Soloaga A, Veiga M P, García-Segura L M, Ostolaza H, Brasseur R, Goñi F M
Departamento de Bioquímica, Universidad del País Vasco, Bilbao, Spain.
Mol Microbiol. 1999 Feb;31(4):1013-24. doi: 10.1046/j.1365-2958.1999.01225.x.
alpha-Haemolysin is an extracellular protein toxin (approximately 107 kDa) secreted by Escherichia coli that acts at the level of the plasma membranes of target eukaryotic cells. The nature of the toxin interaction with the membrane is not known at present, although it has been established that receptor-mediated binding is not essential. In this work, we have studied the perturbation produced by purified alpha-haemolysin on pure phosphatidylcholine bilayers in the form of large unilamellar vesicles, under conditions in which the toxin has been shown to induce vesicle leakage. The bilayer systems containing bound protein have been examined by differential scanning calorimetry, fluorescence spectroscopy, differential solubilization by Triton X-114, and freeze-fracture electron microscopy. All the data concur in indicating that alpha-haemolysin, under conditions leading to cell lysis, becomes inserted in the target membrane in the way of intrinsic or integral proteins. In addition, the experimental results support the idea that inserted alpha-haemolysin occupies only one of the membrane phospholipid monolayers, i.e. it is not a transmembrane protein. The experimental data are complemented by structure prediction studies according to which as many as ten amphipathic alpha-helices, appropriate for protein-lipid interaction, but no hydrophobic transmembrane helices are predicted in alpha-haemolysin. These observations and predictions have important consequences for the mechanism of cell lysis by alpha-haemolysin; in particular, a non-transmembrane arrangement of the toxin in the target membrane is not compatible with the concept of alpha-haemolysin as a pore-forming toxin.
α-溶血素是由大肠杆菌分泌的一种细胞外蛋白质毒素（约107 kDa），作用于靶真核细胞的质膜水平。目前尚不清楚毒素与膜相互作用的本质，尽管已经确定受体介导的结合并非必需。在这项工作中，我们研究了纯化的α-溶血素在大单层囊泡形式的纯磷脂酰胆碱双层膜上产生的扰动，此时毒素已被证明会诱导囊泡泄漏。通过差示扫描量热法、荧光光谱法、Triton X-114差异溶解法和冷冻断裂电子显微镜对含有结合蛋白的双层系统进行了检测。所有数据都一致表明，在导致细胞裂解的条件下，α-溶血素以内在或整合蛋白的方式插入靶膜。此外，实验结果支持这样一种观点，即插入的α-溶血素仅占据膜磷脂单层之一，即它不是跨膜蛋白。根据结构预测研究对实验数据进行了补充，该研究预测α-溶血素中多达十个适合蛋白质-脂质相互作用的两亲性α-螺旋，但没有疏水跨膜螺旋。这些观察结果和预测对α-溶血素导致细胞裂解的机制具有重要意义；特别是，毒素在靶膜中的非跨膜排列与α-溶血素作为成孔毒素的概念不相符。
