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

Abstract

The cellulosome components are assembled into the cellulosome complex by the interaction between one of the repeated cohesin domains of a scaffolding protein and the dockerin domain of an enzyme component. We prepared five recombinant cohesin polypeptides of the Clostridium thermocellum scaffolding protein CipA, two dockerin polypeptides of C. thermocellum Xyn11A and Xyn10C, four cohesin polypeptides of Clostridium josui CipA, and two dockerin polypeptides of C. josui Aga27A and Cel8A, and qualitatively and quantitatively examined the cohesin-dockerin interactions within C. thermocellum and C. josui, respectively, and the species specificity of the cohesin-dockerin interactions between these two bacteria. Surface plasmon resonance (SPR) analysis indicated that there was a certain selectivity, with a maximal 34-fold difference in the K D values, in the cohesin-dockerin interactions within a combination of C. josui, although this was not detected by qualitative analysis. Affinity blotting analysis suggested that there was at least one exception to the species specificity in the cohesin-dockerin interactions, although species specificity was generally conserved among the cohesin and dockerin polypeptides from C. thermocellum and C. josui, i.e. the dockerin polypeptides of C. thermocellum Xyn11A exceptionally bound to the cohesin polypeptides from C. josui CipA. SPR analysis confirmed this exceptional binding. We discuss the relationship between the species specificity of the cohesin-dockerin binding and the conserved amino acid residues in the dockerin domains.

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.…”

“…Initial studies on the cohesin-dockerin interaction from different species indicated a general lack of intraspecies specificity (4 -6), whereas between species the interaction appeared to be selective (5,7). This rule usually prevails within a given scaffoldin, but not among different scaffoldins, even those produced by the same species (26 -30).…”

“…Within a given species, the cohesin-dockerin interaction generally appears to lack specificity in that the various cohesins of the primary scaffoldin tend to generally recognize the dockerins derived from the different enzyme subunits (4 -6). On the other hand, in several cases the cohesins from different species generally fail to recognize dockerins from another bacterium (5,7). The biological significance of the observed intraspecies fidelity versus cross-species variance remains to be elucidated.…”

An enhanced protein–protein interaction based on enzymatic complex through replacement of the recognition site

“…The interaction was determined by the addition of 100 l of the desired competitor samples (the ExgS wild-type dockerin protein, up to a maximum of 1.3 M), immediately followed by the addition of 100 l of a solution of wild-type and modified EngH, ExgS and EngE to a final concentration of 47 nM. The running buffer contained 10 mM CaCl 2 because calcium ions are required for the binding of cellulosomal enzymes to the cohesin modules [12]. Dilutions of the competitor were obtained using TrisNC buffer containing BSA to maintain a constant protein concentration.…”

Transcriptomic Analysis of Clostridium thermocellum in Cellulolytic Consortium after Artificial Reconstruction to Enhance Ethanol Production

“…The known number of dockerin-bearing enzymes in C. thermocellum was reported to be approximately eight times higher than the number of cohesins in the scaffoldin subunit (Shoham et al 1999). A few reports have demonstrated that several cellulosomal catalytic units containing dockerin I modules can bind to cohesin I modules from other Clostridiums (Jindou et al 2004;Pinheiro et al 2009;Sakka et al 2009). This may also contribute to the integral increase in FPA.…”