The α2β1 integrin inhibitor rhodocetin binds to the A-domain of the integrin α2 subunit proximal to the collagen-binding site

Abstract

Rhodocetin is a snake venom protein that binds to α2β1 integrin, inhibiting its interaction with its endogenous ligand collagen. We have determined the mechanism by which rhodocetin inhibits the function of α2β1. The interaction of α2β1 with collagen and rhodocetin differed: Ca2+ ions and slightly acidic pH values increased the binding of α2β1 integrin to rhodocetin in contrast with their attenuating effect on collagen binding, suggesting that rhodocetin preferentially binds to a less active conformation of α2β1 integrin. The α2A-domain [von Willebrand factor domain A homology domain (A-domain) of the integrin α2 subunit] is the major site for collagen binding to α2β1. Recombinant α2A-domain bound rhodocetin, demonstrating that the A-domain is also the rhodocetin-binding domain. Although the interaction of α2β1 with rhodocetin is affected by altering divalent cations, the interaction of the A-domain was divalent-cation-independent. The rhodocetin-binding site on the α2A-domain was mapped first by identifying an anti-α2 antibody that blocked rhodocetin binding and then mapping the epitope of the antibody using human–mouse α2A-domain chimaeras; and secondly, by binding studies with α2A-domain, which bear point mutations in the vicinity of the mapped epitope. In this way, the rhodocetin-binding site was identified as the α3–α4 loop plus adjacent α-helices. This region is known to form part of the collagen-binding site, thus attaining a mainly competitive mode of inhibition by rhodocetin.