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

E Leibovitz,P Béguin

doi:10.1128/jb.178.11.3077-3084.1996

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.

Highlights

The mode of attachment of the catalytic subunits to CipA has been elucidated
Their restriction maps were consistent with the restriction fragments revealed by Southern blotting in C. thermocellum DNA
Within the region covered by the cloned fragments, a 1.6-kb segment between the PstI site and the left boundary of the insert borne by pCT1801 (Fig. 1) was necessary and sufficient to encode a polypeptide able to bind the dockerin domain of CipA

Summary

Introduction

The mode of attachment of the catalytic subunits to CipA has been elucidated. Each catalytic subunit contains a conserved duplicated segment of 23 residues [3], which has been termed the dockerin domain [2]. Besides the reiterated cohesin domains, the CipA polypeptide includes a cellulose-binding domain and a C-terminal duplicated segment of 23 residues [11]. This segment resembles the dockerin domains of the catalytic subunits, its amino acid sequence is more divergent from the consensus. We report the molecular cloning and sequence analysis of a gene whose product binds the dockerin domain borne by CelC-DSCipA. Segments of the gene were subcloned and expressed separately in order to identify the region of the polypeptide responsible for binding the dockerin domain of CipA. One of the OlpB segments was shown to bind CelCDSCipA

Methods

Results

Conclusion