Structural and Functional Insights into pH-Dependent Dynamics of Ostrinia furnacalis Pheromone-Binding Protein 3...

Male moths of Ostrinia furnacalis (Asian corn borer) rely on a highly sensitive olfactory system to detect female-emitted sex pheromones essential for mating. Pheromone-binding proteins (PBPs), expressed in male antennae, transport hydrophobic pheromones through the aqueous sensillar lymph to olfactory receptors. OfurPBP3, a male-biased PBP, plays a key role in species-specific pheromone detection. Here, we report the cloning, recombinant expression, refolding, purification, and structural characterization of OfurPBP3. Fluorescence binding assays revealed high nanomolar affinity for both E- and Z-isomers of tetradec-12-en-1-yl acetate pheromone. Circular dichroism and NMR studies demonstrated that although OfurPBP3 retains a predominantly α-helical secondary structure under acidic conditions, its tertiary structure is perturbed, exhibiting increased conformational dynamics and features consistent with partial unfolding. Upon restoration to pH 6.5, the native tertiary structure is recovered, indicating that the pH-induced structural changes are reversible. Homology modeling predicted a conserved PBP fold containing a hydrophobic pocket. Molecular docking studies identified residues potentially involved in ligand recognition. The behaviors of OfurPBP3 in acidic pH are reminiscent of the molten-globule-like states previously reported for other pheromone-binding proteins from Ostrinia species. Collectively, these findings suggest that ligand release in Ostrinia PBPs is associated with pH-dependent structural destabilization and partial unfolding, rather than a canonical two-state (open ⇌ closed) conformational switch as described for several other lepidopteran PBPs, such as ApolPBP1.