INTERACTION OF SOLUBLE CELLOOLIGOSACCHARIDES WITH THE N-TERMINAL CELLULOSE-BINDING DOMAIN OF CELLULOMONAS-FIMI CENC .2. NMR AND ULTRAVIOLET-ABSORPTION SPECTROSCOPY

The N-terminal cellulose-binding domain (CBDN1) from Cellulomonas fimi beta-1,4-glucanase CenC binds amorphous but not crystalline cellulose . To investigate the structural and thermodynamic bases of cellulose b inding, NMR and difference ultraviolet absorbance spectroscopy were us ed in parallel with calorimetry (Tomme, P., Creagh, A. L., Kilburn, D. G., & Haynes, C. A., (1996) Biochemistry 35, 13885-13894] to characte rize the interaction of soluble cellooligosaccharides with CBDN1 Assoc iation constants, determined from the dependence of the amide H-1 and N-15 chemical shifts of CBDN1 upon added sugar, increase from 180 +/- 60 M(-1) for cellotriose to 4 200 +/- 720 M(-1) for cellotetraose, 34 000 +/- 7 600 M(-1) for cellopentaose, and an estimate of 50 000 M(-1) for cellohexaose. This implies that the CBDN1 cellulose-binding site spans approximately five glucosyl units, On the basis of the observed patterns of amide chemical shift changes, the cellooligosaccharides bi nd along a five-stranded beta-sheet that forms a concave face of the j elly-roll beta-sandwich structure of CBDN1. This beta-sheet contains a strip of hydrophobic side chains flanked on both sides by polar resid ues, NMR and difference ultraviolet absorbance measurements also demon strate that tyrosine, but not tryptophan, side chains may be involved in oligosaccharide binding. These results lead to a model in which CBD N1 interacts with soluble cellooligosaccharides and, by inference, wit h single polysaccharide chains in regions of amorphous cellulose, prim arily through hydrogen bonding to the equatorial hydroxyl groups of th e pyranose rings. Van der against the apolar side chains may augment b inding. CBDN1 stands in marked contrast to previously characterized CB Ds that absorb to crystalline cellulose via a flat binding surface dom inated by exposed aromatic rings.