NMR Methods to decipher the structural and dynamics aspects of TCR mechanobiology

破译 TCR 力学生物学结构和动力学方面的 NMR 方法

• 批准号: 10655350
• 负责人: GERHARD WAGNER
• 金额: $ 40.49万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-29 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10655350
• 关键词: 3-Dimensional; Address; Adopted; Affect; Aromatic Amino Acids; Binding; Biology; Carbon; Cell Lineage; Cell Nucleus; Cell physiology; Cell surface; Cells; Communities; Complex; Crowding; Cryoelectron Microscopy; Data; Detection; Development; Disease; Engineering; Escherichia coli; Extracellular Domain; Feedback; Goals; Histocompatibility; In Vitro; Isotope Labeling; Label; Ligand Binding; Ligands; Ligation; Link; Measurement; Measures; Mechanoreceptors; Methods; Modeling; Molecular Conformation; Molecular Weight; Motion; Mus; Mutation; Nature; Outcome; Pattern; Peptide Receptor; Peptides; Population; Procedures; Property; Proteins; Pyruvate; Receptor Activation; Recombinants; Relaxation; Resolution; Retinal blind spot; Scheme; Side; Signal Transduction; Site; Source; Stable Isotope Labeling; Structure; Surface; System; T cell response; T-Cell Receptor; T-Lymphocyte; Techniques; Technology; Tertiary Protein Structure; Time; Variant; Vertebral column; X-Ray Crystallography; adaptive immunity; design; detection method; dynamic system; experimental study; forging; in silico; in vivo; in vivo evaluation; insight; mechanotransduction; molecular dynamics; mutant; novel; peptide structure; programs; receptor; receptor function; restraint; simulation; single molecule; synergism; tool

ABSTRACT To protect us from myriad diseases, adaptive immunity requires T cell recognition of protein-derived peptides of foreign, mutant, or otherwise anomalous origin expressed on the surface of aberrant cells. Central to T cell function is the cell surface  T cell receptor (TCR), which recognizes these various peptides bound to major histocompatibility molecules (pMHC). While a great deal is known about the static conformations of TCR molecules and their pMHC ligands as well as the resultant ligation complexes, it is still unknown precisely what happens within the TCR-pMHC complex to generate the diverse signaling outcomes which drive T cell responses. Recent experiments have highlighted the dynamic nature of TCR-pMHC ligation and signaling, with a critical input of force necessary to generate T cell responses. This implies a dynamic system, poised to signal with the input of piconewton (pN) amounts of force to generate signaling-ready TCR proteins. We propose to develop NMR methods for studies of large extracellular domains involved in TCR mechanobiology, including the TCR and its developmental precursor, the preTCR, as well as the pMHC ligands that are at the limit of NMR observation. This includes protein domains that cannot be expressed in bacterial systems and thus cannot readily be perdeuterated, a current standard labeling strategy for addressing high molecular weight protein systems. Thus, in Aim 1 we employ direct 15N-detection methods, which do not require protein perdeuteration for backbone resonance assignment. Further, we will develop new 13C-detected experiments with TROSY enhancement that yield highly resolved spectra of aromatic side chains. To augment the state of the art NMR technology above, in Aim 2 we will establish new labeling schemes to aid in deciphering the structure and dynamics of preTCR, TCR and pMHC. To tackle the resonance assignment of these large proteins we will pursue the use of “mixed pyruvate” as a carbon source to label proteins to obtain residue specific patterns. In tandem with Aim 1 we will produce isolated 13C and 13C-19F labeled aromatic amino acids through chemoenzymatic synthesis. Since some protein components of the TCR systems we intend to study cannot be recombinantly expressed in E.Coli, we will pursue expression in eukaryotic systems, where complete deuteration is a challenge. The labeling technology developed here will be transferred to Core B. In Aim 3, we will use the NMR technologies and labeling methods, described above, to obtain information about structure and dynamics of TCR-pMHC complexes. In particular relaxation dispersion and CEST, we will leverage 19F nuclei as probe to access dynamics in the low microsecond time scale. The extracted dynamics information will be utilized with the MD Core C to observe dynamics in silico and link the dynamics and conformational states measured in NMR to those that are observed under force. Functional impact of the atomistic findings will be assessed in Projects 1 and 2 through mutational iterations. The NMR methods forged here will illuminate the proverbial blind spot in mechanistic understanding of TCR activation, already previewed via exciting preliminary hidden state data in Aim 3.

抽象的 为了保护我们免受无数疾病的侵害，适应性免疫学需要T细胞识别蛋白质衍生的肽 在异常细胞表面表达的异物，突变或其他异常起源。中心的T细胞 功能是细胞表面T细胞接收器（TCR），它识别与主要结合的各种肽 组织相容性分子（PMHC）。虽然对TCR的静态构型有很多了解 分子及其PMHC配体以及由此产生的连接复合物，仍然是未知的 发生在TCR-PMHC复合物中，以生成驱动T细胞的潜水员信号传导结果 回答。最近的实验强调了TCR-PMHC连接和信号传导的动态性质 产生T细胞反应所需的力的关键输入。这意味着动态系统中毒以发出信号 随着Piconnewton（PN）的输入，要产生信号就绪的TCR蛋白的力量。我们建议 开发用于研究参与TCR机械生物学的大型细胞外域的研究，包括 TCR及其发育的前体Pretcr以及NMR极限的PMHC配体 观察。这包括在细菌系统中无法表达的蛋白质结构域，因此不能 容易被渗透，这是一种针对高分子量蛋白的当前标准标记策略 系统。在AIM 1中，我们采用了直接的15N检测方法，不需要蛋白质perdeuteration 用于骨干共振分配。此外，我们将开发新的13C检测实验 增强芳香侧链的高度分辨光谱的增强。增强最先进的NMR 上面的技术，在AIM 2中，我们将建立新的标签方案，以帮助破译结构和 Pretcr，TCR和PMHC的动力学。为了解决这些大蛋白的共振分配，我们将追求 使用“混合丙酮酸”作为碳源来标记蛋白质以获得保留特定模式。串联 使用AIM 1，我们将通过化学酶产生孤立的13c和13c-19f标记为芳族氨基酸的标记 合成。由于我们打算研究的TCR系统的某些蛋白质成分不能重组 在E.Coli中表达，我们将在真核系统中实践表达，在真核系统中，完全申请是一个挑战。 这里开发的标签技术将转移到核心B。在AIM 3中，我们将使用NMR Technologies 上面描述的标记方法，以获取有关TCR-PMHC结构和动力学的信息 复合物。特别是放松分散和cest，我们将利用19F核作为探测器来访问动态 在低微秒的时间尺度上。提取的动态信息将与MD Core C一起使用 观察计算机中的动力学，并将NMR中测量的动力学和构象状态连接到那些 在力下观察到。原子观察结果的功能影响将在项目1和2到2中评估 突变迭代。这里忘记的NMR方法将阐明机械的谚语盲点 了解TCR激活，已经通过AIM 3中的令人兴奋的初步隐藏状态数据预览。