progenitors transduced with wild-type or mutant preTCRs. Aim 3 will employ in silico molecular dynamics simulation to identify unfolding pathways of TCR-pMHC or preTCR-pMHC complexes and their impact on the dynamic adaptability of the interface under load. We will thereby reveal atomistic mechanisms for mechanosensing.

抽象的 哺乳动物适应性免疫系统可保护其宿主免受传染病以及肿瘤的影响 高度具体的方式。 AB T淋巴细胞识别的核心是自我与非歧视，A 克隆细胞表面T细胞受体（TCR）赋予功能。数百万个不同的TCR在 哺乳动物的胸腺创建了一个精致的曲目，以消除不必要的自动反应性特异性 导出到外周淋巴室。到达那里后，成熟的ABT细胞扫描其环境 在免疫监视期间，在巨大的PN-NN力上产生拉伸和剪切应力。直接的 提供了TCR作为机械传感器的证据，并解释了其精致的特异性和 在没有物理负荷的情况下，灵敏度却低的亲和力。最近，我们展示了基于力的 ABTCR歧视扩展到其发育前体，PTA-B异二聚体。而且， 在力下观察到加强配体结合所需的可逆结构重排 TCR和Pretcr。在此提案中，我们将结合单分子（SM）和单细胞（SMSC） 使用光学陷阱，结构功能突变分析，重组蛋白表达和 分子动力学模拟，以探测TCR和PMHC的探针和pphc络合物，以提供清晰的 理解机械感应的结构基础。我们的假设是，绑定是“封闭”的 卸载的TCR，但当力加载，延伸并稳定时，TCRS进入“绑定阅读状态” 寿命延长的键可以促进信号传导，或者，或者很快从无关的配体中释放出来。 在AIM 1中，我们将阐明临界TCR A和B亚基变量（V）和常数（C）域结构 包括与机械调节加载TCR-PMHC强度有关的CB FG环中的元素 互动。将评估拓扑稳定的结构以及去稳定的突变。 改变PMHC键寿命和构象变化以及影响AB T细胞激活的能力 通过细胞因子产生测量。我们将利用新开发的单分子和单个系绳测定法 为了直接比较分离的TCRAB-PMHC复合物和AB T细胞系上负载TCR的强度。目的 2将检查Pretcr的机械调整以及Pretcr PTA-B结构与TCRAB的结构有何不同。 在两个目标1和2中，我们将确定构象过渡，从而导致粘结加强和释放 对于T细胞激活和发育至关重要的途径。假定突变对胸腺细胞的影响 发育进展将使用胸腺基质细胞系OP9-DL4和 胎儿肝造血祖细胞用野生型或突变体prest型转导。 AIM 3将在Silico中使用 分子动力学模拟，以识别TCR-PMHC或Pretcr-PMHC复合物的展开途径和 它们对负载下界面的动态适应性的影响。我们将因此揭示原子 机械感应机制。