Crucial roles of single residues in binding affinity, specificity, and promiscuity in the cellulosomal cohesin-dockerin interface.

Interactions between cohesin and dockerin modules play a crucial role in the assembly of multienzyme cellulosome complexes. Although intraspecies cohesin and dockerin modules bind in general with high affinity but indiscriminately, cross-species binding is rare. Here, we combined ELISA-based experiments with Rosetta-based computational design to evaluate the contribution of distinct residues at the Clostridium thermocellum cohesin-dockerin interface to binding affinity, specificity, and promiscuity. We found that single mutations can show distinct and significant effects on binding affinity and specificity. In particular, mutations at cohesin position Asn(37) show dramatic variability in their effect on dockerin binding affinity and specificity: the N37A mutant binds promiscuously both to cognate (C. thermocellum) as well as to non-cognate Clostridium cellulolyticum dockerin. N37L in turn switches binding specificity: compared with the wild-type C. thermocellum cohesin, this mutant shows significantly increased preference for C. cellulolyticum dockerin combined with strongly reduced binding to its cognate C. thermocellum dockerin. The observation that a single mutation can overcome the naturally observed specificity barrier provides insights into the evolutionary dynamics of this system that allows rapid modulation of binding specificity within a high affinity background.

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

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.

Complexity of the Ruminococcus flavefaciens FD-1 cellulosome reflects an expansion of family-related protein-protein interactions.

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.

High-throughput screening of cohesin mutant libraries on cellulose microarrays.

The specificity of cohesin-dockerin interactions is critically important for the assembly of cellulosomal enzymes into the multienzyme cellulolytic complex (cellulosome). In order to investigate the origins of the observed specificity, a variety of selected amino acid positions at the cohesin-dockerin interface can be subjected to mutagenesis, and a library of mutants can be constructed. In this chapter, we describe a protein-protein microarray technique based on the high affinity of a carbohydrate-binding module (CBM), attached to mutant cohesins. Using cellulose-coated glass slides, libraries of mutants can be screened for binding to complementary partners. The advantages of this tool are that crude cell lysate can be used without additional purification, and the microarray can be used for screening both large libraries as initial scanning for "positive" plates, and for small libraries, wherein individual colonies are printed on the slide. Since the time-consuming step of purifying proteins can be circumvented, the approach is also appropriate for providing molecular insight into the multicomponent organization of complex cellulosomes.