Solution conformation of Lewis a--derived selectin ligands is unaffected by anionic substituents at the 3'- and...

The selectins are a family of proteins that mediate leukocyte tethering and rolling along the vascular endothelium. E-, P-, and L-selectin recognize various derivatives of the Lewis(a) and Lewis(x) trisaccharides. The distribution of negative charges on the Lewis(a) and Lewis(x) oligosaccharides appears to be an important factor in their binding by the selectins. Previous work exploring this electrostatic dependence found that a series of synthetic anionic trisaccharides, 3'-sulfo, 3'-phospho, 6'-sulfo, and 3',6'-disulfo Lewis(a)-(Glc), exhibited differing selectin inhibitory efficacies. To explore the possibility that these differences arise from conformational differences between the sugars, the solution structures of these trisaccharides were determined using NMR and molecular dynamics simulations. Interproton distances and interglycosidic torsion angles were determined at 37 degrees C using NOESY buildup curves and 1D LRJ experiments, respectively. Data from both experiments agreed well with predictions made from 2000 picosecond unrestrained molecular dynamics simulations. We found that 3'-sulfation did not alter the core Lewis(a) conformation, a finding that reaffirms the results of previous study. In addition, we found that sulfation at the 6' position also leaves the trisaccharide conformation unperturbed. This is significant because the proximity of the 6'-sulfate group to the fucose ring might have altered the canonical Lewis (a) structure. The disulfate exhibited greater flexibility than the other derivatives in dynamics simulations, but not so much as to affect NOE and heteronuclear coupling constant measurements. Taken together, our findings support the use of Lewis(a) as a template onto which charged groups may be added without significantly altering the trisaccharide's structure.