Structural and Functional Characterization of European Corn Borer, Ostrinia nubilalis, Pheromone Binding Protein 3

Abstract

Ostrinia nubilalis, a lepidopteran moth, also known as the European corn borer, has a major impact on the production of economically important crops in the United States and Europe. The female moth invites the male moth for mating through the release of pheromones, a volatile chemical signal. Pheromone binding proteins (PBPs) present in the male moth antennae are believed to pick up the pheromones, transport them across the aqueous sensillum lymph, and deliver them to the olfactory receptor neurons. Here we report for the first time the cloning, expression, refolding, purification, and structural characterization of Ostrinia nubilalis PBP3 (OnubPBP3). The recombinant protein showed nanomolar affinity to each isomer of the Ostrinia pheromones, E- and Z-11-tetradecenyl acetate. In a pH titration study by nuclear magnetic resonance, the protein exhibited an acid-induced unfolding at pH below 5.5. The molecular dynamics simulation study demonstrated ligand-induced conformational changes in the protein with both E- and Z-isomers of the Ostrinia pheromone. The simulation studies showed that while protein flexibility decreases upon binding to E-pheromone, it increases when bound to Z-pheromone. This finding suggests that the OnubPBP3 complex with E-pheromone is more stable than with Z-pheromone.

Ostrinia furnacalisPBP2 solution NMR structure: Insight into ligand binding and release mechanisms

“…Recombinant OfurPBP2 for the binding assay was expressed in bacteria and purified following the previously reported method 14,17 . Delipidation was carried out to remove the lipid bound to the protein from the bacterial cells 21,24 . The successful delipidation of OfurPBP2 was verified by 2D { 1 H, 15 N} HSQC (Figure S1).…”

“…14,17 Delipidation was carried out to remove the lipid bound to the protein from the bacterial cells. 21,24 The successful delipidation of OfurPBP2 was verified by 2D { 1 H, 15 N} HSQC (Figure S1).…”

“…Indeed, Ostrinia nubilalis PBP3(OnubPBP3) behaves in a similar manner 24 . It seems likely that at acidic pH, the protonated His88 would disrupt the cation–π interactions with Arg70.…”

“…Indeed, Ostrinia nubilalis PBP3(OnubPBP3) behaves in a similar manner. 24 It seems likely that at acidic pH, the protonated His88 would disrupt the cation-π interactions with Arg70. In addition, the repulsion between the positively charged His88 (α5) and His95 (α5) would disrupt the helix, causing it to unwind and unfold partially at pH 4.5.…”

“…OfurPBP2 was delipidated using a protocol modified from prior work. 21,24 Briefly, OfurPBP2 in 50 mM sodium citrate buffer (buffer A) at pH 4.5 was loaded to a column packed with Lipidex-1000 and equilibrated in buffer A. The column loaded with the protein was incubated at 37 C for 30 min.…”