N-Terminal α-Helices in Domain I of <i>Bacillus thuringiensis</i> Vip3Aa Play Crucial Roles in Disruption of Liposomal Membrane
Ensi Shao,
Hanye Huang,
Jin Yuan,
Yaqi Yan,
Luru Ou,
Xiankun Chen,
Xiaohong Pan,
Xiong Guan,
Li Sha
Affiliations
Ensi Shao
China National Engineering Research Center of JUNCAO Technology, College of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Hanye Huang
State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops & Key Laboratory of Biopesticide and Chemical Biology of Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Jin Yuan
China National Engineering Research Center of JUNCAO Technology, College of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Yaqi Yan
China National Engineering Research Center of JUNCAO Technology, College of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Luru Ou
China National Engineering Research Center of JUNCAO Technology, College of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Xiankun Chen
China National Engineering Research Center of JUNCAO Technology, College of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Xiaohong Pan
State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops & Key Laboratory of Biopesticide and Chemical Biology of Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Xiong Guan
State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops & Key Laboratory of Biopesticide and Chemical Biology of Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Li Sha
China National Engineering Research Center of JUNCAO Technology, College of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China
Bacillus thuringiensis Vip3 toxins form a tetrameric structure crucial for their insecticidal activity. Each Vip3Aa monomer comprises five domains. Interaction of the first four α-helices in domain I with the target cellular membrane was proposed to be a key step before pore formation. In this study, four N-terminal α-helix-deleted truncations of Vip3Aa were produced and, it was found that they lost both liposome permeability and insecticidal activity against Spodoptera litura. To further probe the role of domain I in membrane permeation, the full-length domain I and the fragments of N-terminal α-helix-truncated domain I were fused to green fluorescent protein (GFP), respectively. Only the fusion carrying the full-length domain I exhibited permeability against artificial liposomes. In addition, seven Vip3Aa-Cry1Ac fusions were also constructed by combination of α-helices from Vip3Aa domains I and II with the domains II and III of Cry1Ac. Five of the seven combinations were determined to show membrane permeability in artificial liposomes. However, none of the Vip3Aa-Cry1Ac combinations exhibited insecticidal activity due to the significant reduction in proteolytic stability. These results indicated that the N-terminal helix α1 in the Vip3Aa domain I is essential for both insecticidal activity and liposome permeability and that domain I of Vip3Aa preserved a high liposome permeability independently from domains II–V.
