Functional and Computational Analysis of the Role of the Adjacent to Metal Ion-Dependent Adhesion Site (ADMIDAS) of Integrin αIIbβ3.

The role of the β3 MIDAS in αIIbβ3 ligand binding is well established, but the role of the nearby ADMIDAS is less well defined. Thus, we studied HEK293 cells expressing normal αIIbβ3 (normal cells) or the ADMIDAS mutants β3 D126A and D127A (mutant cells). Both mutant cells adhered as well or better than normal cells to immobilized fibrinogen under static conditions in the presence of either Ca2+/Mg2+ or Mn2+. Under low shear flow conditions (0.15 dyne/cm2), adhesion of normal cells and D126A mutant cells to fibrinogen was similar in the presence of either Ca2+/Mg2+ or Mn2+. Adherent D126A mutant cells, however, demonstrated greater resistance to detachment at increasing shear rates in the presence of Ca2+/Mg2+ (e.g., at 20.4 dynes/cm², only 40 ± 10% of normal cells remained vs 85 ± 8% of D126A mutant cells; mean ± SD; p<0.001). Substituting Mn2+ for Ca2+/Mg2+ increased the resistance to detachment of the normal cells (60 ± 20% remaining at 20.4 dynes/cm2; p=0.01), but the value was still less than the mutant cells in the presence of either Ca2+/Mg2+ (see above; p<0.01) or Mn2+ (84 ± 4%; p<0.01). The increased strength of adhesion we observed in the αIIbβ3 ADMIDAS mutant cells is similar to that found in α4β7 ADMIDAS mutant cells (Chen et al, JBC 2004) and is consistent with the findings in isolated β3 βA (I-like) domains (Pesho et al. JBC 2006). The binding of 7E3, whose epitope is near the ADMIDAS, to the D126 mutant cells was similar to its binding to the normal cells, but 7E3 binding to the D127A mutant cells was reduced by 89 ± 7% (n = 4; p<0.001). 7E3 decreased adhesion of normal cells to fibrinogen by 88 ± 4%, but it only decreased adhesion of D126A mutant cells by 3 ± 9%, and it did not inhibit adhesion of D127A cells at all. To provide a structural context for the role of the ADMIDAS in ligand binding to αIIbβ3, we compared results from nanosecond time-scale molecular dynamics (MD) simulations of the cyclic peptide ligand eptifibatide in complex with either the fully hydrated normal αIIbβ3 or the D126A mutant in the presence of Ca2+/Mg2+. Calculations were carried out using the OPLS all-atom force-field of the GROMACS simulation package. With respect to normal αIIbβ3, the mutant receptor demonstrated reduced fluctuations in the β3 207–210 and 335 regions and increased fluctuations in the β3 282–284 region. In addition, the ADMIDAS metal ion moved ~3 Å away from the MIDAS and became more solvent exposed. Rearrangements of the coordination of the ADMIDAS involving S123, D251, and D127 were also observed in the D126A mutant compared to normal αIIbβ3. Steered MD simulations were used to investigate the unbinding of eptifibatide from its binding site. The unbinding force for the D126A mutant was similar to that for the normal αIIbβ3. Quantitative estimations of the binding energies of eptifibatide to normal and D126A mutant αIIbβ3 from Molecular Mechanics/Poisson Boltzman Surface Area analysis of the MD trajectories also yielded similar results. Thus, the much greater resistance of D126A mutant cells to detachment from fibrinogen at increasing shear rates does not appear to be explained by differences in fibrinogen-αIIbβ3 interactions at the sites involved in the binding of eptifibatide. Potential alternative mechanisms involve differences in fibrinogen’s access to the binding site, interactions with other sites, or changes in fibrinogen avidity due to receptor clustering.