SURPRISING LEADS FOR A CHOLERA-TOXIN RECEPTOR-BINDING ANTAGONIST - CRYSTALLOGRAPHIC STUDIES OF CTB MUTANTS

Background: Because agents which inhibit the receptor binding of chole ra toxin constitute possible lead compounds for the structure-based de sign of anti-cholera drugs, detailed investigation of the toxin's rece ptor-binding site is of key importance. The substitution Gly-->Asp at residue 33 of the cholera toxin B subunit (CTB) has been reported to a bolish receptor-binding ability. The substitution Arg35-->Asp has been reported to result in deficient assembly of the AB(5) holotoxin. The molecular basis for these effects was not readily apparent from analys is of an earlier crystal structure of the wild-type toxin B pentamer i n a complex with the receptor pentasaccharide. Results: We now report at a resolution of 2.0 Angstrom the crystal structure of a recombinant CTB pentamer containing the Gly33-->Asp substitution. The observed co nformation of the Asp33 side chain suggests that the loss in binding a ffinity is due to a steric clash with atoms C9 and O9 of the sialic ac id moiety of the receptor, ganglioside G(M1). The crystal structure al so reveals an unexpected mode of pentamer-pentamer interaction in whic h pairs of toxin pentamers are joined by reciprocal insertion of the i midazole ring of His13 from one subunit of each pentamer into one of t he receptor-binding sites on the other. The surface of interaction at each pentamer-pentamer interface is on the order of 500 Angstrom(2) an d primarily involves contact of residues 10-14 with the receptor-bindi ng site on the associated pentamer. This same pentamer-pentamer intera ction 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 Angstrom resolution. Conclusions: These structures suggest that analogs to all or part of the pentapeptide Ala-Glu-Tyr-Hi s-Asn, corresponding to residues 10-14 of CTB, may constitute lead com pounds for the design of binding-site inhibitors.