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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
5P01HL103526-04
Application #
8638059
Study Section
Heart, Lung, and Blood Initial Review Group (HLBP)
Project Start
Project End
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
4
Fiscal Year
2013
Total Cost
$371,869
Indirect Cost
$92,846
Name
Children's Hospital Boston
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
02115
Springer, Timothy A (2014) von Willebrand factor, Jedi knight of the bloodstream. Blood 124:1412-25
Liu, Ying; Zhang, Yan; Wang, Jia-Huai (2014) Crystal structure of human Ankyrin G death domain. Proteins 82:3476-82
Blenner, Mark A; Dong, Xianchi; Springer, Timothy A (2014) Structural basis of regulation of von Willebrand factor binding to glycoprotein Ib. J Biol Chem 289:5565-79
OuYang, Bo; Chou, James J (2014) The minimalist architectures of viroporins and their therapeutic implications. Biochim Biophys Acta 1838:1058-67
Yu, Yamei; Schurpf, Thomas; Springer, Timothy A (2013) How natalizumab binds and antagonizes *4 integrins. J Biol Chem 288:32314-25
Yu, Yamei; Zhu, Jianghai; Huang, Po-Ssu et al. (2013) Domain 1 of mucosal addressin cell adhesion molecule has an I1-set fold and a flexible integrin-binding loop. J Biol Chem 288:6284-94
Zhu, Jieqing; Zhu, Jianghai; Springer, Timothy A (2013) Complete integrin headpiece opening in eight steps. J Cell Biol 201:1053-68
Wang, Rui; Zhu, Jianghai; Dong, Xianchi et al. (2012) GARP regulates the bioavailability and activation of TGFýý. Mol Biol Cell 23:1129-39
Eng, Edward T; Smagghe, Benoit J; Walz, Thomas et al. (2011) Intact alphaIIbbeta3 integrin is extended after activation as measured by solution X-ray scattering and electron microscopy. J Biol Chem 286:35218-26