ORIENTATION OF CHOLERA-TOXIN BOUND TO MODEL MEMBRANES

The orientation of cholera toxin bound to its cell-surface receptor, ganglioside G(M1), 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 G(M1). 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 (Lend.). 351:371-377). The putative G(M1) binding sites are located on the opposite face of the B-pentamer. Cholera toxin, therefore appears to bind to a model membrane with its G(M1) 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.