桃小食心虫化学感受蛋白CSP16配体结合特性

【目的】桃小食心虫（Carposina sasakii）是我国北方果树生产中的重要害虫之一,本文通过体外表达桃小食心虫化学感受蛋白CsasCSP16,明确其与寄主挥发物分子和性信息素分子的结合特性,并通过生物信息学预测CsasCSP16与挥发物分子结合的关键氨基酸位点,为揭示桃小食心虫嗅觉的分子机理提供理论依据。【方法】通过原核表达系统获得CsasCSP16蛋白,并使用Ni-NTA柱纯化重组蛋白。采用荧光竞争结合的方法,以N-苯基-1-萘胺（N-pheny-1-naphthylamine,1-NPN）为荧光探针,从33种寄主挥发物和2种性信息素分子中筛选出与CsasCSP16结合亲和性高的配体分子。通过对CsasCSP16进行同源建模获得其三维结构模型,以CSPMbraA6（PDB ID：1N8U）为模板成功构建CsasCSP16的三维结构模型。使用Autodock Vina软件将CsasCSP16与亲和性高的配体分子进行对接,构建蛋白-配体复合物。然后使用GROMACS（2019.3）软件对复合物进行动力学模拟,从动力学模拟的平衡状态中选取100个构象,使用g_mmpbsa软件计算CsasCSP16与气味分子的结合能,并通过能量分解的方法预测关键氨基酸残基。【结果】成功构建了CsasCSP16的克隆表达载体,通过大肠杆菌原核表达系统获得高纯度的重组蛋白。与35种配体分子的荧光竞争结合表明,CsasCSP16与水杨酸甲酯、6-甲基-5-庚烯-2-酮、十五烷、庚酸丁酯和α-蒎烯有较强的结合活性,其Ki值分别为6.59、6.25、3.50、6.73和4.47 μmol·L^-1。分子对接的结果表明,CsasCSP16与水杨酸甲酯、6-甲基-5-庚烯-2-酮、十五烷、庚酸丁酯和α-蒎烯的Vina Score值分别为-6.1、-5.3、-5.8、-5.2和-6.6。动力学模拟结果表明,CsasCSP16与气味分子复合物在50 ns内达到平衡状态,通过g_mmpbsa计算复合物的结合能,CsasCSP16-水杨酸甲酯、CsasCSP16-6-甲基-5-庚烯-2-酮、CsasCSP16-十五烷和CsasCSP16-庚酸丁酯的结合自由能分别为-50.264、-65.551、-136.035和-93.805 kJ·mol^-1;最后,通过分解每个氨基酸贡献的结合自由能表明异亮氨酸49（Ile49）、缬氨酸71（Val71）、异亮氨酸72（Ile72）和酪氨酸90（Tyr90）贡献的结合能>3 kJ·mol^-1,因此推测这4种氨基酸在CsasCSP16结合气味分子的过程中发挥关键作用。【结论】桃小食心虫CsasCSP16能与寄主植物的多种气味分子结合,推测其可能在桃小食心虫对寄主植物的定位过程中发挥重要作用。异亮氨酸49（Ile49）、缬氨酸71（Val71）、异亮氨酸72（Ile72）和酪氨酸90（Tyr90）可能是CsasCSP16与配体分子结合的关键氨基酸位点。

Ligands Binding Characteristics of Chemosensory Protein CsasCSP16 of Carposina sasakii

【Objective】Carposina sasakii is one of the most destructive pests on orchards in north China. The objectives of this study are to get the recombinant CsasCSP16 protein, characterize the binding profiles of CsasCSP16 with some small molecular volatiles from host plants, and predict the key residue between CsasCSP16 and putative ligands. In overall this research possibly lays a theoretical and practical foundation for the understanding of olfactory mechanism in C. sasakii.【Method】The recombinant protein CsasCSP16 was induced to express by constructing prokaryotic expression system, and purified by using the Ni-NTA agarose affinity column. Then, the fluorescence competitive assay was applied, and N-phenyl-1-naphthylamine (1-NPN) was selected as the fluorescence probe to measure the binding profiles of CsasCSP16 recombinant with 33 host plant volatiles and 2 sex pheromone compounds. The Modeller software was used to build three-dimensional model. CSPMbraA6 (PDB ID: 1N8U) was used as the template for homology modeling of CsasCSP16 protein structure. The modeled structure of CsasCSP16 was docked with 35 selected ligands by Autodock Vina. Then, the complexes from above step were introduced to GROMACS (2019.3) to conform the stability. Moreover, the binding energies of the complexes were calculated by g_mmpbsa and the key residues interaction between CsasCSP16 and host volatiles were predicted.【Result】The recombinant expression vector was successfully constructed, and the recombinant protein of CsasCSP16 with high purity was obtained by bacterial system. Further, competitive fluorescence binding assays with 35 candidate ligands showed that CsasCSP16 had high binding affinities against methyl salicylate, 6-methyl-5-hepten-2-one, pentadecane, butyl heptanoate and α-pinene, and the recorded Ki values were 6.59, 6.25, 3.50, 6.73 and 4.47 μmol·L^-1, respectively. Ligand docking results revealed that the Vina Scores of CsasCSP16-methyl salicylate, CsasCSP16-6-methyl-5-hepten-2-one, CsasCSP16-pentadecane, CsasCSP16-butyl heptanoate and CsasCSP16-α-pinene were -6.1, -5.3, -5.8, -5.2 and -6.6, respectively. Finally, the g_mmpbsa analysis demonstrated that the binding energies of CsasCSP16-methyl salicylate, CsasCSP16-6-methyl-5- hepten-2-one, CsasCSP16-pentadecane and CsasCSP16-butyl heptanoate were -50.264, -65.551, -136.035 and -93.805 kJ·mol^-1, respectively. Ile49, Val71, Ile72 and Tyr90 contribute energy more than 3 kJ·mol^-1, suggesting their key involvement in the function of CsasCSP16. 【Conclusion】The CsasCSP16 has a certain binding capacity with several odors from host plant volatiles, suggesting that it may play an important role in the localization of host plants. Ile49, Val71, Ile72, and Tyr90 may be the key residues involved in the function of CsasCSP16.