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

Abstract

The family IV cellulose-binding domain of Clostridium thermocellum CelK (CBD(CelK)) was expressed in Escherichia coli and purified. It binds to acid-swollen cellulose (ASC) and bacterial microcrystalline cellulose (BMCC) with capacities of 16.03 and 3.95 micromol/g of cellulose and relative affinities (K(r)) of 2.33 and 9.87 liters/g, respectively. The CBD(CelK) is the first representative of family IV CBDs to exhibit an affinity for BMCC. The CBD(CelK) also binds to the soluble polysaccharides lichenin, glucomannan, and barley beta-glucan, which are substrates for CelK. It does not bind to xylan, galactomannan, and carboxymethyl cellulose. The CBD(CelK) contains 1 mol of calcium per mol. The CBD(CelK) has three thiol groups and one disulfide, reduction of which results in total loss of cellulose-binding ability. To reveal amino acid residues important for biological function of the domain and to investigate the role of calcium in the CBD(CelK) four highly conserved aromatic residues (Trp(56), Trp(94), Tyr(111), and Tyr(136)) and Asp(192) were mutated 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 CBD(CelK), W56A and W94A totally lost the ability to bind to cellulose, Y136A bound to both ASC and BMCC but with significantly reduced binding capacity and K(r) and Y111A bound weakly to ASC and did not bind to BMCC. Mutations of the aromatic residues in the CBD(CelK) led to structural changes revealed by studying solubility, circular-dichroism spectra, dimer formation, and aggregation. Calcium content was drastically decreased in D192A. The results suggest that Asp192 is in the calcium-binding site of the CBD(CelK) and that calcium does not affect binding to cellulose. The 14 amino acids from the N terminus of the CBD(CelK) are not important for binding. Tyr136, corresponding to Cellulomonas fimi CenC CBD(N1) Y85, located near the binding cleft, might be involved in the formation of the binding surface, while Y111, W56A, and W94A are essential for the binding process by keeping the CBD(CelK) correctly folded.

FIG. 1

SDS-PAGE of the purified CBD_CelK and its deletion and mutation variants overexpressed in E. coli. Lanes: 1, CBD_CelK; 2, W56A; 3, W94A; 4, Y111A; 5, Y136A; 6, CBD-N_CelK; 7, D192A; M, low-range protein standards (Bio-Rad Laboratories, Richmond, Calif.), including phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), and trypsin inhibitor (21.5 kDa).

FIG. 2

DTNB titration of thiol groups in the CBD_CelK. A, denaturing buffer; B, native buffer; C, DTNB titration of thiol groups in denaturing buffer after carboxymethylation of free SH groups, followed by disulfide bridge reduction (see Materials and Methods for details).

FIG. 3

Affinity electrophoresis of CelK containing a CBD (CelK94) and without a CBD (CelK74) in the absence (−) and presence (+) of barley β-glucan (0.1%, wt/vol) (A), glucomannan (0.1%, wt/vol) (B), lichenin (0.1%, wt/vol) (C), galactomannan (0.1%, wt/vol) (D), CMC (0.1%, wt/vol) (E), and birch wood xylan (0.1%, wt/vol) (F).

FIG. 4

CD spectra of the wild-type CBD_CelK and mutant CBDs. The proteins were in 10 mM Tris-HCl buffer (pH 7.5) at a concentration of 10 μM. Measurements were performed with a J710 spectrometer (Jasco).

FIG. 5

Alignment of family IV CBDs. A and B, subgroups IVa and IVb, respectively. Abbreviations: Cel1_Strre, Streptomyces reticuli Cel1 (accession no. X65616); E1_Thfu, Thermomonospora fusca E1 (accession no. L20094); CenC_Celfi, Cellulomonas fimi CenC (accession no. X57858); CelK_Clotm, Clostridium thermocellum CelK (accession no. AF039030); CbhA_Clotm, C. thermocellum CbhA (accession no. X80993); CelE_Ccellu, C. cellulolyticum CelE (accession no. PC1140); Xyn1_Rhoma, Rhodothermomonas marinus Xyn1 (accession no. Y11564); LicA_Clotm, C. thermocellum LicA (accession no. X89732); LamA_Thne, Thermotoga neapolitana LamA (accession no. Z47974). Conserved amino acid residues mutated in the CBD_CelK are marked with asterisks.