Structures and Conformational Equilibria of Integrin alpha5 beta1

整合素α5β1的结构和构象平衡

基本信息

批准号：
9079774
负责人：
TIMOTHY A SPRINGER
金额：
$ 44.25万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-20 至 2020-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9079774
关键词：
Affect Affinity Antibodies Antibody Binding Sites Bacteria Binding Binding Sites Blood Platelets Blood Vessels C-terminal Calorimetry Carbohydrates Cell Adhesion Cell Surface Proteins Cell membrane Cell surface Cells Complex Coupled Cryoelectron Microscopy Crystallization Cultured Cells Cytoplasmic Tail Development Disease Electron Microscopy Epithelium Equilibrium Extracellular Matrix Family Fibrinogen Fibronectins Flow Cytometry Fluorescence Fluorescence Polarization Free Energy Funding Gastroenteritis Grant Hybrids Immune Infection Integrin Binding Integrin alpha5 Integrin alpha5beta1 Integrin beta Chains Integrins K562 Cells Knee Laminin Leg Ligand Binding Ligands Link Malignant Neoplasms Mannose Measurable Measurement Measures Mediating Membrane Membrane Proteins Methods Modeling Molecular Conformation Negative Staining Oncogenic Output Pathologic Neovascularization Peptides Pharmaceutical Preparations Plague Play Polysaccharides Population Proteins Quinine RGD (sequence)Regulation Resolution Role Shapes Signal Transduction Site Specificity Structure Testing Therapeutic Thigh structure Thrombocytopenia Thrombocytopenic Purpura Time Tissues Titrations Transmembrane Domain Vascular System Work Yersinia adhesion receptor angiogenesis base cell transformation design extracellular fibrinopeptides gamma glycosylation inhibitor/antagonist insight interest novel public health relevance receptor binding stereochemistry tumor tumor progression

项目摘要

DESCRIPTION (provided by applicant): Integrins αIIbβ3 and α5β1 are cell adhesion receptors that bind extracellular ligands, transduce signals bidirectionally across plasma membranes, and play important roles in the vasculature. Ligand binding and integrin activation are coupled to two distinct, large conformational changes, extension at the integrin knees and opening of the ligand-binding headpiece. On cell surfaces, integrins dynamically equilibrate between two low affinity conformations, bent-closed and extended-closed, and a high affinity extended-open conformation. Investigating integrin conformational equilibria, which have never been measured for any integrin, is paramount for understanding how the conformational ensemble dictates the functional output of integrins. Furthermore, how ligands bind integrins and drive headpiece opening remain incompletely understood. Aim 1 continues work on integrin αIIbβ3, which recognizes an Arg-Gly-Asp (RGD) motif. We characterize binding of an AGDV peptide from fibrinogen, which lacks the Arg of RGD, and demonstrate that its engagement of the MIDAS in the β-subunit is sufficient to open the integrin headpiece. We also complete work on the structural basis for quinine-dependent antibody binding to platelet integrin αIIbβ3, which causes drug-induced immune thrombocytopenia (DITP). Aim 2 focuses on another RGD-binding integrin, α5β1, which binds its primary ligand fibronectin (Fn) and directs its assembly into the extracellular matrix. Their interaction is important for angiogenesis, vascular development, and cancer progression. Intriguingly, α5β1 also binds the non-RGD ligand Invasin (Inv) to mediate bacterial internalization of Yersenia spp. that cause plague and gastroenteritis. We characterize the conformational states of α5β1 ectodomain by negative stain electron microscopy. We examine complexes of α5β1 with function-perturbing Fabs and Inv to define their binding sites and the integrin conformations they stabilize. Aim 3 investigates crystal structures of the α5β1 headpiece and its complexes with Fn and Inv fragments. Structures will illustrate how RGD in Fn3 module 10 and the synergy site in Fn3 module 9 bind α5β1 and the extent to which the non-RGD ligand Inv mimics Fn binding, and provide insight into how binding of different ligands affect headpiece opening. Aim 4 measures conformational equilibria for α5β1, and how conformational equilibria mix with the intrinsic affinity of a specific integrin conformation for ligand to yield the apparent affinity measured for an integrin ectodomain fragment or an intact integrin on the cell surface. For the first time, the conformational equilibria for integrin extenson and headpiece opening will be separately measured, and related to regulation of affinity for fibronectin. The effects of transmembrane domain association, glycosylation state, and oncogenic cell transformation on conformational equilibria of α5β1 are also studied. Results from our work will guide the design of higher affinity and novel non-RGD based α5β1-inhibitors as therapeutics for pathological angiogenesis and cancer.

描述（由适用提供）：整联蛋白αIIBβ3和α5β1是结合细胞外配体的细胞粘附受体，在跨质膜上双向转移信号，并在脉管系统中起重要作用。配体结合和整联蛋白激活与两个不同的大构象变化，整联蛋白膝盖的伸展以及配体结合头饰的打开。在细胞表面上，整联蛋白在两个低亲和力构象之间动态等效，弯曲和扩展闭合以及高亲和力扩展构象。研究整联蛋白构象等效物（从未为任何整合素）进行测量，这对于理解构象合奏如何决定整联蛋白的功能输出至关重要。此外，配体如何结合整联蛋白和驱动头置开口仍然不完全理解。 AIM 1继续对整合蛋白αIIBβ3进行工作，该整联蛋白αIIBβ3识别Arg-Gly-Asp（RGD）基序。我们表征了缺乏RGD的纤维蛋白原的AGDV胡椒的结合，并证明其在β-亚基中的互动足以打开整联蛋白头饰。我们还以奎宁依赖性抗体结合与血小板整合素αIIBβ3结合的结构基础完成工作，这会引起药物诱导的免疫血小板减少症（DITP）。 AIM 2专注于另一个RGD结合整合素α5β1，该整合素α5β1结合其主要配体纤连蛋白（FN）并将其组装引导到细胞外基质。它们的相互作用对于血管生成，血管发育和癌症进展至关重要。有趣的是，α5β1还结合了非RGD配体侵入蛋白（INV），以介导细菌的内在化Yersenia spp。这会导致瘟疫和胃肠炎。我们通过负染色电子显微镜表征了α5β1外生域的构象状态。我们检查了α5β1的复合物与功能扰动的晶圆厂，并INV定义其结合位点以及它们稳定的整合素组成。 AIM 3研究了α5β1头饰的晶体结构及其具有FN和INV片段的复合物。结构将说明FN3模块10中的RGD和FN3模块9中的协同位点结合α5β1以及非RGD配体INV模拟FN结合的程度，并提供了对不同配体结合的洞察力。 AIM 4测量α5β1的构象平衡，以及构象平衡如何与特定整联蛋白构象的固有亲和力混合在一起，以产生对整联蛋白外构域片段的明显亲和力或在细胞表面上完整的整合素的明显亲和力。首次将分别测量整联蛋白伸素蛋白的构象平衡，并与纤连蛋白的亲和力调节有关。跨膜结构域关联，糖基化态和致癌细胞转化对α5β1构象当量的影响也是研究的。我们工作的结果将指导较高亲和力和新型非RGD的α5β1抑制剂作为病理血管生成和癌症的治疗剂的设计。