Heterologous expression, structural analysis, and functional characterization of Ostrinia nubilalis pheromone-binding...

The European corn borer (Ostrinia nubilalis) is a major lepidopteran pest, causing annual maize losses of 5.5-8.5 million tons worldwide. Successful mating relies on the male's ability to detect species-specific, hydrophobic sex pheromones released by females. Pheromone-binding proteins (PBPs), abundantly expressed in male antennae, capture these pheromones and transport them through the sensillar lymph to olfactory receptor neurons. Here, we report the first successful heterologous expression, purification, and functional characterization of O. nubilalis PBP2 (OnubPBP2). Fluorescence binding assays show that recombinant OnubPBP2 is active and binds both E- and Z-isomers of 11-tetradecenyl acetate with nanomolar affinity at pH 6.5. The calculated binding energies based on molecular docking simulation are in good agreement with the binding affinities measured using fluorescence displacement assays. High-resolution NMR titrations reveal a reversible pH-dependent conformational transition, with OnubPBP2 adopting a partially unfolded, molten-globule-like state under acidic conditions. Such conformational flexibility, observed across several Ostrinia PBPs, likely represents an adaptive mechanism that balances structural stability with ligand release efficiency, defining a divergent PBP subclass with unique pH-dependent dynamics likely contributing to enhanced functional versatility.