Molecular Principles of TCR Recognition and Activation by peptide-MHC

肽-MHC识别和激活TCR的分子原理

• 批准号: 7532779
• 负责人: Kenan Christopher GARCIA
• 金额: $ 27.56万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-12-15 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7532779
• 关键词: Affinity; Allogenic; Architecture; Award; Binding; CD3 Antigens; Cell surface; Cells; Chemicals; Code; Complex; Crystallization; Crystallography; Detergents; Docking; Electron Microscopy; Engineering; Epitopes; Foundations; Haplotypes; Image; Immunoglobulin Variable Region; Insecta; Length; Ligands; Maps; Membrane; Methods; Micelles; Molecular; NMR Spectroscopy; Peptide Library; Peptide Phage Display Library; Peptide/MHC Complex; Peptides; Play; Population; Receptor-CD3 Complex, Antigen, T-Cell; Recombinants; Research Personnel; Resolution; Role; Series; Signal Transduction; Solutions; Structure; Surface; T-Lymphocyte; TCR Activation; Techniques; Variant; base; cross reactivity; flexibility; programs; reconstitution; structural biology

Structural biology has elucidated several general principles for how the ap TCR interacts with peptide- MHC ligands, but several fundamental questions, pertaining to both the recognition and activation of the TCR, remain unanswered. First, we do not yet understand the structural basis of TCR bias for MHC. Is there a recognition code underlying the loosely convergent TCR/pMHC diagonal docking orientations, or "footprints" so far seen in the different complexes ? A recent TCR/pMHC complex structure we determined suggests there may be limited sets of docking motifs that could be elucidated through structural studies of a series of related complexes. These docking topologies are likely the result of a complex interplay between junctionally-encoded TCR CDR3 interactions with peptide, and the germline- encoded Variable region interactions with the MHC helices. Therefore, we are engineering molecules for structural studies that will isolate the contributions of each of these components. Second, what is the scope of TCR cross-reactivity, and to what extent does TCR CDR conformational flexibility play a role in expanding the T cell repertoire ? Our previous studies suggest that T cell recognition is not broadly promiscuous, and yet there is a prevailing notion that CDR3 induced fit is a mechanism to expand the repertoire of pMHC recognized by the TCR. In order to better understand the role of conformational dynamics in TCR/pMHC interactions, we are determining TCR and pMHC structures, and solution interactions, using Nuclear Magnetic Resonance spectroscopy (NMR). Finally, we currently do not know how TCR and CDS interact in the TCR-CD3 complex. How is recognition of pMHC by the TCR structurally communicated to the associated CD3 subunits for subsequent signaling ? We propose to reconstitute and purify a recombinant form of a full-length, membrane-bound TCR-CD3 complex for biophysical imaging studies. In summary, during the previous term of this award we developed robust methods for expression of TCR, and peptide-MHC that now enable us to carry out a multi-disciplinary analysis of a focused set of TCR/pMHC interactions using x-ray crystallography, NMR, peptide libraries, phage display, and electron microscopy (EM).

结构生物学已经阐明了 ap TCR 如何与肽相互作用的几个一般原则 MHC 配体，但有几个基本问​​题，与 MHC 配体的识别和激活有关 TCR，仍然没有答案。首先，我们还不了解 MHC 的 TCR 偏差的结构基础。是 松散收敛的 TCR/pMHC 对角对接背后有一个识别代码 迄今为止在不同综合体中看到的方向或“足迹”？最近的 TCR/pMHC 复合体 我们确定的结构表明可能存在有限的对接基序可以被阐明 通过一系列相关复合体的结构研究。这些对接拓扑可能是以下结果 连接编码的 TCR CDR3 与肽的相互作用以及种系之间的复杂相互作用 编码可变区与 MHC 螺旋的相互作用。因此，我们正在工程分子 结构研究将分离出每个组成部分的贡献。二、范围是什么 TCR 交叉反应性的影响，以及 TCR CDR 构象灵活性在多大程度上发挥作用 扩大 T 细胞库 ?我们之前的研究表明 T 细胞识别并不广泛 混杂，但有一个普遍的观点认为 CDR3 诱导的拟合是一种扩大 TCR 认可的 pMHC 库。为了更好地理解构象的作用 TCR/pMHC 相互作用的动力学，我们正在确定 TCR 和 pMHC 结构以及解决方案 相互作用，使用核磁共振波谱（NMR）。最后，目前我们还不知道 TCR 和 CDS 如何在 TCR-CD3 复合物中相互作用。 TCR 如何识别 pMHC 在结构上与相关的 CD3 亚基进行后续信号传导 ?我们建议 重建并纯化全长膜结合 TCR-CD3 复合物的重组形式 生物物理成像研究。总之，在该奖项的上一任期内，我们开发了稳健的 TCR 和肽-MHC 的表达方法现在使我们能够开展多学科研究 使用 X 射线晶体学、NMR、肽库分析一组集中的 TCR/pMHC 相互作用， 噬菌体展示和电子显微镜（EM）。