Conformer selection and differential restriction of ligand mobility by a plant lectin

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Abstract
To study conformational parameters of ligands before and after complex formation with the galactoside‐binding agglutinin of Viscum album L. (VAA) in solution, combined computer‐assisted random walk molecular mechanics (RAMM) calculations extended by conformational clustering analysis (CCA), molecular dynamics (MD) simulations as well as two‐dimensional rotating‐frame nuclear Overhauser effect (ROE) and two‐dimensional nuclear Overhauser effect (NOE) spectroscopy NMR experiments were employed. Derivatives of the naturally occurring disaccharides Galβ1‐3GlcNAcβ1‐R and Galβ1‐3GalNAcβ1‐R as well as of a synthetic high‐affinity binding partner, i.e. the disaccharide Galβ1‐2Galβ1‐R′, were chosen as ligands in this study. The disaccharides displayed inherent flexibility in the valley of the global minimum between KY combinations of (40+/60+) and (40+/−60+). Calculations of the de‐N‐acetylated sugars revealed that presence of this group did not markedly influence the distribution of low‐energy conformers in the KY, E plot. Occupation of side minima at KY (180+/0+) or (0+/180+) is either unlikely or low according to the results of MD simulations and RAMM calculations extended by CCA. Notably, these side minima define conformations which are not stable during a MD simulation. Transitions to other minima occur already a few picoseconds after the start of the simulation. NMR experiments of the free‐state ligand confirmed the validity of the data sets obtained by the calculations. Following the description of the conformational space in the free‐state NMR experiments were performed for these disaccharides complexed with VAA. They yielded two interresidual contacts for Galβ1‐3GlcNAcβ1‐R and Galβ1‐2Galβ1‐R′. The ligand conformations in the complex did not deviate markedly from those of a minimum conformation in the free state. One‐ and two‐dimensional transferred nuclear Overhauser enhancement (TRNOE) experiments at different mixing times excluded the influence of spin‐diffusion effects. When the NOE build‐up curves in the three studied cases were compared, the residual mobility of the penultimate carbohydrate unit of Galβ1‐3GalNAcβ1‐R was observed to be higher than that of the respective hexopyranose unit of the other two bound ligands. Due to the availability of the conformational parameters of Galβ1‐2Galβ1‐R′ in association with a galectin, namely the β‐galactoside‐binding protein from chicken liver, it is remarkable to note that this ligand displays different conformations in the binding sites of either the plant or the animal lectin. They correspond to local energy‐minimum conformations in the KY, E plot and substantiate differential conformer selection by these two lectins with identical nominal monosaccharide specificity.

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