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: 7155516
• 负责人: Kenan Christopher GARCIA
• 金额: $ 28.27万
• 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/7155516
• 关键词: Affinity; Allogenic; Architecture; Award; Binding; CD3 Antigens; Cell surface; Cells; Chemicals; Class; Code; Complex; Crystallization; Crystallography; Detergents; Docking; Electron Microscopy; Engineering; Epitopes; Foundations; Haplotypes; Helix (Snails); 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; Pliability; 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; ear helix; programs; reconstitution; structural biology

DESCRIPTION (provided by applicant): Structural biology has elucidated several general principles for how the a¿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).

描述(申请人提供)：结构生物学已经阐明了α？TCR如何与多肽-MHC配体相互作用的几个一般原则，但与TCR的识别和激活有关的几个基本问题仍然没有答案。首先，我们还不了解MHC TCR偏向的结构基础。在松散收敛的TCR/pMHC对角对接方向或迄今在不同复合体中看到的“足迹”之下，是否有识别代码？我们最近测定的TCR/pMHC络合物结构表明，可能存在有限的对接基序集，可以通过对一系列相关络合物的结构研究来阐明这些基序。这些对接拓扑可能是连接编码的TCR CDR3与多肽的相互作用以及生殖线编码的可变区与MHC螺旋之间复杂相互作用的结果。因此，我们是用于结构研究的工程分子，将分离出这些成分的每一种贡献。第二，TCR交叉反应的范围是什么，TCR CDR构象灵活性在多大程度上起到了扩大T细胞谱系的作用？我们以前的研究表明，T细胞识别并不是广泛混杂的，但有一种流行的观点认为，CDR3诱导的FIT是一种扩大TCR识别的pMHC谱系的机制。为了更好地了解构象动力学在TCR/pMHC相互作用中的作用，我们使用核磁共振(NMR)确定了TCR和pMHC结构以及溶液相互作用。最后，我们目前不知道TCR和CDS在TCR-CD3复合体中是如何相互作用的。TCR对pMHC的识别在结构上是如何传递到相关的CD3亚基以进行后续信号传递的？我们建议重组和纯化全长、膜结合的TCR-CD3复合体，用于生物物理成像研究。总而言之，在本奖项的前一期，我们开发了强大的TCR和多肽-MHC表达方法，现在使我们能够使用X射线结晶学、核磁共振、多肽库、噬菌体展示和电子显微镜(EM)对一组TCR/pMHC相互作用进行多学科分析。