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（由申请人提供）：整合素 αIIbβ3 和 α5β1 是细胞粘附受体，可结合细胞外配体，跨膜质双向转导信号，并在脉管系统中发挥重要作用。配体结合和整合素激活与两种不同的大构象变化相结合。整合素膝盖处的延伸和配体结合头的开口在细胞表面上，整合素在两个低亲和力之间动态平衡。研究整合素构象平衡（从未对任何整合素进行过测量）对于了解构象整体如何决定整合素的功能输出至关重要。结合整合素和驱动头件打开仍不完全清楚，目标 1 继续研究整合素 αIIbβ3，它识别 Arg-Gly-Asp。 (RGD) 基序。我们表征了纤维蛋白原中缺乏 RGD 精氨酸的 AGDV 肽的结合，并证明其与 β 亚基中的 MIDAS 的结合足以打开整合素头。奎宁依赖性抗体与血小板整合素 αIIbβ3 结合的基础，这会导致药物诱导的免疫性血小板减少症 (DITP) 的重点是另一种 RGD 结合整合素 α5β1，它与其主要配体纤连蛋白 (Fn) 结合并指导其组装到血小板中。有趣的是，α5β1 还与非 RGD 配体 Invasin (Inv) 结合，介导引起鼠疫和胃肠炎的细菌内化。我们通过负染色电子显微镜检查 α5β1 与功能扰动 Fab 和 Inv 的复合物来定义。目标 3 研究 α5β1 头部及其与 Fn 和 Inv 片段的复合物的晶体结构。结构将说明 Fn3 模块 10 中的 RGD 和 Fn3 模块 9 中的协同位点如何结合 α5β1 及其程度。非 RGD 配体 Inv 模拟 Fn 结合，并深入了解不同配体的结合如何影响头件打开。 α5β1 的平衡，以及构象平衡如何与配体的特定整联蛋白构象的内在亲和力混合，以产生针对细胞表面上的整联蛋白胞外域片段或完整整联蛋白测量的表观亲和力。延伸子和头件开口将分别测量，并与纤连蛋白亲和力的调节、糖基化状态和纤连蛋白的影响相关。还研究了 α5β1 构象平衡的致癌细胞转化，我们的工作结果将指导设计更高亲和力和新型非 RGD α5β1 抑制剂作为病理性血管生成和癌症的治疗方法。