Structure and binding specificity of the second N-terminal cellulose-binding domain from Cellulomonas fimi endoglucanase C

The 1,4-beta-glucanase CenC from Cellulomonas fimi contains two cellulose-b inding domains, CBDN1 and CBDN2, arranged in tandem at its N-terminus. Thes e homologous CBDs are distinct in their selectivity for binding amorphous a nd not crystalline cellulose. Multidimensional heteronuclear nuclear magnet ic resonance (NMR) spectroscopy was used to determine the tertiary structur e of CBDN2 in the presence of saturating amounts of cellopentaose. A total of 1996 experimental restraints were used to calculate an ensemble of 21 fi nal structures for the protein. CBDN2 is composed of 11 beta-strands, folde d into two antiparallel beta-sheets, with a topology of a jellyroll beta-sa ndwich. On the basis of patterns of chemical shift perturbations accompanyi ng the addition of cellooligosaccharides, as well as the observation of int ermolecular protein-sugar NOE interactions, the cellulose-binding site of C BDN2 was identified as a cleft that lies across one face of the beta-sandwi ch. The thermodynamic basis for the binding of cellooligosaccharides was in vestigated using isothermal titration calorimetry and NMR spectroscopy. Bin ding is enthalpically driven and consistent with a structural model involvi ng hydrogen bonding between the equatorial hydroxyls of the glucopyranosyl rings and polar amino acid side chains lining the CBDN2 cleft. Affinity ele ctrophoresis was used to determine that CBDN2 also binds soluble beta-1,4-l inked polymers of glucose, including hydroxyethylcellulose and beta-1,3-1,4 -glucans. This study complements a previous analysis of CBDN1 [Johnson, P. E., Joshi, M. D., Tomme, P., Kilburn, D. G., and McIntosh, L. P. (1996) Bio chemistry 35, 14381-14394] and demonstrates that the homologous CBDs from C enC share very similar structures and sugar binding properties.