State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China.
Insect Mol Biol. 2013 Apr;22(2):172-82. doi: 10.1111/imb.12012. Epub 2013 Jan 7.
Olfaction plays an important role in insects' survival and reproduction. Odorant-binding proteins (OBPs) are considered to be one of the crucial proteins in the insect olfactory pathway. In this study, an antenna-specific OBP of the alfalfa plant bug, Adelphocoris lineolatus AlinOBP5, was expressed and purified in vitro. The binding affinities of AlinOBP5 with sex pheromone analogues of the Miridae and cotton volatiles were investigated by fluorescence competitive binding assays. The binding sites of AlinOBP5 were predicted by three-dimensional structure modelling and molecular docking, and site-directed mutagenesis. AlinOBP5 could not effectively bind with sex pheromone analogues of Miridae but showed high binding abilities with specific cotton volatiles, such as cis-nerolidol, ethyl laurate, β-ionone, β-caryophyllene, 2,3-dimethylbenzoic acid and (E)-farnesol. The strongest binding affinity was to cis-nerolidol, suggesting a role of AlinOBP5 in general odorant chemoreception. Based on the relatively strong binding affinity and the reported physiological activity of cis-nerolidol in other insects, we chose cis-nerolidol for further homology modelling and ligand docking. The results of molecular simulation and site-directed mutagenesis indicated that two amino acids, Lys74 and Pro121, in the protein binding pocket are the key amino acids involved in the binding of cis-nerolidol. The Lys74 residue may participate in specific recognition of ligands, and the Pro121 residue plays a crucial role in ligand binding and release by changing the binding pocket environment and stabilizing the conformation of the C-terminus of AlinOBP5.
嗅觉在昆虫的生存和繁殖中起着重要作用。气味结合蛋白（OBP）被认为是昆虫嗅觉途径中的关键蛋白之一。在这项研究中，苜蓿盲蝽的一个触角特异性 OBP，即 AlinOBP5，在体外被表达和纯化。通过荧光竞争结合测定法研究了 AlinOBP5 与盲蝽科性信息素类似物和棉挥发物的结合亲和力。通过三维结构建模、分子对接和定点突变预测了 AlinOBP5 的结合位点。AlinOBP5 不能有效地与盲蝽科性信息素类似物结合，但对特定的棉挥发物，如顺式-橙花叔醇、月桂酸乙酯、β-紫罗兰酮、β-石竹烯、2,3-二甲基苯甲酸和(E)-法呢醇，表现出较高的结合能力。与顺式-橙花叔醇的结合亲和力最强，表明 AlinOBP5 在一般气味化学感受中起作用。基于顺式-橙花叔醇在其他昆虫中相对较强的结合亲和力和报道的生理活性，我们选择顺式-橙花叔醇进行进一步的同源建模和配体对接。分子模拟和定点突变的结果表明，蛋白质结合口袋中的两个氨基酸，Lys74 和 Pro121，是参与顺式-橙花叔醇结合的关键氨基酸。Lys74 残基可能参与配体的特异性识别，Pro121 残基通过改变结合口袋环境和稳定 AlinOBP5 的 C 末端构象，在配体结合和释放中起着关键作用。
