STRUCTURE OF THE N-TERMINAL CELLULOSE-BINDING DOMAIN OF CELLULOMONAS-FIMI CENC DETERMINED BY NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY

Multidimensional heteronuclear nuclear magnetic resonance (NMR) spectr oscopy was used to determine the tertiary structure of the 152 amino a cid N-terminal cellulose-binding domain from Cellulomonas fimi 1,4-bet a-glucanase CenC (CBDN1) CBDN1 was studied in the presence of saturati ng concentrations of cellotetraose, but due to spectral overlap, the o ligosaccharide was not included in the structure calculations. A total of 1705 interproton nuclear Overhauser effect (NOE), 56 phi, 88 psi, 42 chi 1, 9 chi 2 dihedral angle, and 88 hydrogen-bond restraints were used to calculate 25 final structures. These structures have a rmsd f rom the average of 0.79 +/- 0.11 Angstrom for all backbone atoms exclu ding disordered termini and 0.44 +/- 0.05 Angstrom for residues with r egular secondary structures. CBDN1 is composed of 10 beta-strands, fol ded into two antiparallel beta-sheets with the topology of a jelly-rol l beta-sandwich. The strands forming the face of the protein previousl y determined by chemical shift perturbations to be responsible for cel looligosaccharide binding [Johnson, P. E., Tomme, P., Joshi, M. D., & McIntosh, L. P. (1996) Biochemistry, 35, 13895-13906] are shorter than those forming the opposite side of the protein. This results in a 5-s tranded binding cleft, containing a central strip of hydrophobic resid ues that is flanked on both sides by polar hydrogen-bonding groups. Th e presence of this cleft provides a structural explanation for the uni que selectivity of CBDN1 for amorphous cellulose and other soluble oli gosaccharides and the lack of binding to crystalline cellulose, The te rtiary structure of CBDN1 is strikingly similar to that of the bacteri al 1,3-1,4-beta-glucanases, as well as other sugar-binding proteins wi th jelly-roll folds.