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Human lysozyme (HL) labelled with the 2′,3′-epoxypropyl β-glycoside of Man-β1,4-GlcNAc was crystallized at pH 4.5. The cell dimensions were a = 36.39, b = 116.38, c = 30.91 Å and the space group was P212121. The unit cell contained four molecules (Vm = 2.18 Å3 Da−1). The crystal structure was determined by molecular replacement and refined to an R value of 0.168 for 7060 reflections [|Fo| > 3σ(F)] in the resolution range 8.0–2.1 Å. A prominent shift of the Cα-atom positions by up to 3.8 Å in the region of residues 45–50 was observed compared with wild-type HL. Owing to the conformational change in this region the intermolecular contacts were altered remarkably compared with wild-type HL, explaining the difference in molecular packing. The Man-β1,4-GlcNAc moiety occupied subsites B and C in the substrate-binding site of HL. Several differences in the hydrogen-bonded contacts between the ligand part and the protein part were observed for HL labelled with the 2′,3′-epoxypropyl β-glycoside of Man-β1,4-GlcNAc compared with HL labelled with the corresponding derivatives of GlcNAc-β1,4-GlcNAc and Gal-β1,4-GlcNAc. In contrast to the replacement of GlcNAc with Gal, the replacement of GlcNAc with Man did not sacrifice the stacking interactions with the side-chain group of Tyr63 as determined by the parallelism of the apolar face of the carbohydrate residue and the aromatic plane of the Tyr63 side chain. The 2′,3′-epoxypropyl β-glycoside of Man-β1,4-GlcNAc exhibited almost the same affinity towards HL as Gal-β1,4-GlcNAc, a much lower affinity than that of GlcNAc-β1,4-GlcNAc. The difference in the protein–ligand interactions was discussed in relation to the carbo­hydrate-residue recognition specificity at subsite B of HL. The results suggested that Gln104 was a determinant for the strong recognition of GlcNAc residue at subsite B in HL.