Hou Xiaoyue, Mao Chengjuan, Zhang Wen, Jiang Lei, Li Mengying, Guo Jixin, Yang Chenru, Wang Jinxuan, Fang Yaowei, Jiang Xu, Cai Jun
Jiangsu Key Laboratory of Marine Bioresources and Environment, Jiangsu Ocean University, Lianyungang, China.
Co-Innovation Center of Jiangsu Marine Bio-Industry Technology, Jiangsu Ocean University, Lianyungang, China.
Pest Manag Sci. 2025 Sep;81(9):5686-5695. doi: 10.1002/ps.8922. Epub 2025 May 24.
The widespread use of Bacillus thuringiensis Cry proteins in pest control has led to resistance in some lepidopteran pests. Vip3 proteins, lacking sequence homology with Cry toxins, offer a promising alternative due to distinct insecticidal mechanisms. This study investigates how modulating interactions between domain III and the N-terminal region (P14-G22) of Vip3 proteins influences their activation efficiency and insecticidal activity.
Nine residues in domain III of the Vip3Aa protein were selected for alanine mutation. After testing the membrane perforation activity of the mutants, the results showed that the mutant Vip3Aa-V383A exhibited increased membrane perforation activity compared with the Vip3Aa protein. Structural analysis found that replacing residue V383 with alanine can reduce the hydrogen bonding between domain III and residue Y19. The membrane perforation activity of the disulfide bond mutant Vip3Aa-N21C-T525C was seriously affected. Based on this, the two residues in domain III of the Vip3Ca protein that formed hydrogen bonds with residue Y19 were mutated to alanine respectively. The mutant Vip3Ca-K383A also showed increased membrane perforation activity compared with the Vip3Ca protein. Furthermore, Vip3Aa-V383A and Vip3Ca-K383A showed enhanced insecticidal activity against the four tested lepidopteran pests. In addition, residues K385, K526, and V529 in domain III of the Vip3Aa protein were critical for receptor binding, with mutation diminishing binding affinity and toxicity.
Targeted disruption of hydrogen bonds between residues in domain III and residue Y19 enhances the membrane perforation and insecticidal efficacy of Vip3Aa and Vip3Ca, offering a novel engineering strategy for optimizing biopesticides. © 2025 Society of Chemical Industry.
苏云金芽孢杆菌Cry蛋白在害虫防治中的广泛应用已导致一些鳞翅目害虫产生抗性。Vip3蛋白与Cry毒素缺乏序列同源性，因其独特的杀虫机制而提供了一种有前景的替代方案。本研究调查了调节Vip3蛋白结构域III与N端区域（P14 - G22）之间的相互作用如何影响其激活效率和杀虫活性。
选择Vip3Aa蛋白结构域III中的9个残基进行丙氨酸突变。测试突变体的膜穿孔活性后，结果表明，与Vip3Aa蛋白相比，突变体Vip3Aa - V383A的膜穿孔活性增加。结构分析发现，用丙氨酸取代残基V383可减少结构域III与残基Y19之间的氢键。二硫键突变体Vip3Aa - N21C - T525C的膜穿孔活性受到严重影响。基于此，将Vip3Ca蛋白结构域III中与残基Y19形成氢键的两个残基分别突变为丙氨酸。与Vip3Ca蛋白相比，突变体Vip3Ca - K383A也表现出增加的膜穿孔活性。此外，Vip3Aa - V383A和Vip3Ca - K383A对四种受试鳞翅目害虫表现出增强的杀虫活性。此外，Vip3Aa蛋白结构域III中的残基K385、K526和V529对受体结合至关重要，突变会降低结合亲和力和毒性。
有针对性地破坏结构域III中的残基与残基Y19之间的氢键可增强Vip3Aa和Vip3Ca的膜穿孔和杀虫效果，为优化生物农药提供了一种新的工程策略。© 2025化学工业协会。
