Dramatic and concerted conformational changes enable rhodocetin to block α2β1 integrin selectively

Abstract

The collagen binding integrin α2β1 plays a crucial role in hemostasis, fibrosis, and cancer progression amongst others. It is specifically inhibited by rhodocetin (RC), a C-type lectin-related protein (CLRP) found in Malayan pit viper ( Calloselasma rhodostoma) venom. The structure of RC alone reveals a heterotetramer arranged as an αβ and γδ subunit in a cruciform shape. RC specifically binds to the collagen binding A-domain of the integrin α2 subunit, thereby blocking collagen-induced platelet aggregation. However, until now, the molecular basis for this interaction has remained unclear. Here, we present the molecular structure of the RCγδ-α2A complex solved to 3.0 Å resolution. Our findings show that RC undergoes a dramatic structural reorganization upon binding to α2β1 integrin. Besides the release of the nonbinding RCαβ tandem, the RCγ subunit interacts with loop 2 of the α2A domain as result of a dramatic conformational change. The RCδ subunit contacts the integrin α2A domain in the “closed” conformation through its helix C. Combined with epitope-mapped antibodies, conformationally locked α2A domain mutants, point mutations within the α2A loop 2, and chemical modifications of the purified toxin protein, this molecular structure of RCγδ-α2A complex explains the inhibitory mechanism and specificity of RC for α2β1 integrin.

Author summary

In animals, collagen-mediated platelet aggregation is an essential component of the blood’s clotting response following vascular injury. A small group of snake venom toxins belonging to the C-type lectin protein family exert their harmful effects by directly targeting this pathway. Rhodocetin (RC) is a heterotetrameric protein found in the venom of the Malayan pit viper ( C. rhodostoma). RC specifically binds α2β1 integrin, the key protein required for collagen-mediated platelet aggregation. In this study, we describe the interaction between RC and α2β1 integrin at atomic resolution. This study reveals that RC undergoes a massive structural reorganization upon α2β1 integrin binding, such that RC’s αβ subunit is released from its γδ subunit and a γδ-α2β1 integrin complex is formed. The inhibitory nature of this complex can be readily explained as RC binding along the top surface of the α2β1 integrin and directly above the collagen binding site. As a result, access of collagen to its binding site is blocked, thereby preventing collagen-mediated platelet aggregation.