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A new type of cohesin domain that specifically binds the dockerin domain of the Clostridium thermocellum cellulosome-integrating protein CipA.

ABSTRACT: The cellulosome-integrating protein CipA, which serves as a scaffolding protein for the cellulolytic complex produced by Clostridium thermocellum, comprises a COOH-terminal duplicated segment termed the dockerin domain. This paper reports the cloning and sequencing of a gene, termed sdbA (for scaffoldin dockerin binding), encoding a protein which specifically binds the dockerin domain of CipA. The sequenced fragment comprises an open reading frame of 1,893 nucleotides encoding a 631-amino-acid polypeptide, termed SdbA, with a calculated molecular mass of 68,577 kDa. SAA comprises an NH2-terminal leader peptide followed by three distinct regions. The NH2-terminal region is similar to the NH2-terminal repeats of C. thermocellum OlpB and ORF2p. The central region is rich in lysine and harbors a motif present in Streptococcus M proteins. The COOH-terminal region consists of a triplicated sequence present in several bacterial cell surface proteins. The NH2-terminal region of SdbA and a fusion protein carrying the first NH2-terminal repeat of OlpB were shown to bind the dockerin domain of CipA. Thus, a new type of cohesin domain, which is present in one, two, and four copies in SdbA, ORF2p, and OlpB, respectively, can be defined. Since OlpB and most likely SdbA and ORF2p are located in the cell envelope, the three proteins probably participate in anchoring CipA (and the cellulosome) to the cell surface.

SUBMITTER: Leibovitz E

PROVIDER: S-EPMC178055 | biostudies-other | 1996 Jun

REPOSITORIES: biostudies-other

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A new type of cohesin domain that specifically binds the dockerin domain of the Clostridium thermocellum cellulosome-integrating protein CipA.

Leibovitz E E Béguin P P

Journal of bacteriology 19960601 11

The cellulosome-integrating protein CipA, which serves as a scaffolding protein for the cellulolytic complex produced by Clostridium thermocellum, comprises a COOH-terminal duplicated segment termed the dockerin domain. This paper reports the cloning and sequencing of a gene, termed sdbA (for scaffoldin dockerin binding), encoding a protein which specifically binds the dockerin domain of CipA. The sequenced fragment comprises an open reading frame of 1,893 nucleotides encoding a 631-amino-acid p ...[more]

PMID: 8655483

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Project description: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.

Dataset Information

A new type of cohesin domain that specifically binds the dockerin domain of the Clostridium thermocellum cellulosome-integrating protein CipA.

Publications

A new type of cohesin domain that specifically binds the dockerin domain of the Clostridium thermocellum cellulosome-integrating protein CipA.

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