Binding of cholera toxin B-subunits to derivatives of the natural ganglioside receptor, GM1

In a previous paper we showed that the B-pentamer of cholera toxin (CT-B) b inds with reduced binding strength to different C(1) derivatives of N-acety lneuraminic acid (NeuAc) of the natural receptor ganglioside, GM1, We have now extended these results to encompass two large amide derivatives, butyla mide and cyclohexylmethylamide, using an assay in which the glycosphingolip ids are adsorbed on hydrophobic PVDF membranes. The latter derivative showe d an affinity approximately equal to that earlier found for benzylamide (si milar to 0.01 relative to native GM1) whereas the former revealed a approxi mately tenfold further reduction in affinity. Another derivative with a cha rged C(1)-amide group, aminopropylamide, was not bound by the toxin, Toxin binding to C(7) derivatives was reduced by about 50% compared with the nati ve ganglioside. Molecular modeling of C(1) and C(7) derivatives in complex with CT-B gave a structural rationale for the observed differences in the r elative affinities of the various derivatives. Loss of or altered hydrogen bond interactions involving the water molecules bridging the sialic acid to the protein was found to be the major cause for the observed drop in CT-B affinity in the smaller derivatives, while in the bulkier derivatives, hydr ophobic interactions with the protein were found to partly compensate for t hese losses.