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. 2022 Jul 30;23(15):8456.
doi: 10.3390/ijms23158456.

Key Residues Affecting Binding Affinity of Sirex noctilio Fabricius Odorant-Binding Protein (SnocOBP9) to Aggregation Pheromone

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Key Residues Affecting Binding Affinity of Sirex noctilio Fabricius Odorant-Binding Protein (SnocOBP9) to Aggregation Pheromone

Enhua Hao et al. Int J Mol Sci. .

Abstract

Sirex noctilio Fabricius (Hymenoptera Siricidae) is a major quarantine pest responsible for substantial economic losses in the pine industry. To achieve better pest control, (Z)-3-decen-ol was identified as the male pheromone and used as a field chemical trapping agent. However, the interactions between odorant-binding proteins (OBPs) and pheromones are poorly described. In this study, SnocOBP9 had a higher binding affinity with Z3D (Ki = 1.53 ± 0.09 μM) than other chemical ligands. Molecular dynamics simulation and binding mode analysis revealed that several nonpolar residues were the main drivers for hydrophobic interactions between SnocOBP9 and Z3D. Additionally, computational alanine scanning results indicated that five amino acids (MET54, PHE57, PHE71, PHE74, LEU116) in SnocOBP9 could potentially alter the binding affinity to Z3D. Finally, we used single-site-directed mutagenesis to substitute these five residues with alanine. These results imply that the five residues play crucial roles in the SnocOBP9-Z3D complex. Our research confirmed the function of SnocOBP9, uncovered the key residues involved in SnocOBP9-Z3D interactions, and provides an inspiration to improve the effects of pheromone agent traps.

Keywords: Sirex noctilio; aggregation pheromone; computational simulation; fluorescence binding assay; odorant-binding proteins; site-directed mutagenesis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The sequences of SnocOBP9 in S. noctilio and the other six PBPs of Hymenoptera were aligned. Sequence conservation was determined based on the overall height of the stack at that position, and the height of the symbols within the stack suggests the relative frequency of each amino acid at that position. Conserved cysteines are marked with purple dots. The insect species and GenBank accession numbers are the following: Formica exsecta PBP (XP_029680467.1), Belonocnema treatae PBP (XP_033213373.1), Nylanderia fulva PBP (XP_029165894.1), Orussus abietinus PBP (XP_012277309.1), Dinoponera quadriceps PBP (XP_014476791.1), Odontomachus brunneus PBP (XP_032663926.1).
Figure 2
Figure 2
(A) Western blot analysis of SnocOBP9 with 6*His-Tag. Lane M represents molecular marker. (B) SDS-PAGE analyses of the expression and purification of recombinant SnocOBP9. Lane SnocOBP9 represents purified recombinant protein; lane M represents molecular marker.
Figure 3
Figure 3
UV (180–260 nm) scan of SnocOBP9. The abscissa represents the scanning wavelength, and the ordinate represents the molar ellipticity [θ] (deg·cm2 dmol−1).
Figure 4
Figure 4
(A) Binding curve for 1-NPN to SnocOBP9 with Scatchard plot. Dissociation constant was Kd = 6.87 ± 0.73 μM. (B) The chemical structure.
Figure 5
Figure 5
Binding curves for host volatiles (A), symbiotic fungal volatiles (B) and insect pheromone volatiles (C) to SnocOBP9. The ligand names are shown on the right side in the curves. The binding data are listed in Table 3.
Figure 6
Figure 6
(A) Structural modeling of SnocOBP9; templates A. mellifera ASP1 (PDB ID: 3cdn_A) and model SnocOBP9. (B) The amino acid sequences alignment of SnocOBP9 and the template A. mellifera pheromone-binding protein (AmelASP1); 3CDN_A represents AmelASP1. Disulfide bridges rendered in green digits “1−3”.
Figure 7
Figure 7
(A) The time-evolution RMSD curves (200 ns) of the SnocOBP9-Z3D complex, SnocOBP9 and Z3D. (B) RMSF curve of SnocOBP9-Z3D complex (50~200 ns).
Figure 8
Figure 8
The hydrophobic cavities of Z3D (ten representations) anchored in SnocOBP9 displayed in surface view.
Figure 9
Figure 9
Representative conformation (Cluster I) for the SnocOBP9-Z3D complex produced based on the MD simulation trajectories. Representative residues, including MET54, PHE57, PHE71, PHE74 and LEU116, are marked on the binding interface.
Figure 10
Figure 10
(A) Each residue for the SnocOBP9-Z3D complex calculated from the equilibrated conformations with 100–200 ns MD. Residues contributing more than −1.00 kcal/mol to the binding free energy are marked by the blue line. (B) Centroid distance between the five key amino acids and Z3D.
Figure 11
Figure 11
(A) Binding curve of SnocOBP9WT and mutant SnocOBP9 proteins. SnocOBP9WT represents the wild-type SnocOBP9 proteins. SnocOBP9M54A, SnocOBP9F57A, SnocOBP9F71A, SnocOBP9F74A and SnocOBP9L116A refer to MET54, PHE57, PHE71, PHE74 and LEU116 substitutions with ALA in SnocOBP9. (B) Comparison of the binding affinities (indicated by 1/Ki) for wild-type (WT) SnocOBP9 and SnocOBP9F57A to Z3D. Different letters indicate significant differences between SnocOBP9WT and SnocOBP9F57A (p < 0.05, ANOVA, Tukey’s HSD).

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