Conformation of sLe^x 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, {k off, K D}, 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.

Ab Initio Conformational Study of Two Lewis X Analogues

“…b The published NOE NMR C-O-C-H torsion angles 5 for sLe x were transformed to C-O-C-C torsion angles using a function of C-O-C-C vs C-O-C-H derived from ab initio results (see text for details). To facilitate the overview, the torsion angles differing more than (6°form the NOE NMR value in the solution are set in bold.…”

“…The sLe x is relatively strongly bound to E-selectin according to the measurement of the equilibrium binding-constant (K D ≈ 0.72 mM at 300 K). 5 This strong binding distorts the glycosidic linkages considerably relative to the free (gas or liquid phase) structures.…”

“…In sLe x bound to P-selectin (K D ≈ 7.8 mM at 300 K), 5 the conformations of the two glycosidic linkages of Le x change only slightly, mostly in the τ1 Fuc torsion. The K D value shows that the binding is very weak between sLe x and P-selectin.…”

“…In sLe x bound to L-selectin (K D ≈ 3.9 mM at 300 K), 5 the orientation of the Fuc-R-1,3-GlcNAc linkage of Le x does not change relative to the most stable gas-phase structures (cf. Table 1 and Figure 3).…”

“…4 The 2-, 3-, and 4-hydroxyl groups of R-L-fucose, the 4-, and 6-hydroxyl groups of D-galactose, and the carboxylate group of sialic acid play essential roles in the E-, P-, and L-selectin recognition of sLe x . 5 It has been found that a CH 2 COO -group on the 3-position of galactose sufficiently mimics NeuAc. 4 The next step was to replace GlcNAc.…”