NMR and molecular modeling studies of the interaction between wheat germ agglutinin and the beta-D-GlcpNAc-(1-->6)-alpha-D-Manp epitope present in glycoproteins of tumor cells.

The beta-D-GlcpNAc-(1-->6)-alpha-D-Manp disaccharide is a constituent of highly branched cell-surface glycoconjugates that are malignancy markers. The conformational preference of the disaccharide beta-D-GlcpNAc-(1-->6)-alpha-D-Manp-OMe in solution has been studied by molecular modeling and NMR spectroscopy including 1D (1)H,(1)H T-ROESY experiments and analysis of (3)J(H,H) of the hydroxymethyl group being part of the glycosidic linkage of the disaccharide, which revealed the relative populations of the omega torsion angle as gt = 0.60, gg = 0.35, and tg = 0.05. Good agreement was obtained between the effective proton-proton distances from the experiment and those obtained by molecular modeling when the flexibility at the omega torsion angle was taken into account. Molecular modeling of the disaccharide in the binding sites of the lectin wheat germ agglutinin indicates that several conformations could be adopted in the bound state. (1)H NMR and transfer NOESY experiments confirmed that binding took place, and trans-glycosidic proton-proton interactions indicated that a conformational preference was present in the bound state, as observed by the relative change of the NOEs from H1' to H6(pro-R) and H6(pro-S). STD NMR experiments showed that binding occurred in the region of the N-acetyl group of the terminal sugar residue. In addition, the O-methyl group received saturation transfer because of the proximity to the protein. (1)H,(1)H NOEs indicated that the two methyl groups were close in space, as observed in only one of the predicted bound conformations. Experimental and theoretical data therefore agree that one conformation with a gt conformation of the hydroxymethyl group and a negative sign for the psi torsion angle is indeed selected by the lectin upon binding.