X-ray Structure of Human Lysozyme Labelled with 2',3'-Epoxypropyl β-Glycoside of Man-β 1,4-GlcNAc. Structural Change and Recognition Specificity at Subsite B

Abstract

Human lysozyme (HL) labelled with the 2',3'-epoxypropyl ,8-glycoside of Man-,81,4-GlcNAc was crystallized at pH 4.5. The cell dimensions were a --36.39, b = 116.38, c= 30.91 ,~, and the space group was P2k2121. The unit cell contained four molecules (V,,, = 2.18 A 3 Da-l). The crystal structure was determined by molecular replacement and refined to an R value of 0.168 for 7060 reflections [IF,,[ > 3o(F)] in the resolution range 8.0-2.1 .~,. A prominent shift of the C"-atom positions by up to 3.8/k 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-,81,4-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 ,8-glycoside of Man-,81,4-GlcNAc compared with HL labelled with the corresponding derivatives of G1cNAc-,81,4-GIcNAc and Gal-,81,4-GlcNAc. In contrast to the replacement of GIcNAc with Gal, the replacement of GIcNAc 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'-epoxypropy1,8-glycoside of Man-,81,4-GlcNAc exhibited almost the same affinity towards HL as Gal-,81,4-GIcNAc, a much lower affinity than that of GIcNAc-,81,4-GIcNAc. 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 Glnl04 was a determinant for the strong recognition of GIcNAc residue at subsite B in HL.

X‐ray structural analysis of the ligand‐recognition mechanism in the dual‐affinity labeling of c‐type lysozyme with 2′,3′‐epoxypropyl β‐glycoside of N‐acetyllactosamine

“…However, to date, only a few cases, in which the three-dimensional structure of the modified enzyme was determined at high resolution, have been reported with regard to glycosidases (Keitel et al, 1993;Chen et al, 1995;Havukainen et al, 1996). The modified human lysozyme (HL) that uncovered the origin of its carbohydrate-recognition specificity has also constituted part of the limited examples (Muraki et al, 1996(Muraki et al, , 1998(Muraki et al, , 1999(Muraki et al, , 2000a. C-type lysozymes including HL and hen egg-white lysozyme (HEWL) are a family of bacteriolytic enzymes of vertebrate origin.…”

Dual Affinity Labeling of the Active Site of Human Lysozyme with an N-Acetyllactosamine Derivative: First Ligand Assisted Recognition of the Second Ligand,

“…The shift slightly widened the substrate binding cleft, which was not observed in the single labeling of HL with Gal-1,4-GlcNAc-Epo. A larger shift of this region has been observed in the labeling of HL with Man-1,4-GlcNAc-Epo (22).…”

“…The structural analyses of the products indicated that all disaccharides in the labeled HLs occupied essentially the same position designated as subsites B and C (21,22). The lysozyme-saccharide complex has been extensively studied by X-ray crystallography (6).…”

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“…The binding mode of N-acetylglucosamine residue at subsite C was totally maintained, therefore the site-specific labeling reactions were considered to proceed via the recognition of N-acetylglucosamine residue at subsite C. The inactivation of lytic activity against M. luteus cells substrate occurred by the conjugation. The half lifetime of residual lytic activity of human lysozyme in the presence of 2', 3'-epoxypropyl β-glycosides of Gal-β1, 4-GlcNAc and Man-β1,4-GlcNAc was prolonged by approximately twice as much compared with the corresponding derivative of GlcNAc-β1, 4-GlcNAc [47]. This clearly presented the weaker affinity of the former two derivatives than the latter one, which should be ascribed to the difference in the affinity at subsite B.…”

“…X-ray structural analysis of site-specifically conjugated derivatives of disaccharides: In order to further examine the functional role of the interactions between the carbohydrate residue bound at subsite B and Tyr63 residue in the ligand recognition event in human lysozyme, human lysozyme derivatives site-specifically conjugated with 2', 3'-epoxypropyl β-glycoside derivatives of three kinds of disaccharides, namely N-acetylchitobiose (GlcNAc-β1, 4-GlcNAc), N-acetyl lactosamine (Gal-β1, 4-GlcNAc) and β-1, 4 linked mannosyl N-acetylglucosamine (Man-β1, 4-GlcNAc), were synthesized. The detailed structures of these three kinds of site-specifically labeled human lysozymes were analyzed by X-ray crystallography [46,47]. All conjugations were conducted by the ester linkage formation with the side-chain carboxylate group of Asp53.…”