Physicochemical and Dynamic Properties of Mosquito Glutathione S-Transferases: A Comparative Analysis of Delta and Epsilon classes

Mosquito glutathione S-transferases (GSTs) are central components of the detoxification machinery underlying metabolic insecticide resistance, a major threat to the control of vector-borne diseases. Among cytosolic GSTs, the delta (δ) and epsilon (ε) classes have undergone lineage-specific expansion and functional diversification in insects. Although sequence and structural analyses have identified class-specific substitutions within the glutathione-binding (G) and hydrophobic substrate-binding (H) sites, the extent to which these differences are reflected in intrinsic protein dynamics has remained unclear. By integrating template-aligned structural models, reference position mapping, physicochemical profiling, and elastic network-based essential dynamics analysis to compare δ and ε GSTs across multiple mosquito species, it is shown that class-specific differences in backbone dynamics are spatially structured rather than uniformly distributed across the fold. Residue-level fluctuation analysis reveals that dynamically divergent positions cluster within the conserved GST core surrounding the G-site and along the distal arm of the H-site cavity, while the proximal H-site arm remains comparatively constrained. This spatial organisation mirrors ligand geometry in ε-class templates, where glutathione occupies a conserved scaffold and hydrophobic substituents extend into a broader distal cavity. Together, these findings support a model in which glutathione handling is dynamically conserved, whereas class-specific flexibility at the distal H-site enables tuning of substrate recognition. More broadly, this study identifies intrinsic protein dynamics as a key dimension of GST functional diversification and provides a mechanistic framework for understanding how mosquito GSTs adapt to chemically diverse insecticides.