Structural and functional roles of domain III in Vip3Aa and Vip3Ca: implications for membrane perforation and...

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.