Genome-wide toxin gene repertoire of the bark scorpion Centruroides sculpturatus and molecular insights into α-toxin interaction with Nav1.5.

Scorpion venoms represent highly diversified biochemical arsenals shaped by long-term evolution, yet a comprehensive genomic understanding of toxin composition and the molecular basis of toxin-ion channel interactions remains incomplete. Here, we performed an integrated genome-wide and structural investigation of the venom system of Centruroides sculpturatus, combining toxin gene mining, evolutionary analysis, and molecular simulations to elucidate both toxin diversity and potential functional mechanisms. We identified 175 toxin-related genes from the genome, with sodium channel toxins (NaTxs) constituting the dominant component (32%), followed by venom metalloproteases and a large repertoire of previously uncharacterized cysteine-rich peptides. Phylogenetic and genomic analyses reveal that tandem gene duplication may be one of the factors driving NaTx diversification, with a striking asymmetry between extensively expanded β-toxins and relatively conserved α-toxins. Despite sequence divergence, these toxins retain a conserved cysteine-stabilized αβ structural scaffold, supporting a "conserved core-variable surface" evolutionary model. To investigate the molecular basis of toxicity, we focused on the representative α-toxin CsE5 and its interaction with the human cardiac sodium channel hNav1.5. Molecular docking followed by all-atom molecular dynamics simulations revealed a stable toxin-channel complex. The calculated MM/GBSA binding free energy (-44.85 kcal/mol) indicates a favorable interaction under the simulation conditions, with energy decomposition analysis indicating that electrostatic and van der Waals interactions act cooperatively to stabilize the toxin-channel complex. Residue-level analysis identified TRP38 as a primary energetic hotspot on the toxin, while multiple residues in the extracellular region of VSD4 contribute to interface stabilization. Notably, ASP1610 may serve as an electrostatic steering site, guiding toxin recognition rather than directly stabilizing binding. Collectively, these results support a two-step binding mechanism, in which long-range electrostatic guidance is followed by hydrophobic anchoring at the interface. This study provides a preliminary computational framework linking toxin gene evolution to predicted interaction mechanisms. Our findings suggest a possible molecular basis for scorpion venom cardiotoxicity and may inform future antivenom development, pending experimental validation.