The specificity and function of the metal-binding sites in the integrin β3 A-domain

Abstract

The A-domains within integrin β subunits contain three metal sites termed the metal ion-dependent adhesion site (MIDAS), site adjacent to the metal ion-dependent adhesion site (ADMIDAS), and ligand-induced metal-binding site (LIMBS), and these sites are involved in ligand engagement. The selectivity of these metal sites and their role in ligand binding have been investigated by expressing a fragment corresponding to the β3 A-domain, β3-(109-352), and single point mutants in which each of the cation-binding sites has been disabled. Equilibrium dialysis experiments identified three Mn2+- and two Ca2+-binding sites with the LIMBS being the site that did not bind Ca2+. Although the ADMIDAS could bind Ca2+, it did not bind Mg2+. These results indicate that the Ca2+-specific site that inhibits ligand binding is the ADMIDAS. Two different assay systems, surface plasmon resonance and a microtiter plate assay, demonstrated that the β3 A-domain fragment bound fibrinogen in the presence of 0.1 mM Ca2+ but not in 3 mM Ca2+. This behavior recapitulated the effects of Ca2+ on fibrinogen binding to αvβ3 but not αIIbβ3. Disabling any of the three cation-binding sites abrogated fibrinogen binding. These results indicate that the specificities of the three metal-binding sites for divalent cations are distinct and that each site can regulate the ligand binding potential of the β3 A-domain. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.