Mapping structural determinants of biological activities in snake venom phospholipases A 2 by sequence analysis and site directed mutagenesis

Abstract

In addition to their catalytic activity, snake venom phospholipases A 2 (vPLA 2) present remarkable diversity in their biological effects. Sequence alignment analyses of functionally related PLA 2 are frequently used to predict the structural determinants of these effects, and the predictions are subsequently evaluated by site directed mutagenesis experiments and functional assays. In order to improve the predictive potential of computer-based analysis, a simple method for scanning amino acid variation analysis (SAVANA) has been developed and included in the analysis of the lysine 49 PLA 2 myotoxins (Lys49-PLA 2). The SAVANA analysis identified positions in the C-terminal loop region of the protein, which were not identified using previously available sequence analysis tools. Site directed mutagenesis experiments of bothropstoxin-I, a Lys49-PLA 2 isolated from the venom of Bothrops jararacussu, reveals that these residues are exactly those involved in the determination of myotoxic and membrane damaging activities. The SAVANA method has been used to analyse presynaptic neurotoxic and anti-coagulant vPLA 2s, and the predicted structural determinants of these activities are in excellent agreement with the available results of site directed mutagenesis experiments. The positions of residues involved in the myotoxic and neurotoxic determinants demonstrate significant overlap, suggesting that the multiple biological effects observed in many snake vPLA 2s are a consequence of superposed structural determinants on the protein surface.