The cellulose-binding domains from Cellulomonas fimi β-1,4-glucanase CenC bind nitroxide spin-labeled cellooligosaccharides in multiple orientations

• Published in: Journal of molecular biology Vol. 287; no. 3; pp. 609 - 625
• Main Authors: Johnson, Philip E., Brun, Emmanuel, MacKenzie, Lloyd F., Withers, Stephen G., McIntosh, Lawrence P.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 02.04.1999
• Subjects: beta-Glucosidase - chemistry; beta-Glucosidase - metabolism; Binding Sites; Carbohydrate Sequence; Cellulomonas fimi; Cellulose - metabolism; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Electrons; Glucan 1,4-beta-Glucosidase; glycosynthase; Gram-Positive Asporogenous Rods - enzymology; Hydrogen Bonding; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Sequence Data; NMR; Oligosaccharides - chemistry; Oligosaccharides - metabolism; Protein Structure, Secondary; protein-carbohydrate interaction; Protons; spin label; Spin Labels; glycosynthase; CBD; NOE; TEMPO-Glc 3; protein-carbohydrate interaction; spin label; CBD N1; CBD N2; NMR; CBD Cex; CBD N1N2; pH; TEMPO-Glc 4; TEMPO; HSQC
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.1999.2627

The N-terminal cellulose-binding domains CBD N1 and CBD N2 from Cellulomonas fimi cellulase CenC each adopt a jelly-roll β-sandwich structure with a cleft into which amorphous cellulose and soluble cellooligosaccharides bind. To determine the orientation of the sugar chain within these binding clefts, the association of TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl-4-yl) spin-labeled derivatives of cellotriose and cellotetraose with isolated CBD N1 and CBD N2 was studied using heteronuclear 1H- 15N NMR spectroscopy. Quantitative binding measurements indicate that the TEMPO moiety does not significantly perturb the affinity of the cellooligo-saccharide derivatives for the CBDs. The paramagnetic enhancements of the amide 1H N longitudinal (Δ R 1) and transverse (Δ R 2) relaxation rates were measured by comparing the effects of TEMPO-cellotetraose in its nitroxide (oxidized) and hydroxylamine (reduced) forms on the two CBDs. The bound spin-label affects most significantly the relaxation rates of amides located at both ends of the sugar-binding cleft of each CBD. Similar results are observed with TEMPO-cellotriose bound to CBD N1. This demonstrates that the TEMPO-labeled cellooligosaccharides, and by inference strands of amorphous cellulose, can associate with CBD N1 and CBD N2 in either orientation across their β-sheet binding clefts. The ratio of the association constants for binding in each of these two orientations is estimated to be within a factor of five to tenfold. This finding is consistent with the approximate symmetry of the hydrogen-bonding groups on both the cellooligosaccharides and the residues forming the binding clefts of the CenC CBDs.