Disintegrin-like and Cysteine-rich Domains Govern Enzymatic Activity and Substrate Recognition in Echis Snake Venom Metalloproteinases
TL;DR
These findings establish accessory-domain-mediated substrate recognition as a key determinant of SVMP function, informing rational antivenom design and support a model in which PIII SVMP accessory domains enhance substrate positioning and catalytic efficiency while selectively restricting access to non-cognate substrates.
Abstract
Snake venom metalloproteinases (SVMPs) are major drivers of pathology following viper envenomation and represent important targets for the development of next-generation recombinant antivenoms. PIII SVMPs are among the most potent haemorrhagic toxins and contain disintegrin-like (Dis) and cysteine-rich (C-rich) accessory domains. Despite their biomedical importance, the mechanistic roles of these accessory domains in substrate recognition and catalysis remain poorly understood. We produced recombinant full-length and domain-deletion variants of two functionally distinct PIII SVMPs: the broadly proteolytic, cytotoxic cPIII and the highly specific prothrombin activator Ecarin. Proteins were expressed as latent zymogens in insect cells, auto-activated by Zn²⁺, and analysed using enzymatic, blood clotting, and cell-based assays. Progressive removal of the C-rich and Dis domains reduced zymogen auto-activation and markedly diminished catalytic activity in both toxins. In cPIII, domain deletion caused a stepwise loss of proteolytic and cytotoxic activity without altering substrate preference. In Ecarin, removal of the accessory domains strongly impaired prothrombin activation, and thus plasma clotting, demonstrating a critical role in substrate recognition. Conversely, deletion of the C-rich domain increased fibrinogenolytic activity, revealing a substrate-dependent gatekeeping function. Deglycosylation showed that N-linked glycans modulate SVMP activity in a construct-dependent manner. Recombinant Ecarin closely recapitulated the biochemical properties of the native venom-derived toxin. Our data support a model in which PIII SVMP accessory domains enhance substrate positioning and catalytic efficiency while selectively restricting access to non-cognate substrates. These findings establish accessory-domain-mediated substrate recognition as a key determinant of SVMP function, informing rational antivenom design.