Overexpression, characterization, and structure-function studies of pheromone binding proteins from European corn borer, Ostrinia nubilalis
Abstract
Ostrinia nubilalis (European corn borer) has a huge impact on production of economically important crops in the USA and Europe. This species, similar to other lepidopterans, relies on olfaction to find their mating partners. The moth male is invited by the female through a chemical signal started with releasing sex pheromones. Pheromone binding proteins (PBPs) are a key component to bind and deliver the received sex pheromones to olfactory receptors in male moth antennae. The detailed structural and mechanistic studies will aid to biorationally design pheromone mimetics for an effective pest management. OnubPBP3 is one of the Ostrinia nubilalis PBPs and it is expressed at higher levels in the antennae of males than in the females. Here, we report the overexpression, refolding, purification, and biophysical characterization of the recombinant OnubPBP3 by using different techniques including fluorescence spectroscopy, circular dichroism (CD), small angle X-ray scattering (SAXS), and high-resolution solution NMR. We determined the binding affinity of the recombinant OnubPBP3 to both Ostrinia nubilalis pheromones, E11-tetradecenyl acetate and Z11- tetradecenyl acetate. The recombinant protein showed nanomolar affinity to each isomer of the Ostrinia nubilalis pheromones. Ligand-induce conformational change was observed in OnubPBP3 by using NMR spectroscopy. The high-resolution three-dimensional structure of OnubPBP3 was determined by using solution NMR. The structure of OnubPBP3 consists of eight a-helices with residues 3-9 (a1a), 15-23 (a1b), 27-35 (a2), 48-57 (a3), 72-79 (a4), 84-98 (a5), 114-116 (a6a), and 120-123 (a6b), held together by three disulfide bridges: 19-54, 50-107 and 97-116 forming a large hydrophobic pocket inside the protein. The pheromone binding site of the protein was detected by molecular docking. Molecular dynamics simulation study demonstrates ligand-induced conformational and flexibility changes in the protein with both E and Z-pheromone. Characterization of pH-dependent conformational changes of OnubPBP3 via CD, NMR spectroscopy, and SAXS were shown a distinguishable behavior as compared to the well-investigated lepidopteran PBPs at the same pH. Our findings suggest that OnubPBP3 in acidic conditions is in a molten globule state.
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- OSU Dissertations [11222]