Structural and dynamic analysis of oligosaccharide binding by CBDn1

This thesis describes the biophysical and structural characterization of CBDN1, the aminoterminal cellulose-binding domain (CBD) from the Cellulomonas fimi (β-1,4-glucanase CenC. Heteronuclear multidimensional nuclear magnetic resonance (NMR) methods were used to determine the three dimensional structure of CBDN1 in the presence of saturating amounts of cellotetraose. It was found that CBDN1 is composed of 10 β-strands, folded into two antiparallel β-sheets with the topology of a jelly-roll β-sandwich. The dominant feature of the CBDN1 structure is a cleft which runs along the length of one face of the molecule. On the basis of perturbations of the NMR spectrum of CBDN1 due to the addition of sugar, it was shown that CBDN1 binds soluble cellooligosaccharides within the cleft. Intermolecular nuclear Overhauser enhancements (NOEs) between residues in the cleft and the bound sugar confirmed that this face is responsible for ligand binding. Association constants, determined from the dependence of the amide ¹H and 15N chemical shifts upon added sugar, were found to increase with increasing sugar length, reaching a maximum at a ligand length of five glucose units. This corresponds approximately to the length of the binding cleft. The binding cleft is composed of a strip of hydrophobic residues, flanked by hydrophilic residues. It is proposed that the pyranose rings of the sugar lie over the hydrophobic residues while the polar side-chains are involved in hydrogen bonding interactions with the equatorial hydroxyl groups of the glucose rings. CBDN1 binds a calcium ion at a site opposite the oligosaccharide binding face. The binding affinity of oligosaccharides is unaffected by the presence of calcium. CBDN1 binds nitroxide spin-labelled oligosaccharides in multiple orientations. In one orientation the spin-label group lies near residue alanine 18, in the other near glycine 86. This ability to bind the same ligand in several orientations indicates that different hydrogen bonding combinations between the sugar and protein must occur. This is likely related to the motional disorder observed for residues present in the binding face as determined by NMR relaxation methods. [Scientific formulae used in this abstract could not be reproduced.]Science, Faculty ofChemistry, Department ofGraduat