Cabral-Lilly D, Sosinsky G E, Reed R A, McDermott M R, Shipley G G
Biophysics Department, Boston University School of Medicine, Massachusetts 02118.
Biophys J. 1994 Apr;66(4):935-41. doi: 10.1016/S0006-3495(94)80894-X.
The orientation of cholera toxin bound to its cell-surface receptor, ganglioside GM1, in a supporting lipid membrane was determined by electron microscopy of negatively stained toxin-lipid samples. Image analysis of two dimensional crystalline arrays has shown previously that the B-subunits of cholera toxin orient at the membrane surface as a pentameric ring with a central channel (Reed, R. A., J. Mattai, and G.G. Shipley. 1987. Biochemistry. 26:824-832; Ribi, H. O., D. S. Ludwig, K. L. Mercer, G. K. Schoolnik, and R. D. Kornberg. 1988. Science (Wash, DC). 239:1272-1276). We recorded images of negatively stained cholera toxin and isolated B-pentamers oriented perpendicular to the lipid surface so that the pentamer ring is viewed from the side. The pentamer dimensions, estimated from the average of 100 molecules, are approximately 60 by 30 A. Images of side views of whole cholera toxin clearly show density above the pentamer ring away from the lipid layer. On the basis of difference maps between averages of side views of whole toxin and B-pentamers, this density above the pentamer has been identified as a portion of the A-subunit. The A-subunit may also extend into the pore of the pentamer. In addition, Fab fragments from a monoclonal antibody to the A-subunit were mixed with the toxin prior to binding to GM1. Density from the Fab was localized to the region of toxin above the pentamer ring confirming the location of the A-subunit. The structure determined for the homologous heat-labile enterotoxin from Escherichia coli shows that the A-subunit lies mostly on one face of this pentamer with a small region penetrating the pentamer pore (Sixma, T. K., S. E. Pronk, K. H. Kalk, E. S. Wartna, B. A. M. van Zanten, B. Witholt,and W. G. J. Hol. 1991. Nature (Lond.). 351:371-377). The putative GM1 binding sites are located on the opposite face of the B-pentamer. Cholera toxin, therefore appears to bind to a model membrane with its GM1 binding surface adjacent to the membrane. Low resolution density maps were constructed from the x-ray coordinates of the E. coli toxin and compared with the electron microscopy-derived maps.
通过对负染毒素 - 脂质样本进行电子显微镜观察，确定了霍乱毒素与其细胞表面受体神经节苷脂GM1在支持性脂质膜中的取向。二维晶体阵列的图像分析先前已表明，霍乱毒素的B亚基在膜表面以具有中央通道的五聚体环形式取向（里德，R.A.，J.马泰，和G.G.希普利。1987年。《生物化学》。26:824 - 832；里比，H.O.，D.S.路德维希，K.L.默瑟，G.K.斯库尼克，和R.D.科恩伯格。1988年。《科学》（华盛顿特区）。239:1272 - 1276）。我们记录了负染霍乱毒素和垂直于脂质表面取向的分离B五聚体的图像，以便从侧面观察五聚体环。根据100个分子的平均值估算，五聚体的尺寸约为60×30埃。完整霍乱毒素侧面视图的图像清楚地显示，在远离脂质层的五聚体环上方有密度。根据完整毒素和B五聚体侧面视图平均值之间的差异图，已确定五聚体上方的这种密度为A亚基的一部分。A亚基也可能延伸到五聚体的孔中。此外，在与GM1结合之前，将针对A亚基的单克隆抗体的Fab片段与毒素混合。Fab的密度定位于五聚体环上方的毒素区域，证实了A亚基的位置。对来自大肠杆菌的同源热不稳定肠毒素确定的结构表明，A亚基大多位于该五聚体的一个面上，有一小区域穿透五聚体孔（西克斯马，T.K.，S.E.普龙克，K.H.卡尔克，E.S.瓦特纳，B.A.M.范赞滕，B.维霍尔特，和W.G.J.霍尔。1991年。《自然》（伦敦）。351:371 - 377）。推测的GM1结合位点位于B五聚体的相对面上。因此，霍乱毒素似乎以其GM1结合表面邻近膜的方式结合到模型膜上。根据大肠杆菌毒素的X射线坐标构建了低分辨率密度图，并与电子显微镜衍生的图进行了比较。
