ORIENTATION OF CHOLERA-TOXIN BOUND TO MODEL MEMBRANES

The orientation of cholera toxin bound to its cell-surface receptor, g anglioside G(M1), in a supporting lipid membrane was determined by ele ctron microscopy of negatively stained toxin-lipid samples. Image anal ysis of two dimensional crystalline arrays has shown previously that t he B-subunits of cholera toxin orient at the membrane surface as a pen tameric 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 su rface so that the pentamer ring is viewed from the side. The pentamer dimensions, estimated from the average of 100 molecules, are approxima tely 60 by 30 A. images of side views of whole cholera toxin clearly s how 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 ant ibody 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 struct ure determined for the homologous heat-labile enterotoxin from Escheri chia coli shows that the A-subunit lies mostly on one face of this pen tamer 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. Withol t, 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 it s G(M1) binding surface adjacent to the membrane. Low resolution densi ty maps were constructed from the x-ray coordinates of the E. coli tox in and compared with the electron microscopy-derived maps.