Protein−Carbohydrate Interactions in Human Lysozyme Probed by Combining Site-Directed Mutagenesis and Affinity Labeling

Abstract

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.

Aromatic interactions at the catalytic subsite of sucrose phosphorylase: Their roles in enzymatic glucosyl transfer probed with Phe52 → Ala and Phe52 → Asn mutants

“…Glc1P was used at 50 mM. 6 Glc1F was varied at 14 levels between 0.1 and 250 mM for both wild-type enzyme and mutants. Phosphate was constant at 50 mM.…”

“…Not surprisingly, therefore, carbohydrate-p interactions are widely utilized by glycoside hydrolases (e.g. [6,7]), glycosyltransferases (e.g. [8][9][10]) and carbohydrate-binding modules [11,12] to achieve recognition and specificity for their natural substrates.…”

Modelling of carbohydrate–aromatic interactions: ab initio energetics and force field performance

“…These complexes are characterized by parallel orientation of aromatic ring and pyranose or furanose moiety with C-H bonds of a carbohydrate pointing onto the plane of an aromatic ring. The role of aromatic amino acid residues in binding of carbohydrate ligands was studied in a variety of model proteins by site-directed mutagenesis [18][19][20], covalent modification [18], and by calorimetric methods [20]. Mutation of such aromatic amino acid residue (to alanine or other non-aromatic residues) generally leads to a significant drop in affinity or activity towards a carbohydrate ligand/substrate [18][19][20].…”

Contribution of Non‐Canonical Interactions to the Stability of Sm/LSm Oligomeric Assemblies

“…It has been shown that C a ÀH···p interactions are involved in the formation of protein complexes with special ligands, cofactor, nucleotides, carbohydrates and interleukins. [15][16][17] In the literature there is a lack of data on the contributions of C a ÀH···p, to the protein stability. Work of Reeves and Schneider [18] has shown that this interaction is in fact form of weak hydrogen bond.…”