NMR investigations of protein–carbohydrate interactions

Abstract

The specific interaction of the isolated B domain of wheat germ agglutinin (WGA‐B) with N,N′,N″‐triacetylchitotriose has been analyzed by 1H‐NMR spectroscopy. The association constants for the binding of WGA‐B to this trisaccharide have been determined from both 1H‐NMR titration experiments and microcalorimetry methods. Entropy and enthalpy of binding have been obtained. The driving force for the binding process is provided by a negative ΔH which is partially compensated by negative ΔS. 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 327 protein proton‐proton distance constraints. All the experimental constraints were used in a refinement protocol including restrained molecular dynamics in order to determine the refined solution conformation of this protein/carbohydrate complex. With regard to the NMR structure of the free protein, no important changes in the protein NOEs were observed, indicating that carbohydrate‐induced conformational changes are small. The average backbone rmsd of the 35 refined structures was 1.05 Å, while the heavy atom rmsd was 2.10 Å. 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 confer stability to both complexes. A comparison of the three‐dimensional structure of WGA‐B in solution to that reported in the solid state and to those deduced for hevein and pseudohevein in solution has also been performed.

NMR and Modeling Studies of Protein–Carbohydrate Interactions: Synthesis, Three‐Dimensional Structure, and Recognition Properties of a Minimum Hevein Domain with Binding Affinity for Chitooligosaccharides

“…[33][34][35] The entropy of binding, DS 0 , was found to be negative, as has also been observed for a variety of chitooligosaccharides interacting with hevein itself, pseudohevein, WGA, and UDA. [35,36,40,41,49] In the current case, the enthalpy values for the binding of (GlcNAc) 3 to HEV32 amounted to À62.6 kJ mol À1 .…”

“…The hydroxy groups of conserved Ser and Tyr residues (19 and 30 in hevein) are involved in hydrogen-bonding interactions with the carbonyl group of the acetamide moiety and the OH-3 of a GlcNAc residue, respectively. [33][34][35] The same set of interactions is common to complexes of WGA, [38][39][40] pseudohevein, [41] Ac-AMP2, [42] the UDA dimer, [43,44] and pokeweed lectin. [45] In this work we have truncated the C terminus of hevein to obtain a 32-residue peptide (which we call HEV32) by solidphase synthesis with three disulfide bonds in a similar pattern to Ac-AMP2, but with an approximately 50 % sequence homology to this small peptide (Scheme 1).…”

Hyaluronan Recognition Mode of CD44 Revealed by Cross-saturation and Chemical Shift Perturbation Experiments

“…However, in this report, chemical shift perturbation would not be an appropriate method to identify the ligand-binding site of CD44 HABD because of the small chemical shift changes at the contact site and the presence of significant conformational changes in the flanking regions. Several chemical shift perturbation experiments for protein-carbohydrate complexes have been reported (21,(43)(44)(45)(46). A common feature of these experiments is that the chemical shift perturbations of the protons on the putative ligand-binding sites are at most 0.35 ppm.…”

Binding of the AVR4 Elicitor of Cladosporium fulvum to Chitotriose Units Is Facilitated by Positive Allosteric Protein-Protein Interactions

“…the resonances important for binding show shifts exceeding 0.1 ppm at full saturation, whereas shifts Ͻ0.1 ppm are apparently caused by a reorientation of the aromatic side chains influencing other residues as well (30,31,33,34 3 (Fig. 5B).…”