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 是一种表面膜复合物，由非配体结合克隆型 DE 异二聚体组成。 与二聚体 CD3 信号亚基（CD3HJ、CD3HG 和 CD3]]）共价结合，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、动态的 pMHC 配体结合的力响应机械调节。此外，力会诱导不同的受体 与通电和非通电 TCRDE 胞外域状态相关的构象异构体。专注于如何 TCR配体占据信息转入细胞后，我们开始研究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 由动态铰链分段的二分螺旋。第一个跨膜 (TM) 螺旋包含两个带电的 残基 R251 和 K256，从相对侧向外指向，后者控制 TM 深度和所有 CD3 二聚体协会。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，当物理力负载施加到TCRDE-时，它们的处置可能会改变 pMHC 键。因此，这种 DETCR 解离机制意味着，而不是一组稳健的静态 TM 相互作用 段之间和段内存在动态结构转换。在这里我们将利用最新的 NMR 直接检测技术以及弛豫和化学交换（Rex、CEST 等）方面的进步 使用 EPR 直接观察膜模拟物中单个和组装组件的状态， 分布和结构。这些结构观察的生物学相关性应通过评估 将亚基限制为特定构象或消除亚基间相互作用的诱变。修改的 将评估组装成细胞表面 DETCR 复合物的亚基对 pMHC 刺激的影响 T 细胞中的 Ca2+ 通量、IL-2 产生和转录组。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 变体，其铰链 修改为锁定在弯曲或伸直配置时，应与 pTD 进行比较，其 结构，预计是一个直的连续螺旋，负责早期 DN3 的高基础水平激活 胸腺细胞，应测定。 TCRDTM K256 在翻译后修饰过程中的潜力 通过甲基转移酶或位于内部质膜小叶的其他酶进行机械转导 应进行评估。在目标 2 中，将单独使用 TCRE 和 CD3G 或与 其他 DETCR 复杂 TM 组件。对于 CD3G，TM 和 TM 的结构和动态变化 应定义 ITAM Tyr 磷酸化导致的细胞质尾部。鉴于大多数药用 化合物结合蛋白质的跨膜片段，本文收集的信息应具有实用性和实用性 对目标静息或激活构象具有根本意义。