Beyond Fang's fury: a computational study of the enzyme-membrane interaction and catalytic pathway of the snake venom phospholipase A₂ toxin

Snake venom-secreted phospholipases A₂ (svPLA₂s) are critical, highly toxic enzymes present in almost all snake venoms. Upon snakebite envenomation, svPLA₂s 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 svPLA₂s 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 svPLA₂ family, the Myotoxin-I, which is part of the venom of the Central American pit viper terciopelo (Bothrops asper), a ubiquitous but highly aggressive and dangerous species responsible for the most problematic snakebites in its habitat. Furthermore, PLA₂ 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 svPLA₂ 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 svPLA₂s: 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^-1) for POPC hydrolysis, consistent with experimental and computational values obtained for human PLA₂. The reaction mechanisms are similar, albeit not identical, and can be generalized to svPLA₂ 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 sPLA₂ chemistry will help find new, effective small molecule inhibitors with anti-snake venom efficacy.