The leukocyte integrins aM?2 and ax?2 are widely important in inflammatory responses of myeloid cells. They bind ligands including ICAM-1, iC3b, and fibrin. Upon cell activation, integrins change shape and can bind ligand, a phenomenon known as inside-out signaling. Understanding the molecular basis of this structural alteration is of great importance. The long-term goal of this application is to understand at the atomic level 1) how allostery in aM?2 and ax?2 integrins is transmitted from the juxtamembrane regions through the lower legs, upper legs, and ? I domain, to the ligand-binding a I domain, 2) how ligands such as iC3b bind, and 3) how small molecule antagonists inhibit ligand binding by disrupting allostery. Mutations predicted to better stabilize the high affinity, open conformation of ax and aM I domains than current mutations will be examined, and the best ones used in studies of ligand binding and allostery. A structure of the aM I domain bound to domain 3 of ICAM-1 will reveal the mechanism of ligand binding; a ternary complex of the CCL and aM I domains bound to a domain 1-3 fragment will reveal how integrins cooperate to bind a single ligand and how binding sites are organized to allow this. Electron microscopy will reveal how domain-domain orientations change between bent, extended-closed, and extended-open conformations, how allostery is transmitted from one domain to another, and how activating Fab and small molecule antagonists alter allosteric transitions. Structural studies will examine how the overall disposition of the thioester domain in C3 changes from C3 to C3b to iC3b, and how the ax and aM I domain and intact ectodomains specifically recognize iC3b. A crystal structure of the ax?2 ectodomain in the bent conformation will define at the atomic level the structure of the low affinity state. The structure of interfaces between the ? -propeller and a I domain and between the a I domain and ? I domain will reveal important information about how allostery is transmitted. Mutational studies will define the physiologic function of the bent conformation on cell surfaces. Co-crystal structures with small molecule antagonists will reveal binding sites important for development of therapeutics for inflammatory and autoimmune diseases. ? ? ?
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