Binding Properties of General Odorant Binding Proteins from the Oriental Fruit Moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae)
Binding Properties of General Odorant Binding Proteins from the Oriental Fruit Moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae)
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东方果蛾、小食心虫 (Busck)（鳞翅目 Tortricidae）的一般气味结合蛋白的结合特性
DOI：
10.1371/journal.pone.0155096
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发表时间：
2016-05-06
期刊：
影响因子：
3.7
通讯作者：
Wu, Junxiang
中科院分区：
文献类型：
--
作者：
Li, Guangwei;Chen, Xiulin;Wu, Junxiang
东方小食心虫是一种寄主转换害虫。成虫高度依赖嗅觉线索来寻找最佳寄主植物和产卵场所。气味结合蛋白（OBP）被认为负责识别和运输疏水性气味物质穿过水感器淋巴，以刺激触角感器内的气味受体（OR）并激活嗅觉信号转导途径。研究这些OBP的生理功能有助于理解昆虫的化学通讯。molesta的体外表达和纯化。G.用荧光配体结合法测定了梨小食心虫GOBP 1和2（GmolGOBP 1和2）的性信息素组分和寄主植物挥发物。采用整体荧光免疫组织化学（WM-FIHC）方法研究了GmolGOBP 1和2在触角感器中的分布。利用同源模建、分子对接和定点突变技术预测了GmolGOBP 2的结合位点。GmolGOBP 1和2都位于锥形感器中，雄性和雌性触角没有差异。重组GmolGOBP 1（rGmolGOBP 1）对寄主植物挥发物和性信息素组分表现出广泛的结合特性; rGmolGOBP 2不能有效地结合寄主植物挥发物，但对次要的性信息素组分十二烷醇表现出特异性结合亲和力。我们选择GmolGOBP 2和十二烷醇进行进一步的同源建模、分子对接和定点突变。突变体的结合亲和力表明，Thr 9是关键的结合位点，并确认十二烷醇与蛋白质的键合涉及氢键。结合pH值对rGmolGOBP 2结合亲和力的影响，GmolGOBP 2的配体结合和释放与pH依赖性的构象transition.ConclusionTwo rGmolGOBP表现出不同的测试配体的结合特性。rGmolGOBP 1具有识别寄主植物挥发物和性信息素成分的双重功能，而rGmolGOBP 2主要参与次要性信息素成分十二醇的感知。本研究还为重组蛋白配体结合特性中关键氨基酸的预测功能提供了经验证据。
BackgroundThe oriental fruit moth Grapholita molesta is a host-switching pest species. The adults highly depend on olfactory cues in locating optimal host plants and oviposition sites. Odorant binding proteins (OBPs) are thought to be responsible for recognizing and transporting hydrophobic odorants across the aqueous sensillum lymph to stimulate the odorant receptors (ORs) within the antennal sensilla and activate the olfactory signal transduction pathway. Exploring the physiological function of these OBPs could facilitate understanding insect chemical communications.Methodology/Principal FindingTwo antennae-specific general OBPs (GOBPs) of G. molesta were expressed and purified in vitro. The binding affinities of G. molesta GOBP1 and 2 (GmolGOBP1 and 2) for sex pheromone components and host plant volatiles were measured by fluorescence ligand-binding assays. The distribution of GmolGOBP1 and 2 in the antennal sensillum were defined by whole mount fluorescence immunohistochemistry (WM-FIHC) experiments. The binding sites of GmolGOBP2 were predicted using homology modeling, molecular docking and site-directed mutagenesis. Both GmolGOBP1 and 2 are housing in sensilla basiconica and with no differences in male and female antennae. Recombinant GmolGOBP1 (rGmolGOBP1) exhibited broad binding properties towards host plant volatiles and sex pheromone components; rGmolGOBP2 could not effectively bind host plant volatiles but showed specific binding affinity with a minor sex pheromone component dodecanol. We chose GmolGOBP2 and dodecanol for further homology modeling, molecular docking, and site-directed mutagenesis. Binding affinities of mutants demonstrated that Thr9 was the key binding site and confirmed dodecanol bonding to protein involves a hydrogen bond. Combined with the pH effect on binding affinities of rGmolGOBP2, ligand binding and release of GmolGOBP2 were related to a pH-dependent conformational transition.ConclusionTwo rGmolGOBPs exhibit different binding characteristics for tested ligands. rGmolGOBP1 has dual functions in recognition of host plant volatiles and sex pheromone components, while rGmolGOBP2 is mainly involved in minor sex pheromone component dodecanol perception. This study also provides empirical evidence for the predicted functions of key amino acids in recombinant protein ligand-binding characteristics.