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

基于 NMR 的 TCR 跨膜构象状态动态评估与 T 细胞功能相关

• 批准号: 9789827
• 负责人: ELLIS L REINHERZ
• 金额: $ 44.13万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-24 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9789827
• 关键词: Adaptive Immune System; Address; Amino Acids; Antigen-Presenting Cells; Autoimmune Diseases; Autoimmunity; Behavior; Binding; Biological; CD3 Antigens; CD3E gene; Cell membrane; Cell physiology; Cell surface; Cells; Charge; Chemicals; Complex; Cytoplasm; Cytoplasmic Tail; Detection; Disulfides; Environment; Enzymes; Genes; Glean; ITAM; Immunity; Immunotherapy; Individual; Infection; Interleukin-2; Knowledge; Ligand Binding; Ligands; Ligation; Link; Major Histocompatibility Complex; Malignant - descriptor; Malignant Neoplasms; Mammals; Mass Spectrum Analysis; Measures; Mechanics; Mediating; Membrane; Modeling; Molecular Conformation; Mutagenesis; Mutate; Peptide/MHC Complex; Peptides; Pharmaceutical Preparations; Phosphorylation; Post-Translational Protein Processing; Premalignant; Production; Protein Conformation; Protein Dynamics; Proteins; Regulation; Relaxation; Rest; Role; Sensitivity and Specificity; Side; Signal Transduction; Stimulus; Structure; Surface; Surface Antigens; System; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; TRD@ gene cluster; Techniques; Technology; Transmembrane Domain; Variant; alpha-beta T-Cell Receptor; antigen-specific T cells; base; conformer; design; dimer; functional outcomes; insight; irradiation; mechanical force; mechanotransduction; membrane assembly; metaplastic cell transformation; mimetics; optical traps; pathogen; receptor; receptor function; receptor structure function; small molecule; thymocyte; transcriptome

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.

抽象的 DETCR是一种表面膜复合 与二聚体CD3信号亚基（CD3HJ，CD3HG和CD3]]）的共价关联，其灵敏度和 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 PMHC配体结合的力反应性机械调节。此外，力诱导不同的受体 与通电和非功效的TCRDE型域状态相关的构象体。专注于如何 有关TCR配体占用率的信息已转导到细胞中，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，我们观察到该域形成了 两分螺旋由动态铰链分割。第一个跨膜（TM）螺旋包含其两个充电 残留物R251和K256，从相对方向向外指向后者，后者控制TM深度和所有CD3 二聚体关联。机械转导介导的早期T细胞激活的触发是由这些调节的 残留物，相反，当将物理力负荷施加到TCRDE-时，它们的处置可能会改变 PMHC债券。因此，这种Detcr解离机制不是强大的静态TM相互作用集，而是暗示 段之间和内部之间的动态结构转换的存在。在这里，我们将利用最新 NMR直接检测技术的进步，放松和化学交换（Rex，Cest等） 用EPR直接观察膜模拟物中个体和组装成分的状态，它们 分布和结构。这些结构观察的生物学相关性应通过 将亚基限制为定义构象或消除了亚基间相互作用的诱变。修改的 组装到细胞表面提取复合物中的亚基将被评估为对PMHC刺激的影响 T细胞中的Ca2+通量，IL-2产生和转录组。在AIM 1中，我们将衡量的动态行为 单独使用TCRDTMC或与TCRE，CD3]和CD3G TM段中的复合物。 TCRDTMC变体的铰链 修改以锁定弯曲或拉直的配置，应与PTD的配置进行比较 结构，预计将是一个拉直的连续螺旋，负责在早期DN3中高基础水平激活 胸腺细胞应确定。 TCRDTM K256的潜力在后翻译后进行了修改 通过甲基转移酶或其他位于质膜内部膜叶片的甲基转移酶或其他酶的机械转导 应评估。在AIM 2中，仅使用TCRE和CD3G进行类似的研究或与 其他DETCR复合物TM组件。关于CD3G，TM和TM的结构和动态变化 应定义造成ITAM磷酸化的细胞质尾巴。鉴于大多数药用 化合物结合蛋白质的跨膜片段，本文收集的信息既是实用的，又应 目标静息或活化构象的基本意义。