Cohesin-dockerin interactions of cellulosomal subunits of Clostridium cellulovorans

Abstract

The cellulosome of Clostridium cellulovorans consists of three major subunits: CbpA, EngE, and ExgS. The C. cellulovorans scaffolding protein (CbpA) contains nine hydrophobic repeated domains (cohesins) for the binding of enzymatic subunits. Cohesin domains are quite homologous, but there are some questions regarding their binding specificity because some of the domains have regions of low-level sequence similarity. Two cohesins which exhibit 60% sequence similarity were investigated for their ability to bind cellulosomal enzymes. Cohesin 1 (Coh1) was found to contain amino acid residues corresponding to amino acids 312 to 453 of CbpA, which contains a total of 1,848 amino acid residues. Coh6 was determined to contain amino acid residues corresponding to residues 1113 to 1254 of CbpA. By genetic construction, these two cohesins were each fused to MalE, producing MalE-Coh1 and MalE-Coh6. The abilities of two fusion proteins to bind to EngE, ExgS, and CbpA were compared. Although MalE-Coh6 could bind EngE and ExgS, little or no binding of the enzymatic subunits was observed with MalE-Coh1. Significantly, the abilities of the two fusion proteins to bind CbpA were similar. The binding of dockerin-containing enzymes to cohesin-containing proteins was suggested as a model for assembly of cellulosomes. In our examination of the role of dockerins, it was also shown that the binding of endoglucanase B (EngB) to CbpA was dependent on the presence of EngB's dockerin. These results suggest that different cohesins may function with differing efficiency and specificity, that cohesins may play some role in the formation of polycellulosomes through Coh-CbpA interactions, and that dockerins play an important role during the interaction of cellulosomal enzymes and cohesins present in CbpA.

FIG. 1

Sequence similarity between Coh1 and Coh6. Identical amino acid residues are indicated by stars. The numbers refer to amino acid residues of cellulose-binding protein A (CbpA) (17).

FIG. 2

Plasmids used in these studies. (A) Construction of plasmids pMalE-Coh1 and pMalE-Coh6 to investigate the function of different cohesins. The MalE-Coh1 fusion protein contains the peptide from amino acids 312 to the 453 of CbpA (17). The MalE-Coh6 fusion protein contains residues corresponding to amino acids 1113 to 1254 of CbpA (17). (B) Construction of MalE fusion proteins for investigation of binding by the dockerin from EngB. Plasmid pMalE-316EngB441 was constructed for the production of a fusion protein that contained MalE fused with the peptide corresponding to amino acids 316 to 441 in the dockerin of EngB (4). Plasmid pMalE-256-EngB383 was constructed for the production of a fusion protein comprising MalE and a peptide corresponding to amino acids 265 to 383 of CbpA but lacking the dockerin of EngB (4). The XbaI site was from the engB gene. To obtain the PstI site used for subcloning into pMal-c2, the XbaI-BstI107I fragment of pC2-engB (4) was subcloned into the XbaI and HincII sites of M13mp18 (22). The SmaI site and TAA (translation stop codon)-SalI site were constructed by site-directed mutagenesis.

FIG. 3

Effect of SDS concentration on binding of MalE-Coh6 to ExgS and EngE, as tested by slot blotting. Purified ExgS (A) and EngE (B) were blotted onto nitrocellulose membrane disks. Binding ability was monitored by using binding buffer containing MalE-Coh6 and different concentrations of SDS. The interaction Western blot assay (19) was employed to measure the interaction of Coh6 with ExgS and EngE, which is evidenced by dark bands on the disks. The disks blotted with ExgS and EngE were treated with anti-MalE to confirm that the proteins were not recognized by antibody alone (no. 0) but required the binding of Coh6. The numbers above the blots indicate the concentration of SDS: 1, 0%; 2, 0.001%; 3, 0.005%; 4, 0.01%; 5, 0.05%; 6, 0.1%; and 7, 0.5% SDS.

FIG. 4

Interaction of ExgS, EngE, and CbpA with Coh1 and Coh6. (A) Coomassie blue staining of SDS-PAGE gel of CbpA. (B) Interaction Western blot prepared using MalE. (C) Interaction Western blot prepared using MalE-Coh1. (D) Interaction Western blot prepared using MalE-Coh6. Lanes: M, molecular mass markers; 1, ExgS; 2, EngE; 3, CbpA.

FIG. 5

Alignment of the duplicated segments of EngB, EngE, ExgS, and EngH of C. cellulovorans. Shaded-boxed amino acids are identical or have similar chemical properties. The numbers refer to amino acid residues of each enzyme; all sequences are numbered from Met-1 of the peptide. Similar residues are as follows: V, L, I, M, and F; R and K; D and E; N and Q; Y, F, and W; and S and T.

FIG. 6

Interactions between CbpA and the dockerin of EngB. (A) Coomassie blue staining of CbpA on an SDS-PAGE gel. The CbpA band is located at the position corresponding to 190 kDa. (B) Ordinary Western blot prepared using anti-CbpA immunoglobulin G. (C) Interaction Western blot with MalE as a control and without dockerin. (D) MalE-EngB with dockerin. (E) MalE-EngB without dockerin. Lane M, molecular mass markers.