Springer, Timothy A. Project 2 This project examines the specialized molecular features of integrins alpha4B7 and alpha4B1 and IgSF family members MAdCAM-1 and VCAM-1 that enable tethering, rolling, and firm adhesion of leukocytes on vascular endothelium in shear flow. The interaction between alpha4B1 and VCAM-1 is also important in development of the vasculature and the heart. The long-term goal is to acquire a deep understanding of adhesion in vascular flow, enhance treatment of vascular diseases including atherosclerosis, and accelerate development of therapeutics directed to alpha4 integrins.
Four aims address these needs. 1. Structures of MAdCAM-1's integrin-binding loop in domain 1 will address a novel fold for domain 1, and the hypothesis that the unique flexibility of this loop relates to MAdCAM-1's unusual ability to support rolling adhesion through integrins, and that this loop adopts distinct conformations to support rolling and firm adhesion. 2. Crystal and EM structures will define the structure of the integrin alpha4B7 headpiece, how it binds to function blocking antibodies that are approved or in clinical trials to treat multiple sclerosis and inflammatory bowel disease, how small molecule antagonists bind, and the different conformational states adopted by the headpiece and ectodomain in EM. Structures reveal unique features of the ligand binding pocket at the interface between the alpha4 B-propeller and B7 I domains, unique metal-binding residues in B7, and how selectivity and affinity of small molecules can be further improved. 3. To examine how alpha4B7 and MAdCAM-1 can mediate both rolling and firm adhesion, we will obtain crystal structures demonstrating the distinct way in which MAdCAM-1, its peptides, and representative small molecule inhibitors, bind to the closed and open a4P7 headpiece conformations. Structures of MAdCAM-1 and/or MAdCAM-1-derived peptides complexed with the closed and open alpha4B7 headpieces will reveal how macromolecular ligands bind, and further test the hypothesis that the closed and open headpieces mediate low-affinity rolling and high-affinity firm adhesion, respectively. 4. Complex structures with the alpha4B1 headpiece will reveal how it binds to VCAM-1, fibronectin, and small molecule antagonists. Comparisons to alpha4B7 complexes will examine how an integrin B-subunit influences ligand specificity, functional correlates to rolling through alpha4B7 and alpha4B1and how differences between alpha4B17and alpha4B1 can be exploited to develop selective antagonists for the different diseases in which these integrins are important. Complexes with Fab relate function to alpha4B1 conformation.
R E L E V A N C E (See instructions) structures of molecules on the surface of cells are important to understand how white blood cells migrate to sites of immune responses and disease. This application will determine structures of molecules with important functions in health, and which also contribute to diseases including malformation of the heart and vasculature, and atherosclerosis. Structures with antibodies or small molecules in development should accelerate the understanding of these diseases, and contribute to cures for multiple sclerosis, asthma, and inflammatory bowel disease.
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