Plasticity in the primary binding site of galactose/N-acetylgalactosamine-specific lectins - Implication of the C-H center dot center dot center dot O hydrogen bond at the specificity-determining C-4 locus of the saccharide in 4-methoxygalactose recognition by jacalin and winged bean (basic) agglutinin I

It is currently believed that an unsubstituted axial hydroxyl at the specif icity-determining C-4 locus of galactose is indispensable for recognition b y galactose/N-acetylgalactosamine-specific lectins. Titration calorimetry d emonstrates that 4-methoxygalactose retains binding allegiance to the Morac eae lectin jacalin and the Leguminosae lectin, winged bean (basic) agglutin in (WBA I). The binding reactions were driven by dominant favorable enthalp ic contributions and exhibited significant enthalpy-entropy compensation. P roton NMR titration of C-methoxygalactose with jacalin and WBA I resulted i n broadening of the sugar resonances without any change in chemical shift. The alpha-and beta-anomers of 4-methoxygalactose were found to be in slow e xchange with free and lectin-bound states. Both the anomers experience magn etically equivalent environments at the respective binding sites. The bindi ng constants derived from the dependence of NMR line widths on 4-methoxygal actose concentration agreed well with those obtained from titration calorim etry. The results unequivocally demonstrate that the loci corresponding to the axially oriented C-4 hydroxyl group of galactose within the primary bin ding site of these lectins exhibit plasticity. These analyses suggest, for the first time, the existence of C-H ... O-type hydrogen-bond(s) in protein -carbohydrate interactions in general and between the C-4 locus of galactos e derivative and the lectins jacalin and WBA I in particular.