Structural Analysis of the TCR-CD3 Receptor Complex

描述（由申请人提供）：T细胞受体(TCR)-CD3复合物由不同的TCR异源二聚体组成，与不变的CD3二聚体CD3、CD3和CD3非共价结合。TCR介导肽- mhc （pMHC）识别，而相关的CD3分子将激活信号转导到T细胞。尽管我们对下游T细胞信号通路了解甚多，但pMHC参与TCR启动信号通路的机制仍然是一个谜。解决这一问题的关键是建立TCR-CD3复合物的空间组织。虽然已知TCR和CD3的细胞外区域相互作用，但之前通过x射线晶体学研究TCR-CD3受体的所有尝试都因TCR-CD3在溶液中相互作用的亲和力非常低而受挫，这妨碍了形成稳定的组装以进行结构分析。为了克服这一障碍，本项目采用了一种新的策略，将核磁共振光谱与酵母表面显示（YSD）体外定向进化相结合，绘制溶液中的对接位点，以稳定TCR-CD3配合物的结晶。我们的目标是：1；野生型TCR-CD3络合物的核磁共振溶液结构测定。我们将采用化学位移微扰和顺磁弛豫增强（PRE）来确定CD3和TCR之间的结合表位。这些数据将用于确定溶液中TCR-CD3复合物的结构，并指导YSD实验的设计。初步的核磁共振谱支持在溶液中定义野生型TCR-CD3界面的可行性。2. TCR与CD3结合相互作用的亲和成熟。大量的TCR突变文库（107-108个独立克隆）将在酵母上展示，并用CD3或CD3四聚体流式细胞术进行分类，以分离出高亲和力的TCR变体。相反，CD3和CD3的突变文库将与TCR四聚体进行亲和选择。为了证明这种方法的力量，我们最近使用YSD来稳定人类CD4和MHC II类（KD > 400 M）之间极弱的相互作用，这使我们能够确定TCR- pMHC-CD4三元配合物的第一个晶体结构。3. 亲和成熟TCR-CD3配合物的组装与结构分析。我们将追求高亲和力TCR-CD3复合物的结晶。二元和三元配合物将被靶向：TCR-CD3、TCR-CD3和TCR-CD3 -CD3。如果不能获得晶体，我们将使用核磁共振来确定亲和成熟的TCR-CD3复合物的结构。通过x射线或核磁共振确定这些复合物的结构信息，将确定多亚基TCR-CD3受体的整体空间组织。

DESCRIPTION (provided by applicant): The T cell receptor (TCR)-CD3 complex is composed of a diverse TCR heterodimer non-covalently associated with the invariant CD3 dimers CD3, CD3 , and CD3 . The TCR mediates peptide-MHC (pMHC) recognition, while the associated CD3 molecules transduce activation signals to the T cell. Whereas much is known about downstream T cell signaling pathways, the mechanisms whereby TCR engagement by pMHC initiates signaling remain a mystery. A key to solving this problem is establishing the spatial organization of the TCR-CD3 complex. Although the extracellular regions of TCR and CD3 are known to interact, all previous attempts to study the TCR-CD3 receptor by X-ray crystallography have been frustrated by the very low affinity of TCR-CD3 interactions in solution, which precludes the formation of stable assemblies for structural analysis. To overcome this obstacle, this project employs a novel strategy combining NMR spectroscopy to map docking sites in solution with in vitro directed evolution by yeast surface display (YSD) to stabilize TCR-CD3 complexes for crystallization. Our objectives are: 1. Determination of the NMR solution structure for the wild-type TCR-CD3 complex. We will employ chemical shift perturbation and paramagnetic relaxation enhancement (PRE) to determine binding epitopes between CD3 and TCR. These data will be used to determine a structure for the TCR-CD3 complex in solution and to guide the design of YSD experiments. Preliminary NMR spectra support the feasibility of defining the wild-type TCR-CD3 interface in solution. 2. Affinity maturation of binding interactions between TCR and CD3. Large mutant libraries of TCR (107-108 independent clones) will be displayed on yeast and sorted by flow cytometry with CD3 or CD3 tetramers to isolate high-affinity TCR variants. Conversely, mutant libraries of CD3 and CD3 will be affinity-selected with TCR tetramers. As a demonstration of the power of this approach, we recently employed YSD to stabilize the extremely weak interaction between human CD4 and MHC class II (KD > 400 M), which enabled us to determine the first crystal structure of a TCR- pMHC-CD4 ternary complex. 3. Assembly and structural analysis of affinity-matured TCR-CD3 complexes. We will pursue crystallization of high-affinity TCR-CD3 complexes. Both binary and ternary complexes will be targeted: TCR-CD3, TCR-CD3 and TCR-CD3 -CD3 . If crystals cannot be obtained, we will use NMR to determine the structure of the affinity-matured TCR-CD3 complex. Structural information on these complexes, determined either by X-ray or NMR, will define the overall spatial organization of the multisubunit TCR-CD3 receptor.