Structure and Activation of a Multiprotein Signaling Complex

多蛋白信号复合物的结构和激活

基本信息

批准号：
9448073
负责人：
Roy A Mariuzza
金额：
$ 71.1万
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：
2017
资助国家：
美国
起止时间：
2017-09-25 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9448073
关键词：
Address Affinity Antigen-Presenting Cells Antiviral Agents Architecture Autoimmune Process Binding Binding Proteins Biological Biological Assay Biophysical Process CD3 Antigens CRISPR/Cas technology Cell Line Cell Surface Receptors Cell membrane Cell physiology Cells Cellular Assay Chemicals Complex Crystallization Crystallography Data Dimerization Directed Molecular Evolution Docking Epitope Mapping Epitopes Event Flow Cytometry Goals HLA-A2 Antigen HLA-DR4 Antigen Human T-lymphotropic virus 1 Immune response Immune system Immunologic Receptors In Vitro Individual Length Libraries Ligands Ligation Link Lipid Bilayers MHC Class I Genes MHC Class II Genes MHC binding peptide Major Histocompatibility Complex Malignant Neoplasms Maps Mature T-Lymphocyte Mediating Microbe Molecular Molecular Immunology Multiprotein Complexes Mus Mutagenesis Mutate Mutation Myelin Basic Proteins NMR Spectroscopy Peptide/MHC Complex Peptides Receptor Cell Receptor Signaling Relaxation Research Personnel Resolution Retroviral Vector Roentgen Rays Role Signal Transduction Site Structure Surface System T-Cell Activation T-Cell Development T-Cell Receptor T-Lymphocyte Taxes Technology Testing Validation Variant Viral X-Ray Crystallography Yeasts base biophysical model design dimer extracellular genome editing molecular dynamics mutant novel strategies receptor reconstitution response

项目摘要

Project Summary The ability of cells to respond to their microenvironment is mediated by multiprotein signaling complexes. A particularly important type of multiprotein signaling complex involves cell surface receptors that transmit signals upon binding protein ligands on other cells. A premier example of such a receptor is the T cell receptor (TCR)– CD3 complex, a cardinal receptor of the immune system that is essential for protective responses to microbes and cancers. The TCR–CD3 complex is composed of a diverse TCRαβ heterodimer noncovalently associated with the invariant CD3 dimers CD3εγ, CD3εδ, and CD3ζζ. The TCR mediates peptide–MHC (pMHC) recognition, while CD3 transduces activation signals to the T cell. However, the mechanism whereby TCR engagement by pMHC initiates signaling remains a mystery. Our goal is to understand the early events of TCR signaling by obtaining information on: 1) the architecture of the TCR–CD3 complex; and 2) possible allosteric changes in TCR dynamics upon binding pMHC that are relayed to CD3. These studies will be undertaken through a highly integrated project that combines multiple approaches, technologies, and investigators. We will study both an MHC class I-restricted TCR (A6) and an MHC class II-restricted TCR (MS2-3C8). A6 recognizes a viral peptide from HTLV-1 bound to HLA-A2; MS2-3C8 recognizes a self-peptide from myelin basic protein bound to HLA-DR4. Our Aims are: 1. Epitope mapping and NMR-based docking of the wild-type TCR– CD3 complex. Although the extracellular regions of CD3 interact with those of TCR, crystallization of TCR– CD3 complexes has been thwarted by the low affinity of TCR–CD3 interactions. We will employ NMR chemical shift perturbation and PRE to determine binding epitopes between CD3 and TCR. These data will be used to determine a structure for the wild-type TCR–CD3 complex. 2. Structural analysis of affinity-matured TCR– CD3 complexes by X-ray crystallography. We will attempt to overcome the weak association between TCR and CD3 ectodomains by in vitro directed evolution using yeast display to stabilize TCR–CD3 complexes for crystallization. Information on binding epitopes from NMR will be used to design TCR mutant libraries by focusing mutagenesis on regions that contact CD3. 3. Biological validation of the TCR–CD3 structure. We will validate TCR–CD3 interfaces identified by NMR or crystallography by evaluating the effects of structure- guided mutations in TCR–CD3 interfaces on complex assembly, signaling, and T cell development using cellular assays and retrogenic mice expressing mutated TCR and CD3 components. 4. Dynamics analysis of free and pMHC-bound states of TCRαβ ectodomains. We will address the allosteric change hypothesis by carrying out solution NMR analysis of TCR ectodomains in free form and bound to pMHC, for both TCRs A6 and MS2-3C8. We will address the role of the lipid bilayer on TCR dynamics through MD simulations. Our combined NMR and MD results will generate a biophysical model of signaling through the TCR–CD3 complex whose basic features should apply to signaling through other multiprotein complexes and receptors.

项目摘要细胞对其微环境反应的能力是由多蛋白信号复合物介导的。一个尤其重要的多蛋白信号传导复合物涉及传输信号的细胞表面受体在其他细胞上结合蛋白质配体后。这种受体的主要例子是T细胞受体（TCR） - CD3复合物，免疫系统的基本接收器，这对于对微生物的保护性反应至关重要和癌症。 TCR – CD3复合物由发散的TCRαβ异二聚体无共价相关使用不变的CD3二聚体CD3εγ，CD3εδ和CD3ζζ。 TCR介导胡椒-MHC（PMHC）识别，而CD3将激活信号转换为T细胞。但是，TCR的机制 PMHC的参与启动信号仍然是一个谜。我们的目标是了解TCR的早期事件通过获取以下信息来传导：1）TCR – CD3复合物的体系结构； 2）可能的变构结合PMHC后TCR动力学的变化将传递到CD3。这些研究将进行通过一个高度集成的项目，该项目结合了多种方法，技术和研究人员。我们将研究MHC I类限制性TCR（A6）和MHC II限制性TCR（MS2-3C8）的MHC类。 A6认识从HTLV-1与HLA-A2结合的病毒肽； MS2-3C8识别髓磷脂碱性蛋白的自肽绑定到HLA-DR4。我们的目的是：1。野生型TCR –基于EpiTope映射和基于NMR的扩展坞 CD3复合物。尽管CD3的细胞外区域与TCR相互作用，但TCR –结晶 TCR – CD3相互作用的低亲和力挫败了CD3复合物。我们将使用NMR化学移动扰动和预先确定CD3和TCR之间的结合表位。这些数据将用于确定野生型TCR – CD3复合物的结构。 2。亲和成熟TCR –的结构分析 CD3复合物通过X射线晶体学。我们将尝试克服TCR之间的弱关联和CD3外生域通过体外定向进化，使用酵母显示器稳定TCR – CD3复合物结晶。 NMR的结合表位的信息将用于设计TCR突变库将诱变集中在接触CD3的区域上。 3。TCR – CD3结构的生物学验证。我们通过评估结构的影响，将验证NMR或晶体学鉴定的TCR – CD3接口使用复合组装，信号传导和T细胞开发的TCR – CD3界面中的引导突变使用细胞测定和表达突变的TCR和CD3成分的术后小鼠。 4。动态分析 TCRαβ胞外域的自由和PMHC结合状态。我们将通过对于两个TCRS A6 和MS2-3C8。我们将通过MD模拟来解决脂质双层在TCR动力学上的作用。我们的 NMR和MD的组合结果将通过TCR – CD3复合物生成信号传导的生物物理模型其基本特征应通过其他多蛋白复合物和接收器适用于信号。