Microbial Biotechnology Unit, Department of Microbiology, Faculty of Liberal Arts and Science, Kasetsart University, Nakornpathom 73140, Thailand.
Division of Biology, Department of Science, Faculty of Science and Technology, Prince of Songkla University, Pattani 94000, Thailand.
Toxins (Basel). 2019 Jan 23;11(2):62. doi: 10.3390/toxins11020062.
Although the C-terminal domain (DIII) of three-domain Cry insecticidal toxins from has been implicated in various biological functions, its exact role still remains to be elucidated. Here, the 21-kDa isolated DIII fragment of the 65-kDa Cry4Ba mosquito-specific toxin was analyzed for its binding characteristics toward lipid-bilayer membranes. When the highly-purified Cry4Ba-DIII protein was structurally verified by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, it revealed the presence of a distinct β-sheet structure, corresponding to its structure embodied in the Cry4Ba crystal structure. Binding analysis via surface plasmon resonance (SPR) spectroscopy revealed that the 21-kDa Cry4Ba-DIII truncate displayed tight binding to immobilized liposome membranes in a two-step manner, exhibiting a dissociation rate constant () comparable to the 65-kDa full-length toxin. Also similar to the Cry4Ba full-length toxin, its isolated DIII truncate was able to anchor a part of its molecule into the immobilized membrane as the SPR signal was still detected after prolonged treatment with proteinase K. However, unlike the full-length active toxin, the DIII truncate was unable to induce membrane permeability of calcein-loaded liposomes or ion-channel formation in planar lipid bilayers. Together, our present data have disclosed a pivotal role of C-terminal DIII in serving as a membrane anchor rather than a pore-forming moiety of the Cry4Ba mosquito-active toxin, highlighting its potential mechanistic contribution to the interaction of the full-length toxin with lipid membranes in mediating toxicity.
虽然 来源的三域 Cry 杀虫毒素的 C 端结构域（DIII）已被牵涉到各种生物学功能中，但它的确切作用仍有待阐明。在此，对 65 kDa Cry4Ba 蚊虫特异性毒素的 21 kDa 分离的 DIII 片段进行了分析，以研究其对脂双层膜的结合特性。当通过衰减全反射傅里叶变换红外（ATR-FTIR）光谱对高度纯化的 Cry4Ba-DIII 蛋白进行结构验证时，发现存在明显的β-折叠结构，与 Cry4Ba 晶体结构中体现的结构相对应。通过表面等离子体共振（SPR）光谱进行的结合分析表明，21 kDa 的 Cry4Ba-DIII 截断以两步方式紧密结合到固定化脂质体膜上，其离解速率常数（）与 65 kDa 全长毒素相当。与 Cry4Ba 全长毒素相似，其分离的 DIII 截断能够将其分子的一部分锚定在固定化膜上，因为在长时间用蛋白酶 K 处理后仍检测到 SPR 信号。然而，与全长活性毒素不同，DIII 截断不能诱导载有钙黄绿素的脂质体的膜通透性或平面脂质双层中的离子通道形成。总之，我们目前的数据揭示了 C 端 DIII 在充当膜锚定而不是 Cry4Ba 蚊虫活性毒素的孔形成部分方面的关键作用，突出了其在全长毒素与介导毒性的脂质膜相互作用中潜在的机制贡献。
