Binding affinity of metal ions to the CD11b A-domain is regulated by integrin activation and ligands

Abstract

The divalent cations Mg2+ and Ca2+ regulate the interaction of integrins with their cognate ligands, with Mg2+ uniformly facilitating and Ca2+ generally inhibiting such interactions in vitro. Because both cations are present in mM concentrations in vivo, the physiologic relevance of the in vitro observations is unclear. We measured the affinity of both cations to the inactive and active states of the ligand- and cation-binding A-domain (CD11BA) from integrin CD11b/CD18 in the absence and presence of the single-chain 107 antibody (scFv107), an activation-insensitive ligand-mimetic antibody. Using titration calorimetry, we found that Mg2+ and Ca2+ display equivalent (mM) affinities to inactive CD11bA. Activation induced a ∼10-fold increase in the binding affinity of Mg2+ to CD11bA with no change in that of Ca2+ (106 μM ± 16 and 2.1 mM ± 0.19, respectively, n = 4). This increase is largely driven by favorable enthalpy. scFv107 induced a 50-80-fold increase in the binding affinity of Ca2+ (but not Mg2+ or Mn2+) to either form of CD11bA. Thus the affinity of metal ions to integrins is itself regulated by the activation state of these receptors and by certain ligands. These findings, which we expect will be applicable in vivo, elucidate a new level of regulation of the integrin-metal-ligand ternary complex and help explain some of the discrepant effects of Ca2+ on integrin-ligand interactions.