Carbohydrate binding specificities and crystal structure of the cholera toxin-like B-subunit from Citrobacter freundii

Enterotoxigenic Escherichia coli and Vibrio cholerae are well known causative agents of severe diarrheal diseases. Both pathogens produce AB5 toxins, with one enzymatically active A-subunit and a pentamer of receptor-binding B-subunits. The primary receptor for both B-subunits is the GM1 ganglioside (Galβ3GalNAcβ4(NeuAcα3)Galβ4GlcβCer), but the B-subunits from porcine isolates of E. coli also bind neolacto-(Galβ4GlcNAcβ-)terminated glycoconjugates and the B-subunits from human isolates of E. coli (hLTB) have affinity for blood group A type 2-(GalNAcα3(Fucα2)Galβ4GlcNAcβ-)terminated glycoconjugates.A B-subunit with 73% sequence identity to the B-subunits of cholera toxin and the heat-labile toxin of E. coli is produced by certain strains of enteropathogenic E. coli and by Citrobacter freundii. This C. freundii B-subunit (CFXB) has now been expressed in V. cholerae, and isolated in high yields. Glycosphingolipid binding studies show that CFXB binds to the GM1 ganglioside with high affinity. In addition, CFXB has high affinity for both neolacto-terminated and blood group A type 2-terminated glycoconjugates. The crystal structure of the pentameric arrangement of C. freundii B-subunits display high structural similarity with related proteins from E. coli and V. cholerae and oligosaccharide binding sites can be identified on the protein surface. Small changes in the 88–95 loop connecting the GM1 and blood group A binding sites explains the minor changes in affinity seen for these two ligands. However, the enhanced affinity of CFXB for neolacto-terminated structures can be sought in the Lys34Tyr substitution affording additional hydrogen bond interactions between the tyrosyl side chain and the GlcNAcβ3Galb4Glcβ1 segment of neolactotetraosylceramide via bridging water molecules.