The many dimeric faces of Lys49 PLA2-like proteins: Conformational plasticity and membrane binding drive functional dimer states.

Lys49 secreted phospholipase A2-like proteins (sPLA2s) 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 sPLA2-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.