Experimental identification of a theoretically predicted "left-sided" binding mode for (GlcNAc)6 in the active site of lysozyme.
Using conformational energy calculations, we previously predicted that there are two distinct binding modes for hexasaccharide substrates of hen egg white lysozyme (HEWL), a "left-sided" binding mode and a "right-sided" one. The former involves such residues as Arg-45, Asn-46, and Thr-47, while the latter involves such residues as Asn-113 and Arg-114. The left-sided binding mode was predicted to predominate for (GlcNAc)6. We now present two lines of experimental evidence that indicate that left-sided binding occurs for this substrate. First, we show that ring-necked pheasant lysozyme (RNPL), in which Lys and His replace Asn and Arg at positions 113 and 114, respectively, has the same affinity for (GlcNAc)6 as does HEWL, indicating that the "right" side is not involved in equilibrium binding to the substrate. Second, we show that a monoclonal antibody, HyHEL-5, which binds specifically to an epitope including residues Arg-45, Asn-46, Thr-47, Asp-48, and Arg-68 on the far "left" side of HEWL, is competitively displaced by (GlcNAc)5 and (GlcNAc)6 but not by GlcNAc, (GlcNAc)2, or (GlcNAc)4. Only the former two substrates can bind in site F in the lower active site. Since these two substrates are the only ones that competitively displace HyHEL-5, our results suggest that the terminal saccharide residues of these substrates bind to the left side of the active site cleft, as predicted from theory.
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001665:Binding Sites; D001667:Binding, Competitive; D002236:Carbohydrate Conformation; D002645:Chickens; D004529:Egg White; D005260:Female; D007700:Kinetics; D008958:Models, Molecular; D009113:Muramidase / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D009844:Oligosaccharides; C041162:N-acetylglucosamine hexasaccharide 1-4; D009113:Muramidase