Conformer selection and differential restriction of ligand mobility by a plant lectin -: Conformational behaviour of Galβ1-3GlcNAcβ1-R, Galβ1-3GalNAcβ1-R and Galβ1-2Galβ1-R′ in the free state and complexed with galactoside-specific mistletoe lectin as revealed by random-walk and conformational-clustering molecular-mechanics calculations, molecular-dynamics simulations and nuclear Overhauser experiments

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 Gal beta 1-3GlcNAc beta 1-R and Gal beta 1-3GalNAc beta 1-R as well as of a synthetic high-affinity binding partner, i.e. the disaccharide Gal beta 1-2Gal beta 1-R', were chosen as ligands in this study. The disaccharides displayed inherent flexibility in the valley of the global minimum between Phi/Psi combinations of (40 degrees/60 degrees) and (40 degrees/-60 degrees). 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 Phi, Psi, E plot. Occupation of side minima at Phi Psi(180 degrees/0 degrees) or (0 degrees/180 degrees) 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 foe-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 Gal beta 1-3GlcNAc beta 1-R and Gal beta 1-2Gal beta 1-R'. The ligand conformations in the complex did not deviate markedly from those of a minimum conformation in the foe 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 Gal beta 1-3GalNAc beta 1-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 Gal beta 1-2Gal beta 1-R' in association with a galectin, namely the beta-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 Phi, Psi, E plot and substantiate differential conformer selection by these two lectins with identical nominal monosaccharide specificity.