Properties and mutation analysis of the CelK cellulose-binding domain fromthe Clostridium thermocellum cellulosome

The family IV cellulose-binding domain of Clostridium thermocellum CelK (CB DCelK) was expressed in Escherichia coil and purified. It binds to acid-swo llen cellulose (ASC) and bacterial microcrystalline cellulose (BMCC) with c apacities of 16.03 and 3.95 mu mol/g of cellulose and relative affinities ( K-r) of 2.33 and 9.87 liters/g, respectively. The CBDCelK is the first repr esentative of family IV CBDs to exhibit an affinity for BMCC. The CBDCelK a lso binds to the soluble polysaccharides lichenin, glucomannan, and barley beta -glucan, which are substrates for CelK. It does not bind to xylan, gal actomannan, and carboxymethyl cellulose. The CBDCelK contains 1 mol of calc ium per mel. The CBDCelK has three thiol groups and one disulfide, reductio n of which results in total loss of cellulose-binding ability. To reveal am ino acid residues important for biological function of the domain and to in vestigate the role of calcium in the CBICelK, four highly conserved aromati c residues (Trp(56), Trp(94), Tyr(111), and Tyr(136)) and Asp(192) were mut ated into alanines, giving the mutants W56A, W94A, Y111A, Y136A, and D192A. In addition 14 N-terminal amino acids were deleted, giving the CBD-N-CelK. The CBD-N-CelK and D192A retained binding parameters close to that of the intact CBDCelK, W56A and W94A totally lost the ability to bind to cellulose , Y136A bound to both ASC and BMCC but with significantly reduced binding c apacity and K-r and Y111A bound weakly to ASC and did not bind to BMCC. Mut ations of the aromatic residues in the CBDCelK led to structural changes re vealed by studying solubility, circular-dichroism spectra, dimer formation, and aggregation. Calcium content was drastically decreased in D192A. The r esults suggest that Asp192 is in the calcium-binding site of the CBDCelK an d that calcium does not affect binding to cellulose. The 14 amino acids fro m the N terminus of the CBDCelK are not important for binding. Tyr136, corr esponding to Cellulomonas fimi CenC CBDN1 Y85, located near the binding cle ft, might be involved in the formation of the binding surface, while Y111, W56A, and W94A are essential for the binding process by keeping the CBDCelK correctly folded.