NMR-based dynamic assessment of TCR transmembrane conformational states linked to T cell function

摘要 DETCR是一种表膜复合体，由配体结合的克隆型DE异源二聚体组成。 与二聚体CD3信号亚单位(CD3HJ、CD3HG和CD3]]的共价结合，其敏感性和 特异性是保护性宿主免疫的关键。De TCR已被证明表现出一种不同寻常的、动态的 以及对pMHC配体结合的力响应性机械调节。此外，力诱导不同的感受器 与通电和非通电的TCRDE胞外结构域态相关的构象。专注于如何 关于TCR配体占位的信息被传递到细胞内，我们已经开始研究TCRD 跨膜和细胞质结构域(TMC)结构。出乎意料的是，我们观察到这个域形成了一个 由动态铰链分割的二分螺旋。第一个跨膜(TM)螺旋包含其两个带电的 残基，R251和K256，从对侧指向外，后者控制TM深度和所有CD3 二聚体缔合。机械转导介导的早期T细胞激活的触发受这些因素的调节 当对TCRDE施加物理力载荷时，残留物以及相反地，其处置可能会改变- PMHC键。因此，这种DETCR分离机制不是一组强健的TM交互作用，而是意味着 片断之间和片断内存在动态的结构转换。在这里，我们将利用最新的 核磁共振直接检测技术及松弛和化学交换(REX、CEST等)的研究进展沿着 用EPR直接观察膜模拟中单个和组装的组分的状态，他们的 分布和结构。应通过以下方式评估这些结构观测的生物学相关性 将亚基限制在特定构象或取消亚基间相互作用的突变作用。已修改 将评估组装成细胞表面DETCR复合体的亚基对pMHC刺激的影响 T细胞的CA2通量、IL-2的产生和转录。在目标1中，我们将测量 TCRDTMC单独或与TCRE、CD3]和CD3G TM片段复合。TCRDTMC变种，其铰链 被修改为锁定弯曲或拉直的配置应与pTD进行比较，其 结构，预测是一个伸直的连续螺旋，负责在DN3早期的高基础水平激活 胸腺细胞，应被测定。TCRDTM K256的翻译后修改潜力 由甲基转移酶或其他酶定位于内质膜小叶的机械转导 应予以评估。在AIM 2中，类似的研究将单独使用TCRE和CD3G进行，或与 其他DETCR Complex TM组件。在CD3G方面，TM和CD3G的结构性和动态变化 应定义ITAM Tyr磷酸化所导致的细胞质尾巴。考虑到大多数医学上的 化合物结合蛋白质的跨膜片段，这里收集的信息应该是实用的和 靶标静止或激活构象的基本意义。

ABSTRACT The DETCR is a surface membrane complex consisting of the ligand-binding clonotypic DE heterodimer in non- covalent association with the dimeric CD3 signaling subunits (CD3HJ, CD3HG and CD3]]) whose sensitivity and specificity are critical for protective host immunity. DE TCRs have been shown to manifest an unusual, dynamic and force-responsive mechanical regulation of pMHC ligand binding. Moreover, force induces different receptor conformers associated with energized and non-energized TCRDE ectodomain states. To focus on how information on TCR ligand occupancy is transduced into the cell, we have begun to study the TCRD transmembrane and cytoplasmic domain (TMC) structure. Unexpectedly, we observed that this domain forms a bipartite helix segmented by a dynamic hinge. The first transmembrane (TM) helix contains its two charged residues, R251 and K256, pointing outward from opposite sides with the latter controlling TM depth and all CD3 dimer associations. Mechanotransduction-mediated triggering of early T cell activation is modulated by these residues and, conversely, their disposition may be altered when physical force load is applied to the TCRDE- pMHC bond. Thus, rather than a robust static set of TM interactions, this DETCR dissociative mechanism implies the presence of dynamic structural conversions between and within segments. Here we shall exploit the latest advances in NMR direct detection techniques and relaxation and chemical exchange (Rex, CEST, etc.) along with EPR to directly observe states of individual and assembled components in membrane mimetics, their distributions and structures. The biological relevance of these structural observations shall be assessed through mutagenesis that restricts subunits to defined conformations or abrogates intersubunit interactions. Modified subunits that are assembled into cell surface DETCR complexes will be assessed for impact on pMHC-stimulated Ca2+ flux, IL-2 production and transcriptomes in T cells. In Aim 1, we shall measure the dynamic behavior of the TCRDTMC alone or in complex with TCRE, CD3] and CD3G TM segments. TCRDTMC variants whose hinge is modified to lock in a bent or straightened configuration shall be compared along with that of pTD whose structure, predicted to be a straightened continuous helix responsible for high basal level activation in early DN3 thymocytes, shall be determined. The potential for TCRDTM K256 to be post-translationally modified during mechanotransduction by a methytransferase or other enzyme localized to the inner plasma membrane leaflet shall be assessed. In Aim 2, similar studies will be performed with TCRE and CD3G alone or associated with other DETCR complex TM components. With respect to CD3G, structural and dynamic changes in both TM and cytoplasmic tail consequent to ITAM Tyr phosphorylation shall be defined. Given that the majority of medicinal compounds bind transmembrane segments of proteins, information gleaned herein shall be both of practical and fundamental significance to target resting or activated conformations.