Structural, functional, and bioinformatics studies reveal a new snake venom homologue phospholipase A₂ class

Phospholipases A₂ (PLA₂s) are enzymes responsible for membrane disruption through Ca(2+) -dependent hydrolysis of phospholipids. Lys49-PLA₂s are well-characterized homologue PLA₂s that do not show catalytic activity but can exert a pronounced local myotoxic effect. These homologue PLA₂s were first believed to present residual catalytic activity but experiments with a recombinant toxin show they are incapable of catalysis. Herein, we present a new homologue Asp49-PLA₂ (BthTX-II) that is also able to exert muscle damage. This toxin was isolated in 1992 and characterized as presenting very low catalytic activity. Interestingly, this myotoxic homologue Asp49-PLA₂ conserves all the residues responsible for Ca(2+) coordination and of the catalytic network, features thought to be fundamental for PLA₂ enzymatic activity. Previous crystallographic studies of apo BthTX-II suggested this toxin could be catalytically inactive since a distortion in the calcium binding loop was observed. In this article, we show BthTX-II is not catalytic based on an in vitro cell viability assay and time-lapse experiments on C2C12 myotube cell cultures, X-ray crystallography and phylogenetic studies. Cell culture experiments show that BthTX-II is devoid of catalytic activity, as already observed for Lys49-PLA₂s. Crystallographic studies of the complex BthTX-II/Ca(2+) show that the distortion of the calcium binding loop is still present and impairs ion coordination even though Ca(2+) are found interacting with other regions of the protein. Phylogenetic studies demonstrate that BthTX-II is more phylogenetically related to Lys49-PLA₂s than to other Asp49-PLA₂s, thus allowing Crotalinae subfamily PLA₂s to be classified into two main branches: a catalytic and a myotoxic one.