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 clef ts, the association of TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl-4-yl) sp in-labeled derivatives of cellotriose and cellotetraose with isolated CBDN1 and CBDN2 was studied using heteronuclear H-1-N-15 NMR spectroscopy. Quant itative binding measurements indicate that the TEMPO moiety does not signif icantly perturb the affinity of the cellooligo-saccharide derivatives for t he CBDs. The paramagnetic enhancements of the amide H-1(N) longitudinal (De lta R-1) and transverse (Delta R-2) relaxation rates were measured by compa ring the effects of TEMPO-cellotetraose in its nitroxide (oxidized) and hyd roxylamine (reduced) forms on the two CBDs. The bound spin-label affects mo st significantly the relaxation rates of amides located at both ends of the sugar-binding cleft of each CBD. Similar results are observed with TEMPO-c ellotriose bound to CBDN1. This demonstrates that the TEMPO-labeled cellool igosaccharides, and by inference strands of amorphous cellulose, can associ ate with CBDN1 and CBDN2 in either orientation across their beta-sheet bind ing clefts. The ratio of the association constants for binding in each of t hese 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 t he binding clefts of the CenC CBDs. (C) 1999 Academic Press.