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

摘要 为了保护我们免受无数疾病的侵袭，获得性免疫需要T细胞识别蛋白质衍生的多肽 异源的在异常细胞表面表达的外来的、突变的或其他异常起源的中央到T细胞 功能是细胞表面的T细胞受体(TcR)，它识别这些与主要结合的各种多肽 组织相容性分子(PMHC)。虽然人们对TCR的静态构象有很多了解 分子及其pMHC配体以及由此产生的连接络合物，目前仍不清楚到底是什么 发生在TCR-pMHC复合体内，产生不同的信号结果，驱动T细胞 回应。最近的实验强调了TCR-pMHC连接和信号传递的动态性质， 产生T细胞反应所必需的力量的关键输入。这意味着一个动态的系统，随时准备发出信号 随着微微牛顿(PN)的输入，产生大量的力量来产生信号准备好的TCR蛋白。我们建议 发展核磁共振方法研究与TCR机械生物学有关的大胞外结构域，包括 TCR及其发育前体，前TCR，以及处于核磁共振极限的pMHC配体 观察。这包括不能在细菌系统中表达的蛋白质结构域，因此不能 易过氢，目前用于解决高分子量蛋白质的标准标记策略 系统。因此，在目标1中，我们使用了直接的15N检测方法，它不需要蛋白质过氢 用于主干共振分配。此外，我们将用TROSY开发新的13C检测实验 得到芳香族侧链的高分辨光谱的增强。增强最先进的核磁共振技术 技术，在目标2中，我们将建立新的标签方案来帮助破译结构和 前TCR、TCR和pMHC的动态变化。为了解决这些大蛋白质的共振分配问题，我们将继续 利用“混合丙酮酸”作为碳源标记蛋白质，获得残基特定模式。一起行动 对于目标1，我们将通过化学酶法生产分离的13C和13C-19F标记芳香族氨基酸 综合。由于我们打算研究的TCR系统的某些蛋白质组分不能重组 在E.Coli中表达，我们将寻求在真核系统中表达，在那里完全重离子是一个挑战。 这里开发的标记技术将转移到核心B。在目标3中，我们将使用核磁共振技术 以及上述标记方法，以获得关于TCR-pMHC的结构和动力学的信息 复合体。特别是弛豫色散和CEST，我们将利用19F核作为探针来访问动力学 在低微秒的时间尺度上。提取的动力学信息将与MD Core C一起使用以 观察硅胶中的动力学，并将核磁共振测量的动力学和构象状态与 在武力下观察到的。原子学发现的功能影响将在项目1和2至 突变迭代。这里建立的核磁共振方法将照亮机械学中众所周知的盲点。 对TCR激活的理解，已经通过AIM 3中激动人心的初步隐藏状态数据进行了预览。

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.