NMR INVESTIGATIONS OF PROTEIN-CARBOHYDRATE INTERACTIONS - REFINED 3-DIMENSIONAL STRUCTURE OF THE COMPLEX BETWEEN HEVEIN AND METHYL BETA-CHITOBIOSIDE

The specific interaction of hevein with GlcNAc-containing oligosacchar ides has been analyzed by H-1-NMR spectroscopy. The association consta nts for the binding of hevein to a variety of ligands have been estima ted from IH-NMR titration experiments. The association constants incre ase in the order GlcNAc-alpha(1-->6)-Man < GlcNAc < benzyl-beta-GlcNAc < p-nitrophenyl-beta-GlcNAc < chitobiose < p-nitrophenyl-beta-chitobi oside < methyl-beta-chitobioside < chitotriose. Entropy and enthalpy o f binding for different complexes have been obtained from van't Hoff a nalysis. The driving force for the binding process is provided by a ne gative Delta H-0 which is partially compensated by negative Delta S-0. These negative signs indicate that hydrogen bonding and van der Waals forces are the major interactions stabilizing the complex. NOESY NMR experiments in water solution provided 475 accurate protein proton-pro ton distance constraints after employing the MARDIGRAS program. In add ition, 15 unambiguous protein/carbohydrate NOEs were detected. All the experimental constraints were used in a refinement protocol including restrained molecular dynamics in order to determine the highly refine d solution conformation of this protein-carbohydrate complex. With reg ard to the NMR structure of the free protein, no important changes in the protein nOe's were observed, indicating that carbohydrate-induced conformational changes are small. The average backbone rmsd of the 20 refined structures was 0.055 nm, while the heavy atom rmsd was 0.116 n m. It can be deduced that both hydrogen bonds and van der Waals contac ts confer stability to the complex. A comparison of the three-dimensio nal structure of hevein in solution to those reported for wheat germ a gglutinin (WCA) and hevein itself in the solid state has also been per formed. The polypeptide conformation has also been compared to the NMR -derived structure of a smaller antifungical peptide, Ac-AMP2.