Structures and Conformational Equilibria of Integrin alpha5 beta1

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 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.