China National Engineering Research Center of JUNCAO Technology, College of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China.
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.
Toxins (Basel). 2024 Feb 6;16(2):88. doi: 10.3390/toxins16020088.
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 . 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.
Vip3 毒素形成四聚体结构，这对其杀虫活性至关重要。每个 Vip3Aa 单体由五个结构域组成。据推测，Vip3Aa 结构域 I 中的前四个α-螺旋与靶细胞膜的相互作用是形成孔之前的关键步骤。在本研究中，产生了四个 N 端α-螺旋缺失的 Vip3Aa 截断物，结果发现它们既失去了对脂质体的通透性，也失去了对 的杀虫活性。为了进一步探究结构域 I 在膜渗透中的作用，将全长结构域 I 和 N 端α-螺旋截断结构域 I 的片段分别与绿色荧光蛋白（GFP）融合。只有携带全长结构域 I 的融合蛋白对人工脂质体表现出通透性。此外，还通过 Vip3Aa 结构域 I 和 II 的α-螺旋与 Cry1Ac 的结构域 II 和 III 的组合构建了七个 Vip3Aa-Cry1Ac 融合物。这七种组合中的五种被确定在人工脂质体中具有膜通透性。然而，由于蛋白酶稳定性显著降低，没有一种 Vip3Aa-Cry1Ac 组合表现出杀虫活性。这些结果表明，Vip3Aa 结构域 I 中的 N 端螺旋 α1 对于杀虫活性和脂质体通透性都是必不可少的，并且 Vip3Aa 的结构域 I 独立于结构域 II-V 保持高脂质体通透性。
