Conformation of sLex Tetrasaccharide, Free in Solution and Bound to E-, P-, and L-Selectin,

Abstract

The conformations of the NeuAcα2(I)→3Galβ1(II)→4[Fucα1(III)→3]GlcNAc-O-CH3 tetrasaccharide (sLex), in aqueous solution and bound to E-, P-, and L-selectin have been determined using high resolution NMR spectroscopy. In the free ligand, the conformation of glycosidic linkage I is disordered with {ΦI, ΨI} sampling values close to {−60°, 0°}, {−100°, −50°}, and {180°, 0°}. The trisaccharide portion is rigid and characterized by {ΦII, ΨII; ΦIII, ΨIII} = {46°, 18°; 48°, 24°}. The measured dissociation rates and equilibrium binding constants, {koff, KD}, were {164 ± 24 s-1, 0.72 ± 0.4 mM}, {522 ± 166 s-1, 7.8 ± 1.0 mM}, and {1080 ± 167 s-1, 3.9 ± 0.6 mM} at 300 K for E-, P-, and L-selectin, respectively. The bound conformations of the ligand were calculated from the full relaxation matrix analysis of transferred-NOE spectra for E- and P-selectin or by using a two-spin approximation for the L-selectin complex. Both E- and P-selectin recognize the {−60°, 0°} conformation of sLex while the {−100°, −50°} conformer is probably recognized by L-selectin. The conformation of the branched trisaccharide portion in the bound state remains close to the conformation of the free ligand. In the E-, P-, and L-selectin complexes the GalH4 proton is in the vicinity of protein aromatic protons, most likely Tyr94 and/or Tyr48.