NMR investigations of protein-carbohydrate interactions - Binding studies and refined three-dimensional solution structure of the complex between theB domain of wheat germ agglutinin and N,N ',N ''-triacetylchitotriose

The specific interaction of the isolated B domain of wheat germ agglutinin (WGA-B) with N,N',N"-triacetylchitotriose has been analyzed by H-1-NMR spec troscopy. The association constants for the binding of WGA-B to this trisac charide have been determined from both H-1-NMR titration experiments and mi crocalorimetry methods. Entropy and enthalpy of binding have been obtained. The driving force for the binding process is provided by a negative Delta H which is partially compensated by negative Delta S. These negative signs indicate that hydrogen bonding and van der Waals forces are the major inter actions stabilizing the complex. NOESY NMR experiments in water solution pr ovided 327 protein proton-proton distance constraints. All the experimental constraints were used in a refinement protocol including restrained molecu lar dynamics in order to determine the refined solution conformation of thi s protein/carbohydrate complex. With regard to the NMR structure of the fre e protein, no important changes in the protein NOEs were observed, indicati ng that carbohydrate-induced conformational changes are small. The average backbone rmsd of the 35 refined structures was 1.05 Angstrom, while the hea vy atom rmsd was 2.10 Angstrom. Focusing on the bound ligand, two different orientations of the trisaccharide within WGA-B binding site are possible. It can be deduced that both hydrogen bonds and van der Waals contacts confe r stability to both complexes. A comparison of the three-dimensional struct ure of WGA-B in solution to that reported in the solid state and to those d educed for hevein and pseudohevein in solution has also been performed.