An integrative structural biology approach to the study of T cell signaling -Equipment Supplement

摘要 我建议开发和应用创新的杂交结构生物学工具来研究 通过T细胞受体(TCR)复合体传递信号的分子机制。这是一个 基本适应性免疫途径，细胞毒CD8+T细胞通过该途径检测 体内病毒的存在和肿瘤的发展。免疫功能是通过 持续监测抗原提呈细胞中展示的短肽 细胞表面主要组织相容性复合体(MHC)的分子。启蒙的关键 是一个由克隆型TCR组成的多亚单位膜蛋白组件 识别多肽-MHC的异源二聚体，以及不变的CD3共同受体 六聚体，通过胞浆传递来自细胞表面的激活信号 薄膜。除了基础科学的优点外，在原子的基础上描述这一过程 细节具有重要的临床意义，正如大量的 信号成分及其相互作用的失调所致的免疫缺陷 互动。 尽管有大量的功能和结构研究，但阐明了细胞的3D结构 通过常规方法，信令复合体作为一个整体仍然具有极大的挑战性，因为 它的大小和动态复杂性。因此，TCR和CD3亚基之间的相互作用 信号传递所需的关键构象变化仍未完全确定。 考虑到这些瓶颈，我开发了一种新的方法，将来自 互补的方法，如核磁共振、SANS和CryoEM，以及计算 使用Rosetta程序进行建模，以解决这种具有挑战性的复合体的结构。 在这里，我建议应用这个强大的综合方法来阐明T细胞受体 复杂结构，并研究其在非活动和活动之间的动态转变 构象。我的近期目标是确定TCR/CD3相互作用的分子基础 并描述了抗原上受体复合体排列的关键结构变化 承认。作为一个长期目标，我计划使用一种结构指导的方法来设计小说T 具有定制特异性和信号特性的细胞受体，将用于新兴 免疫疗法的应用。

Summary I propose to develop and apply innovative hybrid structural biology tools to investigate the molecular mechanism of signaling through the T cell receptor (TCR) complex. This is a fundamental adaptive immunity pathway through which cytotoxic CD8+ T cells can detect the presence of viruses and developing tumors in the body. Immune function is achieved through the continuous surveillance of antigen-presenting cells for short peptides displayed within the molecules of the Major Histocompatibility Complex (MHC) on the cell surface. Key to the initiation of signaling, is a multi-subunit membrane protein assembly consisting of a clonotypic TCR heterodimer that recognizes the peptide-MHC, together with the invariant CD3 co-receptor hexamer that relays an activation signal from the cell surface intracellularly, through the plasma membrane. Besides the fundamental basic science merit, characterizing this process at atomic detail has important clinical implications, as is suggested by the large number of immunodeficiencies resulting from dysregulation of the signaling components and their interactions. Despite a large number of functional and structural studies, elucidating the 3D structure of the signaling complex as a whole remains extremely challenging by conventional methods, due to its size and dynamic complexity. As a result, the interactions between the TCR and CD3 subunits and the crucial conformational changes needed for signaling remain incompletely characterized. With these bottlenecks in mind, I have developed a new methodology combining datasets from complementary approaches, such as NMR, SANS and cryoEM, together with computational modeling using the program Rosetta to solve the structures of such challenging complexes. Here, I propose to apply this powerful integrative approach to elucidate the T cell receptor complex structure, and to study its dynamic transitions between inactive and active conformations. My immediate goal is to determine the molecular basis of TCR/CD3 interactions and to characterize the crucial structural changes in receptor complex arrangement upon antigen recognition. As a long-term goal, I plan to use a structure-guided approach to engineer novel T cell receptors with custom specificities and signaling properties, to be used in emerging immunotherapy applications.