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Abstract
AbtractCellulosomes are sophisticated multi-enzymatic nanomachines produced by anaerobes to effectively deconstruct plant structural carbohydrates. Cellulosome assembly involves the binding of enzyme-borne dockerins (Doc) to repeated cohesin (Coh) modules located in a non-catalytic scaffoldin. Docs appended to cellulosomal enzymes generally present two similar Coh-binding interfaces supporting a dual-binding mode, which may confer increased positional adjustment of the different complex components. Ruminococcus flavefaciens’ cellulosome is assembled from a repertoire of 223 Doc-containing proteins classified into 6 groups. Recent studies revealed that Docs of groups 3 and 6 are recruited to the cellulosome via a single-binding mode mechanism with an adaptor scaffoldin. To investigate the extent to which the single-binding mode contributes to the assembly of R. flavefaciens cellulosome, the structures of two group 1 Docs bound to Cohs of primary (ScaA) and adaptor (ScaB) scaffoldins were solved. The data revealed that group 1 Docs display a conserved mechanism of Coh recognition involving a single-binding mode. Therefore, in contrast to all cellulosomes described to date, the assembly of R. flavefaciens cellulosome involves single but not dual-binding mode Docs. Thus, this work reveals a novel mechanism of cellulosome assembly and challenges the ubiquitous implication of the dual-binding mode in the acquisition of cellulosome flexibility.
Highlights
The cellulosome is one of the most intricate nanomachines Nature has evolved
The genome sequence of R. flavefaciens strain FD-1 revealed the presence of 223 dockerin-containing proteins (154 of which were identified as carbohydrate-active enzymes)[8], indicating that this bacterial nanomachine is the most complex cellulosome described to date[9] (Fig. 1)
In an initial attempt to understand the structural determinants of cohesin modules (Cohs)-dockerin modules (Docs) specificity that orchestrate the correct assembly of R. flavefaciens cellulosome, the structure of the third Coh of ScaB, termed RfCohScaB3, was solved by SAD phasing
Summary
The cellulosome is one of the most intricate nanomachines Nature has evolved. Cellulosomes combine an extensive repertoire of enzymes, including glycoside hydrolases, pectate lyases and carbohydrate esterases, into a large multi-enzyme complex (molecular mass >3 MDa) that efficiently deconstructs especially recalcitrant plant structural carbohydrates, such as cellulose and hemicellulose. R. flavefaciens Docs have been organized into six groups based on primary structure homology[10] This classification was recently found to be functionally relevant[11], with the binding of group 1 Docs to the Cohs of scaffoldins ScaA and ScaB providing the major mechanism for cellulosome assembly in R. flavefaciens. This suggests that group 1 Docs may bind to their target Cohs through a single-binding mode To test this hypothesis, we determined the X-ray crystal structure of two R. flavefaciens group 1 Docs, Doc1a and Doc1b, in complex with a ScaB (CohScaB3) and a ScaA Coh, respectively. We determined the X-ray crystal structure of two R. flavefaciens group 1 Docs, Doc1a and Doc1b, in complex with a ScaB (CohScaB3) and a ScaA Coh, respectively These structures together with comprehensive biochemical analyses suggest that integration of a large repertoire of enzymes into the R. flavefaciens cellulosome operates through a single-binding mode
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