Enzyme systems that attack the plant cell wall contain noncatalytic carbohydrate-binding modules (CBMs) that mediate attachment to this composite structure and play a pivotal role in maximizing the hydrolytic process. Although xyloglucan, which includes a backbone of β-1,4-glucan decorated primarily with xylose residues, is a key component of the plant cell wall, CBMs that bind to this polymer have not been identified. Here we showed that the C-terminal domain of the modular Clostridium thermocellum enzyme CtCel9D-Cel44A (formerly known as CelJ) comprises a novel CBM (designated CBM44) that binds with equal affinity to cellulose and xyloglucan. We also showed that accommodation of xyloglucan side chains is a general feature of CBMs that bind to single cellulose chains. The crystal structures of CBM44 and the other CBM (CBM30) in CtCel9D-Cel44A display a β-sandwich fold. The concave face of both CBMs contains a hydrophobic platform comprising three tryptophan residues that can accommodate up to five glucose residues. The orientation of these aromatic residues is such that the bound ligand would adopt the twisted conformation displayed by cello-oligosaccharides in solution. Mutagenesis studies confirmed that the hydrophobic platform located on the concave face of both CBMs mediates ligand recognition. In contrast to other CBMs that bind to single polysaccharide chains, the polar residues in the binding cleft of CBM44 play only a minor role in ligand recognition. The mechanism by which these proteins are able to recognize linear and decorated β-1,4-glucans is discussed based on the structures of CBM44 and the other CBMs that bind single cellulose chains.

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.

Cellulosomes, synthesized by anaerobic microorganisms such as Clostridium thermocellum, are remarkably complex nanomachines that efficiently degrade plant cell wall polysaccharides. Cellulosome assembly results from the interaction of type I dockerin domains, present on the catalytic subunits, and the cohesin domains of a large non-catalytic integrating protein that acts as a molecular scaffold. In general, type I dockerins contain two distinct cohesin-binding interfaces that appear to display identical ligand specificities. Inspection of the C. thermocellum genome reveals 72 dockerin-containing proteins. In four of these proteins, Cthe_0258, Cthe_0435, Cthe_0624 and Cthe_0918, there are significant differences in the residues that comprise the two cohesin-binding sites of the type I dockerin domains. In addition, a protein of unknown function (Cthe_0452), containing a C-terminal cohesin highly similar to the equivalent domains present in C. thermocellum-integrating protein (CipA), was also identified. In the present study, the ligand specificities of the newly identified cohesin and dockerin domains are described. The results revealed that Cthe_0452 is located at the C. thermocellum cell surface and thus the protein was renamed as OlpC. The dockerins of Cthe_0258 and Cthe_0435 recognize, preferentially, the OlpC cohesin and thus these enzymes are believed to be predominantly located at the surface of the bacterium. By contrast, the dockerin domains of Cthe_0624 and Cthe_0918 are primarily cellulosomal since they bind preferentially to the cohesins of CipA. OlpC, which is a relatively abundant protein, may also adopt a ‘warehouse’ function by transiently retaining cellulosomal enzymes at the cell surface before they are assembled on to the multienzyme complex.

During the course of evolution, the cellulosome, one of Nature’s most intricate multi-enzyme complexes, has been continuously fine-tuned to efficiently deconstruct recalcitrant carbohydrates. To facilitate the uptake of released sugars, anaerobic bacteria use highly ordered protein-protein interactions to recruit these nanomachines to the cell surface. Dockerin modules located within a non-catalytic macromolecular scaffold, whose primary role is to assemble cellulosomal enzymatic subunits, bind cohesin modules of cell envelope proteins, thereby anchoring the cellulosome onto the bacterial cell. Here we have elucidated the unique molecular mechanisms used by anaerobic bacteria for cellulosome cellular attachment. The structure and biochemical analysis of five cohesin-dockerin complexes revealed that cell surface dockerins contain two cohesin-binding interfaces, which can present different or identical specificities. In contrast to the current static model, we propose that dockerins utilize multivalent modes of cohesin recognition to recruit cellulosomes to the cell surface, a mechanism that maximises substrate access while facilitating complex assembly.

Protein-protein interactions play a pivotal role in the assembly of the cellulosome, one of natures most intricate nanomachines dedicated to the depolymerisation of complex carbohydrates. The integration of cellulosomal components usually occurs through the binding of type-I dockerin modules, located at the C-terminus of the enzymes to cohesin modules located in the primary scaffoldin subunit. Cellulosomes are typically recruited to the cell surface via type-II cohesin-dockerin interactions established between primary and cell-surface anchoring scaffoldin subunits. In contrast with type-II interactions, type-I dockerins usually display a dual binding mode that may allow increased conformational flexibility during cellulosome assembly. Acetivibrio cellulolyticus produces a highly complex cellulosome comprising an unusual adaptor scaffoldin, ScaB, which mediates the interaction between the primary scaffoldin, ScaA, through type-II cohesin-dockerin interactions and the anchoring scaffoldin, ScaC, via type-I cohesin-dockerin interactions. Here, we report the crystal structure of the type-I ScaB dockerin in complex with a type-I ScaC cohesin in two distinct orientations. The data show that the ScaB dockerin displays structural symmetry, reflected by the presence of two essentially identical binding surfaces. The complex interface is more extensive than those observed in other type-I complexes, which results in an ultra-high affinity interaction (Ka ~ 1012 M). A subset of ScaB dockerin residues were also identified as modulating the specificity of type-I cohesin-dockerin interactions in A. cellulolyticus. This report reveals that recruitment of cellulosomes onto the cell surface may involve dockerins presenting a dual binding mode to incorporate additional flexibility into the quaternary structure of highly populated multi-enzyme complexes.