| Protein-protein interactions play a vital role in many cellular processes as exemplified by the assembly of the cellulosome, a bacterial multi-enzyme complex that efficiently degrades cellulose and hemicellulose. Cellulosome assembly involves the high-affinity binding of type I enzyme-borne dockerins to repeated cohesin modules located on non-catalytic structural proteins termed scaffoldins. In addition, the complex is anchored into the bacterial surface through the binding of a scaffoldin type II dockerin to cell-bound cohesins. Initially, the architecture and organization of cellulosomes was thought to rely uniquely on type I and type II cohesin-dockerin interactions. It was recently suggested that cellulosomes from rumen bacteria are organized through different mechanisms involving a third type of cohesin-dockerin complexes. Thus, the genome of the major ruminal bacterium Ruminococcus flavefaciens FD-1 revealed a particularly elaborate cellulosome system that is assembled from a library of more than 200 different components through divergent cohesin-dockerin pairs. Providing structural insights for the specificity displayed by the increasing repertoire of cohesin-dockerin interaction is not only of fundamental importance but essential for the development of novel cellulosome based tools. The present work aimed to identify the molecular basis for the organization of R. flavefaciens cellulosome by dissecting the structural basis of cohesin-dockerin specificity in cellulosomes of rumen bacteria. The data revealed a collection of unique cohesin-dockerin interactions, supporting the functional relevance of dockerin classification in groups based on primary sequence similarity. In addition, R. flavefaciens cellulosome is assembled through a mechanism involving single but not dual-binding mode dockerins. This contrasts with the majority of the cellulosomes described to date where dockerins generally present two similar cohesin-binding interfaces, supporting a dual-binding mode. To illustrate this, the structures of two cohesin-dockerin complexes containing an Acetivibrio cellulolyticus dual-binding mode dockerin were solved. Finally, structural information was used to engineer a dockerin presenting a dual cohesin specificity, revealing the plasticity of the cohesin-dockerin platform to design novel protein-protein interactions. |
