Studies on the three-dimensional behaviour of the selectin ligands Lewis(a) and sulphated 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 Le(a) 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 3JC,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 Le(a) molecule. Comparison of experimentally and theoretically obtained parameters for SuLe(a) with those for the non-sulphated 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.