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：
10238118
负责人：
Roy A Mariuzza
金额：
$ 69.2万
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 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10238118
关键词：
Address Affinity Antigen-Presenting Cells Antiviral Agents Architecture Autoimmune 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 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 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.

项目概要细胞对其微环境做出反应的能力由多蛋白信号复合物 A 介导。特别重要的多蛋白信号复合物类型涉及传递信号的细胞表面受体这种受体的一个主要例子是 T 细胞受体 (TCR) – CD3 复合物是免疫系统的主要受体，对于微生物的保护性反应至关重要 TCR-CD3 复合物由非共价关联的多种 TCRαβ 异二聚体组成。具有不变的 CD3 二聚体 CD3εγ、CD3εδ 和 CD3δδ TCR 介导肽-MHC (pMHC)。识别，而CD3将激活信号转导至T细胞，然而TCR的机制。 pMHC 参与启动信号传导仍然是一个谜，我们的目标是了解 TCR 的早期事件。通过获取以下信息来发出信号：1) TCR-CD3 复合物的结构；2) 可能的变构；结合 pMHC 后 TCR 动力学的变化，并传递至 CD3，将进行这些研究。我们将通过一个结合多种方法、技术和研究人员的高度集成的项目。研究 MHC I 类限制性 TCR (A6) 和 MHC II 类限制性 TCR (MS2-3C8) 识别。 HTLV-1 的病毒肽与 HLA-A2 结合，识别髓磷脂碱性蛋白的自身肽；与 HLA-DR4 结合我们的目标是： 1. 表位作图和基于 NMR 的野生型 TCR 对接 – 尽管 CD3 的胞外区与 TCR 的胞外区相互作用，但 TCR- 的结晶 CD3 复合物因 TCR-CD3 相互作用的低亲和力而受到阻碍，我们将采用 NMR 化学物质。平移扰动和 PRE 以确定 CD3 和 TCR 之间的结合表位。确定野生型 TCR-CD3 复合物的结构 2. 亲和力成熟的 TCR- 的结构分析。通过 X 射线晶体学检测 CD3 复合物我们将尝试克服 TCR 之间的弱关联。和 CD3 胞外域，通过体外定向进化使用酵母展示来稳定 TCR-CD3 复合物 NMR 中的结合表位信息将用于设计 TCR 突变体文库。将诱变重点放在接触 CD3 的区域。 3. TCR-CD3 结构的生物学验证。将通过评估结构的影响来验证由 NMR 或晶体学鉴定的 TCR-CD3 界面使用 TCR-CD3 界面引导复杂组装、信号传导和 T 细胞发育的突变细胞测定和表达突变TCR和CD3成分的逆转录小鼠4.动力学分析。 TCRαβ 胞外域的游离状态和 pMHC 结合状态我们将通过以下方式解决变构变化假说。对两种 TCR A6 的游离形式和与 pMHC 结合的 TCR 胞外域进行溶液 NMR 分析我们将通过 MD 模拟探讨脂质双层对 TCR 动力学的作用。结合 NMR 和 MD 结果将生成通过 TCR-CD3 复合物信号传导的生物物理模型其基本特征应适用于通过其他多蛋白复合物和受体的信号传导。

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

SARS-CoV-2 infection establishes a stable and age-independent CD8+ T cell response against a dominant nucleocapsid epitope using restricted T cell receptors.

SARS-CoV-2 感染使用限制性 T 细胞受体针对主要核衣壳表位建立稳定且与年龄无关的 CD8 T 细胞反应。

DOI：
发表时间：
2023-10-23
期刊：
影响因子：
16.6
作者：
Choy, Cecily;Chen, Joseph;Li, Jiangyuan;Gallagher, D Travis;Lu, Jian;Wu, Daichao;Zou, Ainslee;Hemani, Humza;Baptiste, Beverly A;Wichmann, Emily;Yang, Qian;Ciffelo, Jeffrey;Yin, Rui;McKelvy, Julia;Melvin, Denise;Wallace, Tonya;Dunn, Christo
通讯作者：
Dunn, Christo

Peptide-MHC (pMHC) binding to a human antiviral T cell receptor induces long-range allosteric communication between pMHC- and CD3-binding sites.

肽-MHC (pMHC) 与人类抗病毒 T 细胞受体结合可诱导 pMHC 和 CD3 结合位点之间的长程变构通讯。

DOI：
发表时间：
2018
期刊：
影响因子：
0
作者：
Rangarajan, Sneha;He, Yanan;Chen, Yihong;Kerzic, Melissa C;Ma, Buyong;Gowthaman, Ragul;Pierce, Brian G;Nussinov, Ruth;Mariuzza, Roy A;Orban, John
通讯作者：
Orban, John

The structural basis of T-cell receptor (TCR) activation: An enduring enigma.

T 细胞受体 (TCR) 激活的结构基础：一个持久的谜。

DOI：
发表时间：
2020
期刊：
影响因子：
0
作者：
Mariuzza, Roy A;Agnihotri, Pragati;Orban, John
通讯作者：
Orban, John

T cell receptors employ diverse strategies to target a p53 cancer neoantigen.

T 细胞受体采用多种策略来靶向 p53 癌症新抗原。

DOI：
发表时间：
2022-03
期刊：
影响因子：
0
作者：
Wu, Daichao;Gowathaman, Ragul;Pierce, Brian G;Mariuzza, Roy A
通讯作者：
Mariuzza, Roy A

Structural assessment of HLA-A2-restricted SARS-CoV-2 spike epitopes recognized by public and private T-cell receptors.

公共和私人 T 细胞受体识别的 HLA-A2 限制性 SARS-CoV-2 刺突表位的结构评估。

DOI：
发表时间：
2022-01-10
期刊：
影响因子：
16.6
作者：
Wu, Daichao;Kolesnikov, Alexander;Yin, Rui;Guest, Johnathan D;Gowthaman, Ragul;Shmelev, Anton;Serdyuk, Yana;Dianov, Dmitry V;Efimov, Grigory A;Pierce, Brian G;Mariuzza, Roy A
通讯作者：
Mariuzza, Roy A