Molecular Principles of TCR Recognition and Activation by peptide-MHC

描述（由申请人提供）：结构生物学已经阐明了a¿TCR如何与肽- mhc配体相互作用的几个一般原理，但是关于TCR的识别和激活的几个基本问题仍然没有答案。首先，我们还不了解MHC的TCR偏倚的结构基础。在松散收敛的TCR/pMHC对角对接方向或迄今为止在不同复合物中看到的“足迹”背后是否存在识别代码？我们最近确定的TCR/pMHC复合物结构表明，可能存在有限的对接基序，可以通过一系列相关复合物的结构研究来阐明。这些对接拓扑结构可能是连接编码的TCR CDR3与肽的相互作用以及种系编码的可变区与MHC螺旋的相互作用之间复杂相互作用的结果。因此，我们正在为结构研究设计分子，以分离出这些成分的贡献。其次，TCR交叉反应性的范围是什么，TCR CDR构象灵活性在多大程度上在扩大T细胞库中发挥作用？我们之前的研究表明，T细胞识别并不是广泛混杂的，然而有一种普遍的观点认为，CDR3诱导的契合是一种机制，可以扩大TCR识别的pMHC的范围。为了更好地理解构象动力学在TCR/pMHC相互作用中的作用，我们正在使用核磁共振波谱（NMR）测定TCR和pMHC结构以及溶液相互作用。最后，我们目前还不知道TCR和CDS如何在TCR- cd3复合体中相互作用。TCR对pMHC的识别如何在结构上传递给相关的CD3亚基以进行后续信号传导？我们建议重建和纯化一种全长、膜结合TCR-CD3复合物的重组形式，用于生物物理成像研究。总之，在该奖项的前一个任期内，我们开发了强大的TCR和肽- mhc表达方法，现在使我们能够使用x射线晶体学，核磁共振，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。

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).