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

摘要 DETCR是一种表面膜复合物，由配体结合克隆型DE异二聚体组成， 与二聚体CD 3信号传导亚基（CD 3 H、CD 3 HG和CD 3）共价缔合，其敏感性和 特异性对于保护性宿主免疫至关重要。DE TCR已被证明表现出一种不寻常的动态 和pMHC配体结合的力响应性机械调节。此外，力诱导不同的受体， 与能量化和非能量化TCRDE胞外域状态相关的构象。关注如何 当TCR配体占有率的信息被转导到细胞中时，我们已经开始研究TCRD 跨膜和胞质结构域（TMC）结构。出乎意料的是，我们观察到这个域形成了一个 由一个动态铰链分割的二分螺旋。第一个跨膜（TM）螺旋含有两个带电荷的 残基R251和K256，从相对侧指向外，后者控制TM深度，所有CD 3 二聚体缔合。机械转导介导的早期T细胞活化的触发由这些调节 当对TCRDE施加物理力负荷时，它们的位置可能会改变。 pMHC键。因此，这种DETCR解离机制意味着， 段之间和段内的动态结构转换的存在。在这里，我们将利用最新的 NMR直接检测技术和弛豫与化学交换的进展（雷克斯，CEST等）沿着 EPR直接观察膜模拟物中单个和组装组分的状态， 分布和结构。这些结构观察结果的生物学相关性应通过以下方式进行评估： 将亚基限制为确定的构象或消除亚基间相互作用的诱变。改性 将评估组装成细胞表面DETCR复合物的亚基对pMHC刺激的细胞增殖的影响。 T细胞中的Ca 2+通量、IL-2产生和转录组。在目标1中，我们将测量 单独的TCRDTMC或与TCRE、CD 3]和CD 3G TM区段的复合物。TCRDTMC变体的铰链 应沿着与pTD进行比较， 结构，预测为拉直的连续螺旋，负责早期DN 3中的高基础水平激活 胸腺细胞，应确定。TCRDTM K256在治疗期间进行后修饰的可能性 通过甲基转移酶或定位于内质膜小叶的其他酶的机械转导 将被评估。在目标2中，将使用单独的TCRE和CD 3G或与TCRE和CD 3G联合使用进行类似的研究。 其他DETCR复合物TM组分。关于CD 3G，TM和CD 3G中的结构和动态变化均与CD 3G的表达有关。 应定义ITAM Tyr磷酸化后的胞质尾。鉴于大多数药物 化合物结合蛋白质的跨膜区段，本文收集的信息应是实用的， 对靶向静止或活化构象具有根本意义。

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.