BACKGROUND: Because agents which inhibit the receptor binding of cholera toxin constitute possible lead compounds for the structure-based design of anti-cholera drugs, detailed investigation of the toxin's receptor-binding site is of key importance. The substitution Gly-->Asp at residue 33 of the cholera toxin B subunit (CTB) has been reported to abolish receptor-binding ability. The substitution Arg35-->Asp has been reported to result in deficient assembly of the AB5 holotoxin. The molecular basis for these effects was not readily apparent from analysis of an earlier crystal structure of the wild-type toxin B pentamer in a complex with the receptor pentasaccharide. RESULTS: We now report at a resolution of 2.0 A the crystal structure of a recombinant CTB pentamer containing the Gly33-->Asp substitution. The observed conformation of the Asp33 side chain suggests that the loss in binding affinity is due to a steric clash with atoms C9 and O9 of the sialic acid moiety of the receptor, ganglioside GM1. The crystal structure also reveals an unexpected mode of pentamer-pentamer interaction in which pairs of toxin pentamers are joined by reciprocal insertion of the imidazole ring of His13 from one subunit of each pentamer into one of the receptor-binding sites on the other. The surface of interaction at each pentamer-pentamer interface is on the order of 500 A2, and primarily involves contact of residues 10-14 with the receptor-binding site on the associated pentamer. This same pentamer-pentamer interaction is also present in the crystal structure of a second recombinant CTB containing an Arg-->Asp substitution at residue 35, which we have determined at 2.1 A resolution. CONCLUSIONS: These structures suggest that analogs to all or part of the pentapeptide Ala-Glu-Tyr-His-Asn, corresponding to residues 10-14 of CTB, may constitute lead compounds for the design of binding-site inhibitors.
BACKGROUND: Because agents which inhibit the receptor binding of cholera toxin constitute possible lead compounds for the structure-based design of anti-cholera drugs, detailed investigation of the toxin's receptor-binding site is of key importance. The substitution Gly-->Asp at residue 33 of the cholera toxin B subunit (CTB) has been reported to abolish receptor-binding ability. The substitution Arg35-->Asp has been reported to result in deficient assembly of the AB5 holotoxin. The molecular basis for these effects was not readily apparent from analysis of an earlier crystal structure of the wild-type toxin B pentamer in a complex with the receptor pentasaccharide. RESULTS: We now report at a resolution of 2.0 A the crystal structure of a recombinant CTB pentamer containing the Gly33-->Asp substitution. The observed conformation of the Asp33 side chain suggests that the loss in binding affinity is due to a steric clash with atoms C9 and O9 of the sialic acid moiety of the receptor, gangliosideGM1. The crystal structure also reveals an unexpected mode of pentamer-pentamer interaction in which pairs of toxin pentamers are joined by reciprocal insertion of the imidazole ring of His13 from one subunit of each pentamer into one of the receptor-binding sites on the other. The surface of interaction at each pentamer-pentamer interface is on the order of 500 A2, and primarily involves contact of residues 10-14 with the receptor-binding site on the associated pentamer. This same pentamer-pentamer interaction is also present in the crystal structure of a second recombinant CTB containing an Arg-->Asp substitution at residue 35, which we have determined at 2.1 A resolution. CONCLUSIONS: These structures suggest that analogs to all or part of the pentapeptide Ala-Glu-Tyr-His-Asn, corresponding to residues 10-14 of CTB, may constitute lead compounds for the design of binding-site inhibitors.