Substitutions of residues in a loop surrounding the active site of a P-I snake venom metalloproteinase abrogates its hemorrhagic activity

Abstract

Snake venom metalolloproteinases (SVMPs) are the main toxins respon- sible for the hemorrhage in viper snakebite envenomation. SVMPs are classified into three classes, according to the presence or absence of different domains (metalloproteinase domain, disintegrin domain, cysteine- rich domain and disintegrin-like domain). The active site of the metalloproteinase domain present in PI, PII and PIII has a consensus zinc- binding sequence (HEXXHXXGXXH). In the different classes of SVMPs there are hemorrhagic and non-hemorrhagic toxins, even though their sequences are highly conserved. The understanding of the structural de- terminants that predict the hemorrhagic potential of the SVMPs of the PI is relevant given the limited knowledge that exists on this subject. Previous studies by molecular dynamic simulation showed higher flexibility in the first part of the U loop surrounding the active site (156-163) in hemor- rhagic SVMPs, as compared to non-hemorrhagic SVMPs. In the present work, an abrogation of the hemorrhagic activity of BaP1, a PI SVMP from the venom of Bothrops asper, was achieved by the substitution of residues in the first part of the U loop by the corresponding residues of a struc- turally-similar non-hemorrhagic PI SVMP from a related venom. The re- sults suggested that the U loop is critical for protein-protein interface and may be involved in the interaction with extracellular matrix proteins, hence influencing the ability of the toxin to bind and hydrolyze basement membrane components. The SVMP with the site mutation completely lost the hemorrhagic activity, and only loss partially the proteinase activity, indicating that this region in the loop plays a key role in the ability to induce hemorrhage. Our findings demonstrate a structural determinant of the hemorrhagic capacity of PI SVMPs.