STUDIES OF THE BOUND CONFORMATIONS OF METHYL ALPHA-LACTOSIDE AND METHYL BETA-ALLOLACTOSIDE TO RICIN-B CHAIN USING TRANSFERRED NOE EXPERIMENTS IN THE LABORATORY AND ROTATING FRAMES, ASSISTED BY MOLECULAR MECHANICS AND DYNAMICS CALCULATIONS

The conformation in solution of methyl beta-galactopyranosyl-(1-->4)-alpha-glucopyranoside (methyl alpha-lactoside) and methyl beta-galactopyranosyl-(1-->6)-beta-glucopyranoside (methyl beta-allolactoside) has been studied through NMR spectroscopy and molecular mechanics calculations. NOE measurements both in the laboratory and rotating frames, have been interpreted in terms of an ensemble average distribution of conformers. Molecular mechanics calculations have been performed to estimate the probability distribution of conformers from the steric energy maps. The experimental results indicate that methyl alpha-lactoside spends about 90% of its time in a broad low-energy region close to the global minimum, while methyl beta-allolactoside presents much higher flexibility. The conformational changes that occur when both disaccharides are bound to the ricin B chain in aqueous solution have been studied using transferred NOE experiments at several protein/ligand ratios. The observed data indicate that the protein causes a conformational variation in the torsion angles of methyl alpha-lactoside changing towards smaller angle values (Phi Psi approximate to -20/-20), although the recognized conformer is still within the lowest energy region. In particular, the torsional changes separate Gal H1 from Glc H3 and Glc H6 protons, with a noticeable decrease in the Intensities of the corresponding NOE cross-peaks, which were clearly observed for the free disaccharide. On the other hand, different conformations around the Phi, Psi, and omega glycosidic bonds of methyl beta-allolactoside are recognized by the lectin. In fact, for the methyl-beta-allolactoside-ricin-B complex, only the NOESY cross-peaks corresponding to the protons of the galactose residue are negative, as expected for a molecule in the slow motion regime. In contrast, the corresponding cross peaks for the glucose residue were about zero, as expected for a molecule whose motion is practically independent of the protein. However, for the methyl-alpha-lactoside - ricin-B complex, all the NOESY cross-peaks for both the galactose and glucose moieties were clearly negative. From the NMR experimental point of view, it is demonstrated that the comparison of longitudinal and transversal transferred NOEs allows one to clearly differentiate direct enhancements from spin diffussion effects, which are of major concern when analysing NOE spectra of macromolecules. Finally, molecular modelling of both disaccharides in the binding site strongly suggests that for methyl alpha-lactoside, apart from the expected contacts between the galactose moiety and different amino acid residues, there are also van der Waals' contacts between the protein and the remote glucose moiety, as previously deduced from binding studies using modified lactoside derivatives [Solis, D., Fernandez, P., Diaz-Maurino, T., Jimenez-Barbero, J. & Martin-Lomas, M. (1993) Eur. J. Biochem. 214, 677-683]. This result is in contrast with the X-ray crystallographic analysis of the ricin-B-lactose and ricin-B - galactose-containing diantennary hexasaccharide complexes [Rutenber, E. & Robertus, J. D. (1991) Proteins 10, 260-269]. On the other hand, for methyl beta-allolactoside, the experimental data and the modelling studies demonstrate that only the galactose moiety is bound by the lectin and, therefore, the conformation around the glycosidic angles can be probably described by a distribution similar to that existing in free solution.