Ostrinia furnacalis PBP2 solution NMR structure: Insight into ligand binding and release mechanisms.

Ostrinia furnacalis is an invasive lepidopteran agricultural pest that relies on olfaction for mating and reproduction. Male moths have an extremely sensitive olfactory system that can detect the sex pheromones emitted by females over a great distance. Pheromone-binding proteins present in the male moth antenna play a key role in the pheromone uptake, transport, and release at the dendritic membrane of the olfactory neuron. Here, we report the first high-resolution NMR structure of a pheromone-binding protein from an Ostrinia species at pH 6.5. The core of the Ostrinia furnacalis PBP2 (OfurPBP2) consists of six helices, α1a (2-14), α1b (16-22), α2 (27-37), α3 (46-60), α4 (70-80), α5 (84-100), and α6 (107-124) surrounding a large hydrophobic pocket. The structure is stabilized by three disulfide bridges, 19-54, 50-108, and 97-117. In contrast to the unstructured C-terminus of other lepidopteran PBPs, the C-terminus of OfurPBP2 folds into an α-helix (α7) at pH 6.5. The protein has nanomolar affinity towards both pheromone isomers. Molecular docking of both pheromones, E-12 and Z-12-tetradecenyl acetate, to OfurPBP2 revealed that the residues Met5, Lys6, Met8, Thr9, Phe12, Phe36, Trp37, Phe76, Ser115, Phe118, Lys119, Ile122, His123, and Ala128 interact with both isomers, while Thr9 formed a hydrogen bond with the acetate head group. NMR structure and thermal unfolding studies with CD suggest that ligand release at pH 4.5 is likely due to the partial unfolding of the protein.

1H, 13C, and 15N resonance assignment and secondary structure of the pheromone-binding protein2 from the agricultural pest Ostrinia furnacalis (OfurPBP2).

Ostrinia furnacalis, a lepidopteran moth, is an invasive pest found in Asia, Australia, Africa, and parts of the United States. The O. furnacalis pheromone-binding protein2 (OfurPBP2), present in the male moth antenna, plays a role in the detection of female-secreted pheromone in a process that leads to mating. To understand the structural mechanism of pheromone binding and release in this pest, we have initiated characterization of OfurPBP2 by solution NMR. Here, we report the backbone resonance assignments and the secondary structural elements of OfurPBP2 at pH 6.5 using uniformly 13C, 15N-labeled protein with various triple resonance NMR experiments. The assignments are 97% completed for backbone and 88% completed for side-chain resonances. The secondary structure of OfurPBP2, based on backbone chemical shifts, consists of eight α-helices, including a well-structured C-terminal helix.

Pheromone Perception: Mechanism of the Reversible Coil-Helix Transition in Antheraea polyphemus Pheromone-Binding Protein 1.

Pheromone-binding protein (PBP) in male moth antennae transports pheromone to the olfactory receptor neuron by undergoing a pH-dependent conformational switch, from PBPB at higher pH to PBPA at lower pH, associated with ligand binding and release, respectively. The characteristic feature of the dramatic protein switch is the pH-dependent reversible coil-helix transition of the C-terminus. In the PBPB conformation at pH >6.0, the C-terminus is exposed to the solvent as a coil while the ligand occupies the hydrophobic pocket. However, in the PBPA conformation at acidic pH, the C-terminus switches to a helix and releases the ligand by outcompeting it for the hydrophobic pocket. In Antheraea polyphemus PBP1 (ApolPBP1), the C-terminus (P129-V142) is composed predominantly of hydrophobic residues except for three strategically located acidic residues: Asp132, Glu137, and Glu141. Here, we report for the first time on the consequences of the mutation of one or more acidic residues in the pH-driven reversible coil-helix transition of the ApolPBP1 C-terminus through biophysical characterization. Mutation of any single acidic residue in the C-terminus to its neutral counterpart destabilizes the helix formation at lower pH; these mutants exist as a mixture of both conformations. However, mutation of the two terminal acidic residues together knocks out the protein switch and adversely affects both ligand binding and release functions. Thus, these mutant proteins remain in the open (PBPB) conformation at all pH levels.

Structure and Function Studies of Asian Corn Borer Ostrinia furnacalis Pheromone Binding Protein2.

Lepidopteran male moths have an extraordinarily sensitive olfactory system that is capable of detecting and responding to minute amounts of female-secreted pheromones over great distances. Pheromone-binding proteins (PBPs) in male antennae ferry the hydrophobic ligand across the aqueous lymph to the olfactory receptor neuron triggering the response. PBPs bind ligands at physiological pH of the lymph and release them at acidic pH near the receptor while undergoing a conformational change. In Anthereae polyphemus PBP1, ligand binding to the hydrophobic pocket and its release is regulated by two biological gates: His70 and His95 at one end of the pocket and C-terminus tail at the other end. Interestingly, in Asian corn borer Ostrinia furnacalis PBP2 (OfurPBP2), critical residues for ligand binding and release are substituted in both biological gates. The impact of these substitutions on the ligand binding and release mechanism in OfurPBP2 is not known. We report here overexpression of soluble OfurPBP2 and structural characterization at high and low pH by circular dichroism (CD) and NMR. Ligand binding and ab initio model development were carried out with fluorescence and small-angle X-ray scattering (SAXS) respectively. OfurPBP2 in solution at pH 6.5 is homogeneous, well-folded and has a compact globular shape.