Cellulosome from Clostridium cellulolyticum: Molecular Study of the Dockerin/Cohesin Interaction

Abstract

Clostridium cellulolyticum produces cellulolytic complexes (cellulosomes) made of 10-13 cell wall degrading enzymes tightly bound to a scaffolding protein (CipC) by means of their dockerin domain. It has previously been shown that the receptor domains in CipC are the cohesin domains and that the cohesin/dockerin interaction is calcium-dependent. In the present study, surface plasmon resonance was used to demonstrate that the free cohesin1 from CipC and dockerin from CelA have the same K(D) (2.5 x 10(-)(10) M) as that of the entire CelA and a larger fragment of CipC, the latter of which contains, in addition to cohesin1, a cellulose binding domain and a hydrophilic domain of unknown function. This demonstrates that neither the catalytic domain of CelA nor the noncohesin domains of CipC have any influence on the interaction. Dockerin domains are composed of two conserved segments of 22 residues: removal of the second segment abolishes the affinity for cohesin1, whereas modified dockerins having twice the first segment, twice the second, or both segments but in a reverse order have K(D) values for cohesin1 in the same range as that observed for wild-type dockerin. These data indicate that if two segments are required for the complexation with the cohesin, segments 1 and 2 are similar enough to replace each other. Calcium overlay experiments revealed that the dockerin domain has one calcium binding site per conserved segment. Circular dichroism performed on wild-type and mutant dockerins indicates that this domain is well structured and that removal of calcium only weakly affects the secondary structure, which remains 40-45% helical.

Pinpoint Mapping of Recognition Residues on the Cohesin Surface by Progressive Homologue Swapping

“…The preference in favor of the C. thermocellum system can be explained by the superior affinity characteristics for the cohe- sin-dockerin interaction of the latter over those of C. cellulolyticum, as observed previously (5,15,(21)(22)(23). In any case, Sw1-2, which contains both the first and last third of the C. thermocellum cohesin, binds strongest to the corresponding dockerin.…”

“…General DNA Manipulation-Genomic DNA was prepared from C. cellulolyticum ATCC 35319 and C. thermocellum YS as described previously (4,7,21) and was used as template for PCR amplification of the carbohydrate-binding module (CBM), 1 cohesin, and dockerin modules. QIAprep Spin Miniprep Kit (Qiagen, Valencia, CA) was used for small-scale plasmid isolation.…”

“…The very high affinity of both cohesins (K D Ͻ 10 Ϫ10 M) to their matching dockerins (15,(21)(22)(23) suggests that numerous intermodular interactions occur between the cohesin and dockerin. On the other hand, we reasoned that not all of the interactions would be necessary for interspecies specificity.…”

Cohesin-Dockerin Interactions within and between Clostridium josui and Clostridium thermocellum

“…All the dockerin polypeptides used in this study contained a linker sequence of only three amino acids. All the cohesin polypeptides were coupled to a carboxymethyl-dextran layer on the surface of the sensor chip in this study, whereas biotinylated recombinant proteins derived from C. cellulolyticum CipA were used for coupling to a streptavidin-dextran layer in the previous experiments (23). Notwithstanding the differences in the experimental conditions for the SPR analysis, the K D values (1.3 ϫ 10 Ϫ10 to 4.4 ϫ 10 Ϫ9 ) for the interactions between cohesin and dockerin polypeptides from C. josui were comparable with the value (2.5 ϫ 10 Ϫ10 ) for the interaction between the first cohesin domain of CipC and the dockerin domain of Cel5A from C. cellulolyticum, suggesting that the experimental conditions adopted here were suitable for SPR analysis.…”

“…HBS buffer (10 mM HEPES, pH 7.4, 0.15 M NaCl) was used as an immobilization buffer, and 10 mM CaCl 2 , 0.005% surfactant P20 (BIAcore), and 50 mM Tris-maleate buffer, pH 6.5, was used as a running buffer, as described previously for SPR analysis of C. cellulolyticum proteins (23). Each of the cohesin polypeptides tested was immobilized on a dextran matrix with free carboxylic groups (CM5 chip, BIAcore) employing conventional carbodiimide coupling chemistry and subsequent deactivation of excess active esters using ethanolamine (EDC/ NHS coupling kit, BIAcore).…”