Abstract
Protein-protein interactions play a vital role in cellular processes as exemplified by assembly of the intricate multi-enzyme cellulosome complex. Cellulosomes are assembled by selective high-affinity binding of enzyme-borne dockerin modules to repeated cohesin modules of structural proteins termed scaffoldins. Recent sequencing of the fiber-degrading Ruminococcus flavefaciens FD-1 genome revealed a particularly elaborate cellulosome system. In total, 223 dockerin-bearing ORFs potentially involved in cellulosome assembly and a variety of multi-modular scaffoldins were identified, and the dockerins were classified into six major groups. Here, extensive screening employing three complementary medium- to high-throughput platforms was used to characterize the different cohesin-dockerin specificities. The platforms included (i) cellulose-coated microarray assay, (ii) enzyme-linked immunosorbent assay (ELISA) and (iii) in-vivo co-expression and screening in Escherichia coli. The data revealed a collection of unique cohesin-dockerin interactions and support the functional relevance of dockerin classification into groups. In contrast to observations reported previously, a dual-binding mode is involved in cellulosome cell-surface attachment, whereas single-binding interactions operate for cellulosome integration of enzymes. This sui generis cellulosome model enhances our understanding of the mechanisms governing the remarkable ability of R. flavefaciens to degrade carbohydrates in the bovine rumen and provides a basis for constructing efficient nano-machines applied to biological processes.
Highlights
R. flavefaciens is a Gram-positive, anaerobic bacterium of the Firmicutes phylum
Cohesins are modular components of scaffoldins, whereas dockerins are borne by individual cellulosomal enzymes that are integrated into the complex through interaction with the cohesins[15,16,17,18]
Some dockerins belong to proteins bearing typical plant cell wall-degrading catalytic modules while others are part of proteins containing structural or functional components (e.g., carbohydrate-binding modules (CBMs), predicted cohesin-bearing scaffoldins, serpins and LRR motifs)
Summary
R. flavefaciens is a Gram-positive, anaerobic bacterium of the Firmicutes phylum. It is the only known bacterium in the rumen shown to possess a definitive cellulosome, i.e., a discrete multi-enzyme complex specialized in the breakdown of cellulose and associated plant cell-wall polysaccharides[12,13,14]. R. flavefaciens strains have in common an enzyme-integrating subunit, ScaB, which carries a C-terminal X module-dockerin (XDoc) dyad that in turn recognizes the single cohesin of the surface-anchored scaffoldin, ScaE28,29. Some enzymes bind directly to ScaA and ScaA-like cohesins on ScaB, whereas others bind via the intermediary ScaC cohesin[32], which acts as a selective “adaptor” scaffoldin that alters enzymatic composition of the cellulosome. These divergent interactions and their significance towards cellulosome organization are presumably governed by the sequence and consequent specificity of the enzyme-borne dockerin. Many of the dockerin-bearing parent proteins appear to be unrelated to traditional cellulosome activities, with predicted functions, such as serpins, peptidases, LRR (leucine-rich repeats) proteins and transglutaminases
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