The Interaction of Hevein with <i>N</i>‐acetylglucosamine‐containing Oligosaccharides

Abstract

The three‐dimensional structure of hevein, a small protein isolated from the latex of Hevea brasiliensis (rubber tree), in water solution has been obtained by using 1 H‐NMR spectroscopy and dynamic simulated annealing calculations. The average root‐mean‐square deviation (rmsd) of the best 20 refined structures generated using DIANA prior to simulated 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 1 H‐NMR. The association constants, K a , for the binding of hevein to GlcNAc, chitobiose [GlcNAc‐β(1→4)‐GlcNAc], chitotriose [GlcNAc‐β(1→4)‐GlcNAc‐β(1→4)‐GlcNAc], and GlcNAc‐α(1→6)‐Man have been estimated from 1 H‐NMR titration experiments. Since the measured K a values for chitobiose binding are almost identical with and without calcium ions, it is shown that these cations are not required for sugar binding. The association increases in the order GlcNAc‐α(1→6)‐Man ≤ GlcNAc &lt; chitobiose &lt; chitotriose. The equilibrium thermodynamic parameters entropy and enthalpy of binding,  S ° and  H °, for the hevein–chitobiose and hevein–chitotriose associations have been obtained from van't Hoff analysis of the temperature dependence of the K a values between 25–40°C. The driving force for the binding process is provided by a negative  H ° which is partially compensated by a negative  S °. These negative signs seem to indicate that hydrogen bonding and van der Waals forces are the major interactions stabilizing the complex. Protein–carbohydrate nuclear Overhauser enhancements have allowed a three‐dimensional model of the hevein‐chitobiose complex to be built. From inspection of this model, a hydrogen bond between Ser19 and the non‐reducing N ‐acetyl 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 residues. In addition, the higher affinities deduced for the β‐linked oligosaccharides with respect to GlcNAc and GlcNAc‐α(1→6)‐Man can be explained by favourable stacking of the second β‐linked GlcNAc moiety and Trp21.