Beyond Fang's fury: a computational study of the enzyme-membrane interaction and catalytic pathway of the snake venom phospholipase A toxin.

Snake venom-secreted phospholipases A (svPLAs) are critical, highly toxic enzymes present in almost all snake venoms. Upon snakebite envenomation, svPLAs hydrolyze cell membrane phospholipids and induce pathological effects such as paralysis, myonecrosis, inflammation, or pain. Despite its central importance in envenomation, the chemical mechanism of svPLAs is poorly understood, with detrimental consequences for the design of small-molecule snakebite antidotes, which is highly undesirable given the gravity of the epidemiological data that ranks snakebite as the deadliest neglected tropical disease. We study a member of the svPLA family, the Myotoxin-I, which is part of the venom of the Central American pit viper terciopelo (), a ubiquitous but highly aggressive and dangerous species responsible for the most problematic snakebites in its habitat. Furthermore, PLA enzymes are a paradigm of interfacial enzymology, as the complex membrane-enzyme interaction is as important as is crucial for its catalytic process. Here, we explore the detailed interaction between svPLA and a 1 : 1 POPC/POPS membrane, and how enzyme binding affects membrane structure and dynamics. We further investigated the two most widely accepted reaction mechanisms for svPLAs: the 'single-water mechanism' and the 'assisted-water mechanism', using umbrella sampling simulations at the PBE/MM level of theory. We demonstrate that both pathways are catalytically viable. While both pathways occur in two steps, the single-water mechanism yielded a lower activation free energy barrier (20.14 kcal mol) for POPC hydrolysis, consistent with experimental and computational values obtained for human PLA. The reaction mechanisms are similar, albeit not identical, and can be generalized to svPLA from most viper species. Furthermore, our findings demonstrate that the sole small molecule inhibitor currently undergoing clinical trials for snakebite is a perfect transition state analog. Thus, understanding snake venom sPLA chemistry will help find new, effective small molecule inhibitors with anti-snake venom efficacy.