The cellulose-binding domains from Cellulomonas fimi beta-1, 4-glucanase CenC bind nitroxide spin-labeled cellooligosaccharides in multiple orientations.
The N-terminal cellulose-binding domains CBDN1 and CBDN2 from Cellulomonas fimi cellulase CenC each adopt a jelly-roll beta-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 CBDN1 and CBDN2 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 1HN longitudinal (DeltaR1) and transverse (DeltaR2) 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 CBDN1. This demonstrates that the TEMPO-labeled cellooligosaccharides, and by inference strands of amorphous cellulose, can associate with CBDN1 and CBDN2 in either orientation across their beta-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.
Johnson
P E
PE
Protein Engineering Network of Centres of Excellence and Department of Chemistry and Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada.
Brun
E
E
MacKenzie
L F
LF
Withers
S G
SG
McIntosh
L P
LP
D001665:Binding Sites; D002240:Carbohydrate Sequence; D002482:Cellulose / Q000378:metabolism*; D003497:Cyclic N-Oxides; D004578:Electron Spin Resonance Spectroscopy; D004583:Electrons; D044643:Glucan 1,4-beta-Glucosidase; D006093:Gram-Positive Asporogenous Rods / Q000201:enzymology*; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011522:Protons; D013113:Spin Labels; D001617:beta-Glucosidase / Q000737:chemistry* / Q000378:metabolism*
D003497:Cyclic N-Oxides; D009844:Oligosaccharides; D011522:Protons; D013113:Spin Labels; D002482:Cellulose; D001617:beta-Glucosidase; D044643:Glucan 1,4-beta-Glucosidase; C003959:TEMPO