Molecular basis for the dynamic strength of the integrin α4β1/VCAM-1 interaction

Intercellular adhesion mediated by integrin alpha(4)beta(1) and vascular cell adhesion molecule-1 (VCAM-1) plays a crucial role in both the rolling and firm attachment of leukocytes onto the vascular endothelium. Essential to the alpha(4)beta(1)/VCAM-1 interaction is its mechanical strength that allows the complex to resist the large shear forces imposed by the bloodstream. Herein we employed single-molecule dynamic force spectroscopy to investigate the dynamic strength of the alpha(4)beta(1)/VCAM-1 complex. Our force measurements revealed that the dissociation of the a4b1/VCAM-1 complex involves overcoming at least two activation potential barriers: a steep inner barrier and a more elevated outer barrier. The inner barrier grants the complex the tensile strength to withstand large pulling forces (>50 pN) and was attributed to the ionic interaction between the chelated Mg2+ ion at the N-terminal A-domain of the beta(1) subunit of alpha(4)beta(1) and the carboxyl group of Asp-40 of VCAM-1 through the use of site-directed mutations. In general, additional mutations within the C-D loop of domain 1 of VCAM-1 suppressed both inner and outer barriers of the alpha(4)beta(1)/VCAM-1 complex, while a mutation at Asp-143 of domain 2 of VCAM-1 resulted in the suppression of the outer barrier, but not the inner barrier. In contrast, the outer barrier of alpha(4)beta(1)/VCAM-1 complex was stabilized by integrin activation. Together, these findings provide a molecular explanation for the functionally relevant kinetic properties of the alpha(4)beta(1)/VCAM-1 interaction.