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 哺乳动物的适应性免疫系统可以保护宿主免受传染病和肿瘤的侵害。 高度具体的方式。 ab T 淋巴细胞识别的核心是自我与非自我歧视， 克隆细胞表面 T 细胞受体 (TCR) 赋予的功能。数以百万计的不同 TCR 表达于 哺乳动物胸腺创造了一套经过改进的库，可以在之前消除不需要的自身反应特异性。 输出到外周淋巴室。一旦到达那里，成熟的 abT 细胞就会扫描它们的环境 在免疫监视过程中，在广泛的 pN-nN 力范围内产生拉应力和剪应力。直接的 已经提供了 TCR 作为机械传感器的证据，解释了其精致的特异性和 在没有物理负荷的情况下，灵敏度高但对配体的亲和力低。最近，我们证明了基于力的 abTCR 歧视延伸至其发育前体 preTCR，即 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。在这个提案中，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，在负载下探测 TCR 和 preTCR 复合物与 pMHC，以提供清晰的结果 了解机械传感的结构基础。我们的假设是结合是“门控”的 卸载 TCR，但当受力加载、延伸或稳定时，TCR 进入“结合读取状态” 具有延长寿命的键以促进信号传导，或者快速从不相关的配体中释放。 在目标 1 中，我们将阐明关键的 TCR a 和 b 亚基变量 (V) 和恒定 (C) 结构域 包括参与机械调节负载 TCR-pMHC 强度的 Cb FG 环在内的元件 互动。将评估拓扑稳定结构以及不稳定突变的作用 改变 pMHC 键寿命和构象变化以及影响 ab T 细胞激活的能力 通过细胞因子的产生来测量。我们将利用新开发的单分子和单链分析 用于直接比较分离的 TCRab-pMHC 复合物和 ab T 细胞系上加载的 TCR 的强度。目的 图 2 将检查 preTCR 的机械调节以及 preTCR pTa-b 结构与 TCRab 的结构有何不同。 在目标 1 和 2 中，我们将确定导致键强化和释放的构象转变 对 T 细胞激活和发育至关重要的途径。 preTCR突变对胸腺细胞的影响 发育进程将使用胸腺基质细胞系 OP9-DL4 进行实验确定，并且 用野生型或突变型 preTCR 转导的胎儿肝脏造血祖细胞。目标 3 将在计算机中使用 分子动力学模拟以确定 TCR-pMHC 或 preTCR-pMHC 复合物的展开途径 它们对负载下界面的动态适应性的影响。从而我们将揭示原子论 机械传感机制。