Protein-carbohydrate interactions in human lysozyme probed by combining site-directed mutagenesis and affinity labeling.
The synergism between apolar and polar interactions in the carbohydrate recognition by human lysozyme (HL) was probed by site-directed mutagenesis and affinity labeling. The three-dimensional structures of the Tyr63-->Leu mutant HL labeled with 2',3'-epoxypropyl beta-glycoside of N,N'-diacetylchitobiose (L63-HL/NAG-NAG-EPO complex) and the Asp102-->Glu mutant HL labeled with the 2',3'-epoxypropyl beta-glycoside of N-acetyllactosamine were revealed by X-ray diffraction at 2.23 and 1.96 A resolution, respectively. Compared to the wild-type HL labeled with the 2', 3'-epoxypropyl beta-glycoside of N,N'-diacetylchitobiose, the N-acetylglucosamine residue at subsite B of the L63-HL/NAG-NAG-EPO complex markedly moved away from the 63rd residue, with substantial loss of hydrogen-bonding interactions. Evidently, the stacking interaction with the aromatic side chain of Tyr63 is essential in positioning the N-acetylglucosamine residue in the productive binding mode. On the other hand, the position of the galactose residue in subsite B of HL is almost unchanged by the mutation of Asp102 to Glu. Most hydrogen bonds, including the one between the carboxylate group of Glu102 and the axial 4-OH group of the galactose residue, were maintained by local movement of the backbone from residues 102-104. In both structures, the conformation of the disaccharide was conserved, reflecting an intrinsic conformational rigidity of the disaccharides. The structural analysis suggested that CH-pi interactions played an important role in the recognition of the carbohydrate residue at subsite B of HL.
Muraki
M
M
Biomolecules Department, National Institute of Bioscience and Human-Technology, 1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan. muraki@nibh.go.jp
Harata
K
K
Sugita
N
N
Sato
K I
KI
D000345:Affinity Labels; D001665:Binding Sites; D002236:Carbohydrate Conformation; D002241:Carbohydrates / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D004187:Disaccharides / Q000737:chemistry; D006801:Humans; D006860:Hydrogen Bonding; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D014961:X-Ray Diffraction
D000345:Affinity Labels; D002241:Carbohydrates; D004187:Disaccharides; D009113:Muramidase