Insights into a type III cohesin–dockerin recognition interface from the cellulose‐degrading bacterium Ruminococcus flavefaciens

Abstract

Cellulosomes are large multicomponent cellulose-degrading assemblies found on the surfaces of cellulolytic microorganisms. Often containing hundreds of components, the self-assembly of cellulosomes is mediated by the ultra-high-affinity cohesin-dockerin interaction, which allows them to adopt the complex architectures necessary for degrading recalcitrant cellulose. Better understanding of how the cellulosome assembles and functions and what kinds of structures it adopts will further effort to develop industrial applications of cellulosome components, including their use in bioenergy production. Ruminococcus flavefaciens is a well-studied anaerobic cellulolytic bacteria found in the intestinal tracts of ruminants and other herbivores. Key to cellulosomal self-assembly in this bacterium is the dockerin ScaADoc, found on the non-catalytic structural subunit scaffoldin ScaA, which is responsible for assembling arrays of cellulose-degrading enzymes. This work expands on previous efforts by conducting a series of binding studies on ScaADoc constructs that contain mutations in their cohesin recognition interface, in order to identify which residues play important roles in binding. Molecular dynamics simulations were employed to gain insight into the structural basis for our findings. A specific residue pair in the first helix of ScaADoc, as well as a glutamate near the C-terminus, was identified to be essential for cohesin binding. By advancing our understanding of the cohesin binding of ScaADoc, this study serves as a foundation for future work to more fully understand the structural basis of cellulosome assembly in R. flavefaciens.

Nanoscale Engineering of Designer Cellulosomes

“…We note that R. flavefaciens also exhibits an unusual dockerin module which binds scaffoldin A on the primary scaffoldin B. Experiments and simulations have probed the details of the cohesion-dockerin interface in various species including very recently in the cellulose-degrading bacterium R. flavefaciens; a particular valine-alanine motif was identified as important but not the sole director of interface formation, with mutation studies showing that the interface can in some cases compensate and form alternative, near iso-energetic interfaces [16] Similar results were found for the type-II cohesindockerin complex of C. thermocellum, and highlighted again the large effect the X-module can have on the cohesin-dockerin interaction [194] Multimodular enzymes, enzymes which embed various functional domains connected by linkers, are responsible for a majority of cellulose hydrolysis in nature, [4] in particular cellobiohydrolases TrCel6A and TrCel7A from T. reesei. Linkers in such enzymes are highly glycosylated, [195] e.g., in Cel6A and Cel7A, linkers (41 and 27 residues, respectively) connect the CBM to the rest of the protein which are mainly O-glycosylated with also some N-linked glycans (Cel6).…”

“…Substantial advances have been made in recent years [16,59,60,75,95,98,99,[127][128][129][130] and models that are generallyaccepted (at least in gross features) exist for cellulosome structure and function, but the fine details of the molecular mechanisms are still poorly understood. For example, even appended to a CBM, it is not clear how an endoglucanase would be capable of pulling a single polysaccharide chain out of its crystalline environment and forcing the chain productively into its active site cleft.…”

Crucial Roles of Single Residues in Binding Affinity, Specificity, and Promiscuity in the Cellulosomal Cohesin-Dockerin Interface

“…Previous studies identified specificity-determining residues in the dockerin module based on sequence conservation within and across different species (8,22). In a gene-swapping experiment, Nakar et al (23) were able to switch the binding specificity of a cohesin from C. cellulolyticum to dockerin of C. thermocellum by the replacement of only three residues.…”