STUDIES ON THE 3-DIMENSIONAL BEHAVIOR OF THE SELECTIN LIGANDS LEWIS(A) AND SULFATED LEWIS(A) USING NMR-SPECTROSCOPY AND MOLECULAR-DYNAMICS SIMULATIONS

Sulphated blood group Lewis(a)/Lewis(x) (Le(a)/Le(x)) type sequences, with sulphate at the 3-position of galactose, have emerged as potent ligands for the endothelial adhesion molecule E-selectin and the leukocyte adhesion molecule L-selectin. As a first step in elucidating the molecular basis of the strong interactions with the selectins, we have performed conformational studies of the sulphated Lea in comparison with the non-sulphated analogue which is less strongly bound by E-selectin and not at all by L-selectin. Experimental NMR parameters [nuclear Overhauser effects (NOE) and interglycosidic (3)J(C,H)] and theoretical values back-calculated from the minimum energy structures are in excellent agreement for both molecules. Molecular dynamics calculations for SuLe(a) depict only minor torsional fluctuations around the glycosidic linkages over the time course of the 500 ps simulations, leading to the conclusion that the conformation of SuLe(a) approximates to a single-rigid structure, as does the previously investigated Lea molecule. Comparison of experimentally and theoretically obtained parameters for SuLe(a) with those for the nonsulphated Le(a) molecule indicate that no detectable changes occur in the three-dimensional structure of the trisaccharide upon sulphation. Thus, the enhanced selectin binding to the sulphated Le(a) is most likely due to favourable electrostatic interactions between the charged sulphate group and corresponding charged groups on the selectin protein.