Expression of Cry1Ac toxin-binding region in Plutella xyllostella cadherin-like receptor and studying their interaction mode by molecular docking and site-directed mutagenesis
Expression of Cry1Ac toxin-binding region in Plutella xyllostella cadherin-like receptor and studying their interaction mode by molecular docking and site-directed mutagenesis
复制标题
小菜蛾钙粘蛋白样受体Cry1Ac毒素结合区的表达及其相互作用模式的分子对接和定点突变研究
DOI：
10.1016/j.ijbiomac.2017.12.135
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发表时间：
2018-05-01
影响因子：
8.2
通讯作者：
Liu, Xianjin
中科院分区：
文献类型：
--
作者：
Hu, Xiaodan;Zhang, Xiao;Liu, Xianjin
钙粘蛋白样蛋白（Cadherin-like protein）是苏云金芽孢杆菌（Bacillus thuringiensis, Bt） Cry毒素的主要受体，在杀虫过程中起关键作用。在本研究中，我们成功地在大肠杆菌BL21 （DE3）中表达了小菜蛾cadherin-like的推测的Cry1Ac毒素结合区（CR7-CR11）。在变性（配体blot）和非变性（ELISA）条件下，表达的CR7-CR11片段显示出与Cry1Ac毒素的结合能力。通过同源性建模构建了CR7-CR11的三维结构。CR7-CR11与Cry1Ac的分子对接结果显示，Cry1Ac的结构域II和结构域III参与了与CR7-CR11的结合，而CR7-CR8则是CR7-CR11与Cry1Ac的相互作用区域。毒素受体复合物的相互作用是由氢键和疏水相互作用引起的。通过计算机辅助丙氨酸突变扫描，预测Cry1Ac （Met341、Asn442和Ser486）和CR7-CR11 （Asp32、Arg101和Arg127）氨基酸残基是参与毒素受体复合物相互作用的热点残基。最后通过结合实验验证了这些关键氨基酸残基的重要作用。这些结果将为进一步阐明Cry毒素的杀虫机制和通过分子修饰增强Cry毒素的杀虫活性奠定基础。(C) 2018 Elsevier B.V.版权所有
Cadherin-like protein has been identified as the primary Bacillus thuringiensis (Bt) Cry toxin receptor in Lepidoptera pests and plays a key role in Cry toxin insecticidal. In this study, we successfully expressed the putative Cry1Ac toxin-binding region (CR7-CR11) of Plutella xylostella cadherin-like in Escherichia colt BL21 (DE3). The expressed CR7-CR11 fragment showed binding ability to Cry1Ac toxin under denaturing (Ligand blot) and non-denaturing (ELISA) conditions. The three-dimensional structure of CR7-CR11 was constructed by homology modeling. Molecular docking results of CR7-CR11 and Cry1Ac showed that domain II and domain III of Cry1Ac were taking part in binding to CR7-CR11, while CR7-CR8 was the region of CR7-CR11 in interacting with Cry1Ac. The interaction of toxin-receptor complex was found to arise from hydrogen bond and hydrophobic interaction. Through the computer-aided alanine mutation scanning, amino acid residues of Cry1Ac (Met341, Asn442 and Ser486) and CR7-CR11 (Asp32, Arg101 and Arg127) were predicted as the hot spot residues involved in the interaction of the toxin-receptor complex. At last, we verified the importance role of these key amino acid residues by binding assay. These results will lay a foundation for further elucidating the insecticidal mechanism of Cry toxin and enhancing Cry toxin insecticidal activity by molecular modification. (C) 2018 Elsevier B.V. All rights reserved.