Biochemical and Structural Studies of Inter-modular Interactions in the Ruminococcus flavefaciens Cellulosom

Insight into the molecular mechanism by which cohesins and dockerins interact is of fundamental importance to the study of cellulosomes. The cellulosome is a bacterial multi-enzyme complex assembled extracellular for the efficient degradation of plant cell-wall polymers. Historically divided into three types according to phylogenetic sequence analysis, the cohesins and dockerins are known to interact with a highly potent protein-protein interaction. The interaction is also species-specific and type-dependent, thereby dictating the overall assembly and architecture of a functional cellulosome complex. Type-I and type-II cohesin-dockerin interactions have been subjected to extensive biochemical and structural studies, however at the onset of this research no structural information was available for either the type-III cohesin or dockerin modules or their interaction.;In view of the above, my major goal in this research was to characterize biochemically and structurally the type-III cohesin and the type-III cohesindockerin (Coh-Doc) interaction from the elaborate cellulosome system of the rumen bacteria Ruminococcus flavefaciens. Rf-ScaECoh was the first detailed description of a type-III cohesin structure. It exhibits a crowning alpha-helix connecting beta-strands 8 and 9, and an elaborate 24-residue N-terminal loop which circumvolved the molecule. This structure contributes deep insight into cohesin diversity and provides a plausible putative binding site for the dockerin. Structural analysis of the type-III cohesin in complex with an X-dockerin modular dyad from R. flavefaciens (RfCohE-XDoc) identified the novel type-III dockerin binding orientation, revealing extensive electrostatic interface chemistry, responsible for the type- and strain-specificity. The structure of the RfCohE-XDoc complex also provides evidence for a calcium-binding site in the second, atypical segment of the dockerin and contributes further insight into the structural divergence of the various dockerin types. The type-III dockerin exhibits a non-conventional second calcium-binding loop, which is disrupted by an extended insert. Furthermore, single- and double homology-swapping experiments provided a rationale for the type-III strain-specific interaction. The X-module is shown to function as an elongated projection whose conformation is stabilized by the three "inserts" of the dockerin through an extensive X-module-type-III dockerin intermodular interaction. The structural effects of calcium binding and cohesin-recognition properties were also investigated in this study. Calcium binding induced a conformational change on the type-III dockerin that stabilized the helical interface for the cohesin-dockerin interaction.;These studies have provided insight into the structural divergence among the different cohesin and dockerin types, their intra- and intermolecular interactions, and the elements that determine their specific high-affinity interaction.