title: Three amino acid residues of an odorant-binding protein are involved in binding odours in Loxostege sticticalis L.
abstract: Odorant-binding proteins (OBPs) play an important role in insect olfactory processes and are thought to be responsible for the transport of pheromones and other semiochemicals across the sensillum lymph to the olfactory receptors within the antennal sensilla. As an important general odorant binding protein in the process of olfactory recognition, LstiGOBP1 of Loxostege sticticalis L. has been shown to have good affinity to various plant volatiles. However, the binding specificity of LstiGOBP1 should be further explored in order to better understand the olfactory recognition mechanism of L. sticticalis. In this study, real-time PCR experiments indicated that LstiGOBP1 was expressed primarily in adult antennae. Homology modelling and molecular docking were then conducted on the interactions between LstiGOBP1 and 1-heptanol to understand the interactions between LstiGOBP1 and their ligands. Hydrogen bonds formed by amino acid residues might be crucial for the ligand-binding specificity on molecular docking, a hypothesis that was tested by site-directed mutagenesis. As predicted binding sites for LstiGOBP1, Thr15, Trp43 and Val14 were replaced by alanine to determine the changes in binding affinity. Finally, fluorescence assays revealed that the mutants Thr15 and Trp43 had significantly decreased binding affinity to most odours; in mutants that had two-site mutations, the binding to the six odours that were tested was completely abolished. This result indicates that Thr15 and Trp43 were involved in binding these compounds, possibly by forming multiple hydrogen bonds with the functional groups of the ligands. These results provide new insights into the detailed chemistry of odours' interactions with proteins.
authors.affiliation: State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China.
authors.forename: J
authors.initials: J
authors.lastname: Yin
authors.affiliation: State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China.
authors.forename: X
authors.initials: X
authors.lastname: Zhuang
authors.affiliation: State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China.
authors.forename: Q
authors.initials: Q
authors.lastname: Wang
authors.affiliation: State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Cao
authors.affiliation: State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China.
authors.forename: S
authors.initials: S
authors.lastname: Zhang
authors.affiliation: College of Plant Protection, Yunnan Agricultural University, Kunming, China.
authors.forename: C
authors.initials: C
authors.lastname: Xiao
authors.affiliation: State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China.
authors.forename: K
authors.initials: K
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D057506:Arthropod Antennae / Q000378:metabolism; D060829:Arthropod Proteins / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D020869:Gene Expression Profiling; D019850:Heptanol / Q000737:chemistry; D008297:Male; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D009036:Moths / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009812:Odorants; D011485:Protein Binding; D018035:Receptors, Odorant / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D060829:Arthropod Proteins; D018035:Receptors, Odorant; C045648:odorant-binding protein; D019850:Heptanol
keywords: Loxostege sticticalis; bioinformatics; fluorescence assays; mutants; odorant binding protein