Structural Analysis of the TCR-CD3 Receptor Complex

TCR-CD3 受体复合物的结构分析

基本信息

批准号：
8512173
负责人：
Roy A Mariuzza
金额：
$ 22.8万
依托单位：
UNIV OF MARYLAND, COLLEGE PARK
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-01-15 至 2014-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8512173
关键词：
Affinity Architecture Binding Biological Testing CD3 Antigens Charge Chemicals Complex Coupled Crystallization Data Docking Elements Epitopes Extracellular Domain Flow Cytometry HLA-DR Antigens Human Immune response Immunology In Vitro Invaded Label Libraries Ligation MHC Class II Genes MHC binding peptide Maps Mature T-Lymphocyte Mediating Methods Molecular Mutagenesis NMR Spectroscopy Peptide T Peptide/MHC Complex Peptides Problem Solving Protein Engineering Relative (related person)Relaxation Resolution Roentgen Rays Signal Pathway Signal Transduction Site Solutions Sorting - Cell Movement Structure Surface T-Cell Activation T-Cell Receptor T-Lymphocyte Variant X-Ray Crystallography Yeasts base design dimer directed evolution extracellular immunoglobulin structure mutant novel strategies pathogen public health relevance receptor research study restraint three dimensional structure

项目摘要

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.

描述（由申请人提供）：T细胞受体（TCR）-CD3复合物由与不变CD3二聚体CD3、CD3 - 和CD3 - 非共价结合的多样化TCR异二聚体组成。 TCR 介导肽-MHC (pMHC) 识别，而相关的 CD3 分子将激活信号转导至 T 细胞。尽管人们对下游 T 细胞信号传导途径了解很多，但 pMHC 参与 TCR 启动信号传导的机制仍然是个谜。解决这个问题的关键是建立TCR-CD3复合物的空间组织。尽管已知 TCR 和 CD3 的胞外区域会相互作用，但之前所有通过 X 射线晶体学研究 TCR-CD3 受体的尝试都因溶液中 TCR-CD3 相互作用的亲和力非常低而受挫，这阻碍了稳定结合体的形成。用于结构分析的组件。为了克服这一障碍，该项目采用了一种新颖的策略，将 NMR 光谱法绘制溶液中的对接位点，并通过酵母表面展示 (YSD) 进行体外定向进化，以稳定 TCR-CD3 复合物的结晶。我们的目标是： 1. 确定野生型 TCR-CD3 复合物的 NMR 溶液结构。我们将采用化学位移扰动和顺磁弛豫增强 (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 NMR 谱支持在溶液中定义野生型 TCR-CD3 界面的可行性。 2.TCR和CD3之间的结合相互作用的亲和力成熟。 TCR 的大型突变文库（107-108 个独立克隆）将在酵母上展示，并通过流式细胞术用 CD3 或 CD3 四聚体进行分选，以分离高亲和力的 TCR 变体。相反，CD3和CD3的突变体文库将通过TCR四聚体进行亲和选择。为了证明这种方法的强大功能，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 的第一个晶体结构三元络合物。 3.亲和力成熟的TCR-CD3复合物的组装和结构分析。我们将追求高亲和力 TCR-CD3 复合物的结晶。二元和三元复合物都将成为目标：TCR-CD3、TCR-CD3 和 TCR-CD3 -CD3 。如果无法获得晶体，我们将使用 NMR 来确定亲和力成熟的 TCR-CD3 复合物的结构。通过 X 射线或 NMR 确定的这些复合物的结构信息将定义多亚基 TCR-CD3 受体的整体空间组织。