Structures and Conformational Equilibria of Integrin alpha5 beta1

整合素α5β1的结构和构象平衡

• 批准号: 9265127
• 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
• 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/9265127
• 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; Drug Modelings; Electron Microscopy; Epithelium; 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; 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; angiogenesis; base; cell transformation; design; extracellular; fibrinopeptides gamma; glycosylation; inhibitor/antagonist; insight; novel; public health relevance; receptor binding; stereochemistry; synergism; tumor; tumor progression

 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，它们结合细胞外配体，跨质膜双向转导信号，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个不同的、大的构象变化耦合，即整合素膝部的延伸和配体结合头的打开。在细胞表面上，整合素在两种低亲和力构象（弯曲闭合和延伸闭合）和高亲和力延伸开放构象之间动态平衡。研究整合素构象平衡（从未测量过任何整合素）对于理解构象整体如何决定整合素的功能输出至关重要。此外，配体如何结合整合素并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。我们表征了来自纤维蛋白原的 AGDV 肽的结合，该肽缺乏 RGD 的 Arg，并证明其与 β 亚基中的 MIDAS 的结合足以打开整合素头。我们还完成了奎宁依赖性抗体与血小板整合素αIIbβ3结合的结构基础的工作，这导致 药物诱导的免疫性血小板减少症（DITP）。目标 2 关注另一种 RGD 结合整合素 α5β1，它结合其主要配体纤连蛋白 (Fn) 并指导其组装到 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很重要。有趣的是，α5β1 还结合非 RGD 配体 Invasin (Inv) 来介导耶尔森氏菌的细菌内化。导致鼠疫和胃肠炎。我们通过负染色电子显微镜表征 α5β1 胞外域的构象状态。我们检查 α5β1 与功能扰动 Fab 和 Inv 的复合物，以确定它们的结合位点和它们稳定的整合素构象。目标 3 研究 α5β1 头件及其与 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 以及非 RGD 配体 Inv 模拟 Fn 结合的程度，并深入了解不同配体的结合如何影响头件打开。目标 4 测量 α5β1 的构象平衡，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。首次单独测量整合素延伸子和头件开口的构象平衡，并与纤连蛋白亲和力的调节相关。还研究了跨膜结构域关联、糖基化状态和致癌细胞转化对 α5β1 构象平衡的影响。我们的工作结果将指导更高亲和力和新型非 RGD α5β1 抑制剂的设计，作为病理性血管生成和癌症的治疗方法。