Binding Affinity of Metal Ions to the CD11b A-domain Is Regulated by Integrin Activation and Ligands

Abstract

The divalent cations Mg(2+) and Ca(2+) regulate the interaction of integrins with their cognate ligands, with Mg(2+) uniformly facilitating and Ca(2+) 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 Mg(2+) and Ca(2+) display equivalent (mm) affinities to inactive CD11bA. Activation induced a approximately 10-fold increase in the binding affinity of Mg(2+) to CD11bA with no change in that of Ca(2+) (106 microm +/- 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 Ca(2+) (but not Mg(2+) or Mn(2+)) 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 Ca(2+) on integrin-ligand interactions.

Dynamic Structural Changes Are Observed upon Collagen and Metal Ion Binding to the Integrin α1 I Domain

“…In ␣ I domains, Ca 2ϩ affinity for MIDAS, and its ability to regulate ligand binding, is both concentration-and activation-state dependent (61,70,71). The affinity of the ␣M I domain for Ca 2ϩ was shown to be low (millimolar) (61)(62)(63), and quantum calculations on the ␣L I domain predicted that the Ca 2ϩ -bound state would have lower affinity than either the Mg 2ϩ -or Mn 2ϩ -bound state for ligand (71).…”

“…In ␣ I domains, Ca 2ϩ affinity for MIDAS, and its ability to regulate ligand binding, is both concentration-and activation-state dependent (61,70,71). The affinity of the ␣M I domain for Ca 2ϩ was shown to be low (millimolar) (61)(62)(63), and quantum calculations on the ␣L I domain predicted that the Ca 2ϩ -bound state would have lower affinity than either the Mg 2ϩ -or Mn 2ϩ -bound state for ligand (71). However, some integrin I domains have shown differences in their relative abilities to bind calcium (70,72) , and Mn 2ϩ were very similar, except for a striking difference in the ␣C helix, a structural element unique to collagen-binding integrins that is believed to be critical for binding and collagen subtype selectivity (6,59,73).…”

“…A true ligand-mimetic antibody would be less desirable as a therapeutic, however, because it could lead to agonistic effects through outside-in integrin signaling. Another related antibody, mAb107, preferentially binds to the ␣M I domain in Ca 2ϩ via a bidentate interaction with Asp-107 (61,77). Similarly to AQC2, ligation of mAb107 did not induce conversion to the open conformation.…”

“…2ϩ does not support ␣1␤1 collagen binding, and it has been shown to bind with poor (mM) affinity to integrin I domains (61)(62)(63). Unlike Mn 2ϩ and Mg 2ϩ , Ca 2ϩ did not stabilize ␣1I toward thermal denaturation at concentrations up to 100 mM (Fig.…”

Ion implantation of titanium based biomaterials

“…This process also generates intracellular signals that lead to the progression of cells through the cell cycle and increased cell proliferation. Since integrin function is critically dependent on the concentration of divalent cations [152] it is possible that the presence of more number of calcium cations might trigger enhanced ligand binding of the integrin receptors and stimulate integrin-mediated activation [65]. To put these ideas in a different perspective, positively charged surfaces are favorable for the adhesion of fibronectin.…”