The leukocyte integrins ?X?2 and ?M?2 are important in inflammatory responses of myeloid and dendritic cells. They bind a range of ligands including iC3b, and are complement receptors CR4 and CR3. Upon cell activation, integrins change shape and bind ligand (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 ?M?2 and ?X?2 is transmitted from the juxtamembrane regions through the lower legs, upper legs, and ?I domain, to the ligand-binding ?I domain, 2) how ligands such as iC3b bind, 3) how small molecule antagonists inhibit ligand binding by disrupting allostery, and 4) how ligand binding and conformation are regulated in terms of the energetics of different conformational states. This proposal builds on our previous progress in determining crystal and EM structures of ?X?2, EM structures of the complement component C3 and its products, and EM structures of ?X?2 bound to iC3b and C3c. A new crystal form of the bent ?X?2 ectodomain will be characterized, in which the ?X I domain is open, ?X Glu-318 binds to the ?2 I MIDAS and other residues move out of contact with ?I and reshape to form an intrinsic ligand structure. The ?2 I domain shifts to an intermediate state. The crystal structure of an ?X?2 headpiece with an ?I-open/?I- open conformation will reveal ?I opening and hybrid domain swing-out. Mutational studies will examine the ?- and ?-genus, features that regulate ?I-?I signal relay, Gly residues in the ?I ?1-helix, an ?I linker disulfide, and specific structural features that regulate the equilibrium between integrin conformational states. Basis for selectivity of ?X?2 for iC3b versus other complement products will be examined using surface plasmon resonance with high affinity mutant ?X?2. The basis of affinity regulation, and the energetics of different ?X?2 conformational states, will be examined by employing different mutations, and measuring affinities for ligand and six Fab specific for the closed headpiece, open headpiece, or extended conformations. Finally, the structure of the ?X?2 headpiece bound to C3c will reveal the basis for specific ligand recognition and selectivity of ?X?2 for iC3b.
Leukocyte integrins are important in defense against infection. This grant deepens our understanding of their structure and function, and is enabling for treatment of disease and improvement of therapeutics for autoimmune and inflammatory diseases.
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