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传染病和肿瘤的侵袭 非常具体的举止。AbT淋巴细胞识别的核心是自我与非自我歧视， 克隆性细胞表面T细胞受体(TCR)所赋予的功能。数以百万计的不同TCR在 哺乳动物胸腺创造了一种经过改进的曲目，以消除先前不需要的自身反应特异性。 输出到外周淋巴间隔室。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，在大范围的PN-NN力上产生拉应力和剪应力。直接 已经提供了TCR作为机械传感器的证据，解释了其精致的特异性和 在没有物理负载的情况下，对配体的敏感度和亲和力都很低。最近，我们展示了基于力的 AbTCR的歧视延伸到它的发育前体，前TCR，一种PTA-b异源二聚体。此外， 观察到在力作用下加强配体结合所需的可逆结构重排。 TCR和前TCR。在这个方案中，我们将结合单分子(SM)和单细胞(SMSC) 方法利用光学陷阱、结构-功能突变分析、重组蛋白表达和 分子动力学模拟探查TCR和前TCR与pMHC的复合物在负荷下提供清晰的 对机械传感的结构基础的理解。我们的假设是，绑定是为 卸载TCR，但当强制加载、伸展或稳定时，TCR进入“结合读取状态” 寿命延长的键，以便于信号传递，或者，替代地，快速从不相关的配体中释放。 在目标1中，我们将阐明临界TCRa和b亚基变量(V)和恒定(C)结构域结构 包括CB FG环在内的元件参与机械调节加载的TCR-pMHC的强度 互动。拓扑稳定的结构以及不稳定的突变将被评估其 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衡量。我们将利用新开发的单分子和单链系链分析 用于直接比较分离的TCRab-pMHC复合体和ab T细胞系上负载的TCR的强度。目标 2将研究前TCR的机械调谐以及前TCR PTa-b结构与TCRab的不同之处。 在目标1和目标2中，我们将确定导致键加强和释放的构象转变 T细胞活化和发育的关键途径。前TCR基因突变对胸腺细胞的影响 将使用胸腺基质细胞系OP9-DL4和 转导野生型或突变型前TCR的胎肝造血祖细胞。AIM 3将在硅胶领域应用 分子动力学模拟确定TCR-pMHC或前TCR-pMHC复合体的展开途径 它们对界面在载荷作用下的动态适应性有影响。我们将由此揭示原子论 机械传感的机械装置。

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.