Structures and Conformational Equilibria of Integrin alpha5 beta1

 描述(申请人提供)：整合素αIIbβ3和α5β1是细胞黏附受体，结合细胞外配体，跨质膜双向传递信号，在血管系统中发挥重要作用。配体结合和整合素激活与两个截然不同的大的构象变化相耦合，即整合素膝部的伸展和配体结合头盔的打开。在细胞表面，整合素在两种低亲和力构象之间动态平衡，即弯曲闭合构象和延伸闭合构象，以及高亲和力延伸开放构象。研究整合素的构象平衡对于理解构象集成如何决定整合素的功能输出是至关重要的。此外，配体如何结合整合素和驱动耳机开口仍然不完全清楚。AIM 1继续研究整合素αIIbβ3，它识别精氨酸-甘氨酸-天冬氨酸(RGd)基序。我们表征了来自纤维蛋白原的AGDV多肽的结合，该多肽缺乏RGDArg，并证明它与β-亚基中的MIDAS的结合足以打开整合素头盔。我们还完成了奎宁依赖的抗体与血小板整合素αIIbβ3结合的结构基础的工作，这导致 药物免疫性血小板减少症(DITP)。目的2重点研究另一种结合RGD的整合素，α5β1，它与其主要配体纤维连接蛋白(FN)结合，并将其组装到 细胞外基质。它们的相互作用对血管生成、血管发育和癌症进展都很重要。有趣的是，α5β1还与非RGD型配体INVAS结合，以介导耶尔森氏菌的细菌内化。会引起鼠疫和胃肠炎。我们用负染色电子显微镜表征了α-5-β-1胞外结构域的构象。我们研究了α5，β1与功能扰动的Fabs和Inv的络合物，以确定它们的结合位置和它们稳定的整合素构象。目的3研究α5β1头盔及其与Fn和Inv片段的络合物的晶体结构。结构将说明Fn3模块10中的Rgd和Fn3模块9中的协同部位如何与α5β1结合，以及非RGD配体Inv模拟Fn结合的程度，并提供对不同配体的结合如何影响耳机开口的洞察。目的4测量α5β1的构象平衡，以及构象平衡如何与特定整合素构象对配体的内在亲和力混合，以产生细胞表面整合素胞外结构域片段或完整整合素的表观亲和力。第一次，将分别测量整合素伸展部分和头部开口的构象平衡，并与纤维连接蛋白亲和力的调节相关。研究了跨膜区结合、糖基化状态和致癌细胞转化对α-5-β-1构象平衡的影响。我们的工作结果将指导设计更高亲和力和新型的非RGD型α5β1抑制剂作为病理性血管生成和癌症的治疗药物。

 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.