THE INTERACTION OF HEVEIN WITH N-ACETYLGLUCOSAMINE-CONTAINING OLIGOSACCHARIDES - SOLUTION STRUCTURE OF HEVEIN COMPLEXED TO CHITOBIOSE

The three-dimensional structure of hevein, a small protein isolated fr om the latex of Hevea brasiliensis (rubber tree), in water solution ha s been obtained by using H-1-NMR spectroscopy and dynamic simulated an nealing calculations. The average root-mean-square deviation (rmsd) of the best 20 refined structures generated using DIANA prior to simulat ed annealing was 0.092 nm for the backbone atoms and 0.163 nm for all heavy atoms (residues 3-41). The specific interaction of hevein with N -acetylglucosamine-containing oligosaccharides has also been analyzed by H-1-NMR. The association constants, K-a, for the binding of hevein to GlcNAc, chitobiose [GlcNAc-beta(1-->4)-GlcNAc], chitotriose [GlcNAc -beta(1-->4)-GlcNAc-beta(1-->4)-GlcNAc], and GlcNAc-alpha(1-->6)-Man h ave been estimated from H-1-NMR titration experiments. Since the measu red K-a values for chitobiose binding are almost identical with and wi thout calcium ions, it is shown that these cations are not required fo r sugar binding. The association increases in the order GlcNAc-alpha(1 -->6)-Man less than or equal to GlcNAc < chitobiose < chitotriose. The equilibrium thermodynamic parameters entropy and enthalpy of binding, Delta S-0 and Delta H-0 for the hevein-chitobiose and hevein-chitotri ose associations have been obtained from van't Hoff analysis of the te mperature dependence of the K-a values between 25-40 degrees C. The dr iving force for the binding process is provided by a negative Delta H- 0 which is partially compensated by a negative Delta S-0. These negati ve signs seem to indicate that hydrogen bonding and van der Waals forc es are the major interactions stabilizing the complex. Protein-carbohy drate nuclear Overhauser enhancements have allowed a three-dimensional model of the hevein-chitobiose complex to be built. From inspection o f this model, a hydrogen bond between Ser19 and the non-reducing N-ace tyl carbonyl group is suggested, as well as between Tyr30 and HO-3 of the same sugar residue. The N-acetyl methyl group of the non-reducing GlcNAc displays non-polar contacts to the aromatic Tyr30 and Trp21 res idues. In addition, the higher affinities deduced for the beta-linked oligosaccharides with respect to GlcNAc and GlcNAc-alpha(1-->6)-Man ca n be explained by favourable stacking of the second beta-linked GlcNAc moiety and Trp21.