Plasticity in the Primary Binding Site of Galactose/N-Acetylgalactosamine-specific Lectins

Abstract

It is currently believed that an unsubstituted axial hydroxyl at the specificity-determining C-4 locus of galactose is indispensable for recognition by galactose/-acetylgalactosamine-specific lectins. Titration calorimetry demonstrates that 4-methoxygalactose retains binding allegiance to the Moraceae lectin jacalin and the Leguminosae lectin, winged bean (basic) agglutinin (WBA I). The binding reactions were driven by dominant favorable enthalpic contributions and exhibited significant enthalpy-entropy compensation. Proton NMR titration of 4-methoxygalactose with jacalin and WBA I resulted in broadening of the sugar resonances without any change in chemical shift. The α- and β-anomers of 4-methoxygalactose were found to be in slow exchange with free and lectin-bound states. Both the anomers experience magnetically equivalent environments at the respective binding sites. The binding constants derived from the dependence of NMR line widths on 4-methoxygalactose concentration agreed well with those obtained from titration calorimetry. The results unequivocally demonstrate that the loci corresponding to the axially oriented C-4 hydroxyl group of galactose within the primary binding 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 galactose derivative and the lectins jacalin and WBA I in particular.