ScaC, an Adaptor Protein Carrying a Novel Cohesin That Expands the Dockerin-Binding Repertoire of the Ruminococcus flavefaciens 17 Cellulosome

Abstract

A new gene, designated scaC and encoding a protein carrying a single cohesin, was identified in the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 as part of a gene cluster that also codes for the cellulosome structural components ScaA and ScaB. Phylogenetic analysis showed that the sequence of the ScaC cohesin is distinct from the sequences of other cohesins, including the sequences of R. flavefaciens ScaA and ScaB. The scaC gene product also includes at its C terminus a dockerin module that closely resembles those found in R. flavefaciens enzymes that bind to the cohesins of the primary ScaA scaffoldin. The putative cohesin domain and the C-terminal dockerin module were cloned and overexpressed in Escherichia coli as His 6 -tagged products (ScaC-Coh and ScaC-Doc, respectively). Affinity probing of protein extracts of R. flavefaciens 17 separated in one-dimensional and two-dimensional gels with recombinant cohesins from ScaC and ScaA revealed that two distinct subsets of native proteins interact with ScaC-Coh and ScaA-Coh. Furthermore, ScaC-Coh failed to interact with the recombinant dockerin module from the enzyme EndB that is recognized by ScaA cohesins. On the other hand, ScaC-Doc was shown to interact specifically with the recombinant cohesin domain from ScaA, and the ScaA-Coh probe was shown to interact with a native 29-kDa protein spot identified as ScaC by matrix-assisted laser desorption ionization-time of flight mass spectrometry. These results suggest that ScaC plays the role of an adaptor scaffoldin that is bound to ScaA via the ScaC dockerin module, which, via the distinctive ScaC cohesin, expands the range of proteins that can bind to the ScaA-based enzyme complex.

Unconventional Mode of Attachment of the Ruminococcus flavefaciens Cellulosome to the Cell Surface

“…flavefaciens strain 17 produces a cellulosome complex that is known to involve the cohesin-containing structural components ScaA, ScaB, and ScaC, together with interacting enzymes and unidentified proteins that carry dockerin domains (1,(36)(37)(38). The assembly of the different components in the R. flavefaciens cellulosome differs from the proposed molecular architecture in the clostridial cellulosomes.…”

“…The three previously characterized cohesin-containing proteins from R. flavefaciens 17 (ScaA, ScaB, and ScaC) are encoded by linked genes within the sca gene cluster (13,36,37). The sequence of this cluster was extended downstream from scaB by genome-walking PCR (see Materials and Methods), revealing two additional open reading frames (Fig.…”

“…We have inferred previously at least two distinct cohesindockerin interactions between enzyme subunits and scaffoldin proteins (36), together with two additional interactions: one between the ScaA dockerin and ScaB cohesins (13) and one between the ScaC cohesin and an uncharacterized set of dockerins (37). The interaction between ScaE and the ScaB C terminus therefore represents the fifth cohesin-dockerin specificity to be identified in this species.…”

“…In addition, ScaC, a small dockerin-bearing protein that also possesses a single divergent cohesin domain, binds both to ScaA via its dockerin and to a range of thus-far-unidentified polypeptides via its cohesin. ScaC has therefore been proposed to serve as an adaptor protein that enhances the repertoire of subunits present in the cellulosome (37). ScaA binds to any of the seven cohesin repeats of ScaB via a specific cohesin-dockerin interaction (13).…”

Action of Designer Cellulosomes on Homogeneous Versus Complex Substrates

“…In addition, these modules are rather divergent from those found in Clostridium spp. and were classified, on the basis of sequence similarities, in a different phylogenetic group, termed type III (27). Cross-reactivity with type-I cohesins or dockerins from either C. cellulolyticum or C. thermocellum is therefore unlikely to occur.…”