Structures and Conformational Equilibria of Integrin alpha5 beta1

 描述（由申请方提供）：整合素αIIbβ3和α5β1是细胞粘附受体，可结合细胞外配体，跨质膜双向传递信号，并在血管系统中发挥重要作用。配体结合和整联蛋白激活耦合到两个不同的，大的构象变化，在整联蛋白的膝盖和配体结合的头部开放的延伸。在细胞表面上，整合素在两种低亲和力构象（闭合和延伸闭合）与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，这从来没有被测量的任何整合素，是至关重要的了解构象合奏如何决定的功能输出的整合素。此外，配体如何结合整合素和驱动头片段打开仍然不完全清楚。目的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，其缺乏RGD的Arg，并证明其与β亚基中的MIDAS的接合足以打开整联蛋白头片段。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， 药物诱导的免疫性血小板减少症（DITP）。目的2关注另一种结合RGD的整合素α5β1，其结合其主要配体纤连蛋白（Fn）并指导其组装成 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是重要的。有趣的是，α5β1还结合非RGD配体侵袭素（Invasin）以介导耶尔森氏菌属的细菌内化。导致瘟疫和肠胃炎我们用负染电子显微镜表征了α5β1胞外域的构象状态。我们研究了α5β1与功能干扰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。目的3研究α5β1头片段及其与Fn和Inv片段复合物的晶体结构。结构将说明Fn 3模块10中的RGD和Fn 3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，并提供对不同配体的结合如何影响头片段打开的见解。目的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对配体的固有亲和力混合，以产生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。第一次，整合素伸展和头片段开放的构象平衡将分别测量，并与调节纤连蛋白的亲和力。研究了跨膜结构域结合、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设计更高亲和力和新型非RGD基α5β1抑制剂作为病理性血管生成和癌症的治疗剂。

 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.