Abstract Lys49 secreted phospholipase A 2 ‐like proteins (sPLA 2 s) are major myotoxins in viperid snake venoms, causing rapid muscle damage in envenomation. Beyond their clinical relevance, these small non‐catalytic proteins provide a model to study how quaternary structure and conformational dynamics enable catalysis‐independent membrane disruption. Using site‐directed mutagenesis, fluorescence anisotropy, and extensive atomistic and coarse‐grained molecular dynamics simulations, we characterized the conformational landscape of Bothropstoxin‐I (BthTx‐I), a prototypical Lys49 sPLA 2 ‐like protein. Our results show that compact and extended dimers coexist in solution but differ in flexibility, with only the extended dimer reproducing experimental FRET efficiencies across wild‐type and mutant proteins. Atomistic MD simulations reveal that the extended dimer undergoes hinge‐like motions that preserve quaternary structure while sampling substates compatible with membrane engagement. Coarse‐grained simulations demonstrate that only geometries similar to the extended crystallographic conformation allow both C‐terminal loops to simultaneously insert into the bilayer, stabilizing the membrane‐bound state required for phospholipid disruption. These findings resolve the long‐standing debate over compact versus extended dimer assemblies by demonstrating that the extended conformation is the functionally competent state, providing a unifying mechanistic framework that links quaternary structure dynamics to the molecular basis of myotoxicity. By pinpointing the structural features essential for productive membrane engagement, this work establishes a predictive platform that is expected to accelerate the rational design of next‐generation inhibitors for more effective treatment of snakebite envenomation.
Lima et al. (2026) studied this question.