The plant cell wall degrading apparatus of anaerobic bacteria includes a large multienzyme complex termed the "cellulosome." The complex assembles through the interaction of enzyme-derived dockerin modules with the multiple cohesin modules of the noncatalytic scaffolding protein. Here we report the crystal structure of the Clostridium cellulolyticum cohesin-dockerin complex in two distinct orientations. The data show that the dockerin displays structural symmetry reflected by the presence of two essentially identical cohesin binding surfaces. In one binding mode, visualized through the A16S/L17T dockerin mutant, the C-terminal helix makes extensive interactions with its cohesin partner. In the other binding mode observed through the A47S/F48T dockerin variant, the dockerin is reoriented by 180 degrees and interacts with the cohesin primarily through the N-terminal helix. Apolar interactions dominate cohesin-dockerin recognition that is centered around a hydrophobic pocket on the surface of the cohesin, formed by Leu-87 and Leu-89, which is occupied, in the two binding modes, by the dockerin residues Phe-19 and Leu-50, respectively. Despite the structural similarity between the C. cellulolyticum and Clostridium thermocellum cohesins and dockerins, there is no cross-specificity between the protein partners from the two organisms. The crystal structure of the C. cellulolyticum complex shows that organism-specific recognition between the protomers is dictated by apolar interactions primarily between only two residues, Leu-17 in the dockerin and the cohesin amino acid Ala-129. The biological significance of the plasticity in dockerin-cohesin recognition, observed here in C. cellulolyticum and reported previously in C. thermocellum, is discussed.

Multifunctional proteins, which play a critical role in many biological processes, have typically evolved through the recruitment of different domains that have the required functional diversity. Thus the different activities displayed by these proteins are mediated by spatially distinct domains, consistent with the specific chemical requirements of each activity. Indeed, current evolutionary theory argues that the colocalization of diverse activities within an enzyme is likely to be a rare event, because it would compromise the existing activity of the protein. In contrast to this view, a potential example of multifunctional recruitment into a single protein domain is provided by CtCel5C-CE2, which contains an N-terminal module that displays cellulase activity and a C-terminal module, CtCE2, which exhibits a noncatalytic cellulose-binding function but also shares sequence identity with the CE2 family of esterases. Here we show that, unlike other CE2 members, the CtCE2 domain displays divergent catalytic esterase and noncatalytic carbohydrate binding functions. Intriguingly, these diverse activities are housed within the same site on the protein. Thus, a critical component of the active site of CtCE2, the catalytic Ser-His dyad, in harness with inserted aromatic residues, confers noncatalytic binding to cellulose whilst the active site of the domain retains its esterase activity. CtCE2 catalyses deacetylation of noncellulosic plant structural polysaccharides to deprotect these substrates for attack by other enzymes. Yet it also acts as a cellulose-binding domain, which promotes the activity of the appended cellulase on recalcitrant substrates. The CE2 family encapsulates the requirement for multiple activities by biocatalysts that attack challenging macromolecular substrates, including the grafting of a second, powerful and discrete noncatalytic binding functionality into the active site of an enzyme. This article provides a rare example of “gene sharing,” where the introduction of a second functionality into the active site of an enzyme does not compromise the original activity of the biocatalyst. Proteins that display multiple activities have typically evolved through the recruitment of different domains, each of which has a specific function. Thus, in a multifunctional protein, the different activities are mediated by spatially distinct domains such that a single domain can provide the specific chemical requirements for one activity. Indeed, current evolutionary theory argues that the colocalization of diverse activities within a single-domain enzyme is likely to be a rare event, as it would compromise the existing activity of the protein when a new function evolves. Nonetheless, a potential example of multifunctional recruitment into a single protein domain is provided by an enzyme that contains a cellulase enzyme module and a discrete noncatalytic cellulose-binding module. In this article, we show that the cellulose-binding module displays esterase activity and that these diverse activities are housed within the same site on the protein. Structural analysis of the enzyme reveals that its catalytic residues also contribute to the noncatalytic cellulose-binding function. This report provides a rare example of “gene sharing,” whereby the introduction of a second functionality into the active site of an enzyme does not compromise the original activity of the catalyst.

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.

The p53 tumor suppressor is widely found to be mutated in human cancer. This protein is regarded as a molecular hub regulating different cell responses, namely cell death. Compelling data have demonstrated that the impairment of p53 activity correlates with tumor development and maintenance. For these reasons, the reactivation of p53 function is regarded as a promising strategy to halt cancer. In the present work, the recombinant mutant p53R280K DNA binding domain (DBD) was produced for the first time, and its crystal structure was determined in the absence of DNA to a resolution of 2.0 Å. The solved structure contains four molecules in the asymmetric unit, four zinc(II) ions, and 336 water molecules. The structure was compared with the wild-type p53 DBD structure, isolated and in complex with DNA. These comparisons contributed to a deeper understanding of the mutant p53R280K structure, as well as the loss of DNA binding related to halted transcriptional activity. The structural information derived may also contribute to the rational design of mutant p53 reactivating molecules with potential application in cancer treatment.