Interaction of polysaccharides with the N-terminal cellulose-binding domain of Cellulomonas fimi CenC .1. Binding specificity and calorimetric analysis

The carbohydrate-binding specificity of the N-terminal cellulose-binding domain (CBDN1) from Cellulomonas fimi beta-1,4-glucanase C (CenC) was investigated using affinity electrophoresis, binding assays and microcalorimetry in parallel with NMR and difference ultraviolet absorbance spectroscopy [Johnson, P. E., Tomme, P., Joshi, M. D., & McIntosh, L. P. (1996) Biochemistry 35, 13895-13906]. Binding of CBDN1 on insoluble cellulose is distinctly different from other cellulose-binding domains. CBDN1 binds amorphous cellulose (phosphoric acid-swollen) with high affinity (K-f = 5.1 L g(-1)), binds Avicel weakly and does not bind highly crystalline bacterial or tunicin cellulose. Moreover, CBDN1 binds soluble cellooligosaccharides and beta-1,4-linked oligomers of glucose such as hydroxyethylcellulose, soluble beta-1,3-1,4-glucans from barley and oat, but has no affinity for alpha-1,4-, beta-1,3-, or beta-1,6-polymers of glucose. This is the first report of a cellulose-binding domain with strong and specific affinity for soluble glycans. The thermodynamics for binding of CBDN1 to oligosaccharides, soluble glycans, and phosphoric acid-swollen cellulose were investigated by titration microcalorimetry. At least four beta-1,4-linked glucopyranosides are required to detect binding. For larger glucans, with five or more glucopyranoside units, the binding constants and standard free energy changes are virtually independent of the glucan chain length, indicating that cellopentaose completely fills the binding site. Binding is moderately strong with binding constants ranging from 3 200 +/- 500 M(-1) for cellotetraose, to 25 000 +/- 3 000 M(-1) for the larger sugars. The reactions are controlled by favorable standard free enthalpy changes which are compensated in a linear fashion by a significant decrease in entropy. A predominance of polar interactions such as hydrogen bonding together with van der Waals interactions provide the major driving forces for the binding event.