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

摘要 哺乳动物的适应性免疫系统保护其宿主免受感染性疾病以及肿瘤的侵害， 非常具体的方式。ab T淋巴细胞识别的核心是自我与非自我识别， 克隆细胞表面T细胞受体（TCR）赋予的功能。细胞中表达的数百万不同TCR 哺乳动物胸腺产生了一个库，该库被精炼以消除不需要的自身反应特异性， 输出到外周淋巴区室。一旦到达那里，成熟的abT细胞就会扫描它们的环境 在免疫监视期间，在宽范围的pN-nN力上产生拉伸和剪切应力。直接 已经提供了TCR作为机械传感器的证据，解释了其精确的特异性， 在没有物理负载的情况下，对配体的灵敏度仍然低亲和力。最近，我们发现基于力的 abTCR辨别延伸至其发育前体preTCR，一种pTa-b异二聚体。此外，委员会认为， 观察到在力作用下加强配体结合所必需的可逆结构重排， TCR和前TCR。在这个提议中，我们将联合收割机单分子（SM）和单细胞（SMSC）结合起来。 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，以探测负载下TCR和preTCR与pMHC的复合物， 理解机械传感的结构基础。我们的假设是，结合是“门控”的， 但是当力加载时TCR进入“结合阅读状态”，延伸并且或者稳定 该键具有延长的寿命以促进信号传导，或者可替代地，从不相关的配体快速释放。 在目的1中，我们将阐明关键的TCR a和B亚基可变（V）和恒定（C）结构域， 元件，包括机械调节负载的TCR-pMHC的强度的Cb FG环 交互.将评估拓扑稳定结构以及去稳定突变的 改变pMHC键寿命和构象变化以及影响ab T细胞活化的能力， 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系链检测 用于直接比较负载的TCR在分离的TCRab-pMHC复合物和ab T细胞系上的强度。目的 2将检查preTCR的机械调谐以及preTCR pTa-b结构与TCRab的结构如何不同。 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细胞活化和发育至关重要的途径。前TCR基因突变对胸腺细胞的影响 将使用胸腺基质细胞系OP 9-DL 4和 用野生型或突变型前TCR转导的胎肝造血祖细胞。Aim 3将采用计算机模拟 分子动力学模拟以鉴定TCR-pMHC或preTCR-pMHC复合物的解折叠途径， 它们对界面在负载下的动态适应性的影响。我们将由此揭示原子论 机械感应的机制。

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.