No abstract

Biocatalysts showcase the upper limit obtainable for high-speed molecular processing and transformation. Efforts to engineer functionality in synthetic nanostructured materials are guided by the increasing knowledge of evolving architectures, which enable controlled molecular motion and precise molecular recognition. The cellulosome is a biological nanomachine, which, as a fundamental component of the plant-digestion machinery from bacterial cells, has a key potential role in the successful development of environmentally-friendly processes to produce biofuels and fine chemicals from the breakdown of biomass waste. Here, the progress toward so-called "designer cellulosomes", which provide an elegant alternative to enzyme cocktails for lignocellulose breakdown, is reviewed. Particular attention is paid to rational design via computational modeling coupled with nanoscale characterization and engineering tools. Remaining challenges and potential routes to industrial application are put forward.

The cellulosome is an intricate multi-enzyme complex, known for its efficient degradation of recalcitrant cellulosic substrates. Its supramolecular architecture is determined by the high-affinity intermodular cohesin-dockerin interaction. The dockerin module comprises a calcium-binding, duplicated 'F-hand' loop-helix motif that bears striking similarity to the EF-hand loop-helix-loop motif of eukaryotic calcium-binding proteins. In the present study, we demonstrate by progressive truncation and alanine scanning of a representative type-I dockerin module from Clostridium thermocellum, that only one of the repeated motifs is critical for high-affinity cohesin binding. The results suggest that the near-symmetry in sequence and structure of the repeated elements of the dockerin is not essential to cohesin binding. The first calcium-binding loop can be deleted entirely, with almost full retention of binding. Likewise, significant deletion of the second repeated segment can be achieved, provided that its calcium-binding loop remains intact. Essentially the same conclusion was verified by systematically mutating the highly conserved residues in the calcium-binding loop. Mutations in one of the calcium-binding loops failed to disrupt cohesin recognition and binding, whereas a single mutation in both loops served to reduce the affinity significantly. The results are mutually compatible with recent crystal structures of the type-I cohesin-dockerin heterodimer, which demonstrate that the dockerin can bind in an equivalent manner to its cohesin counterpart through either its first or second repeated motif. The observed plasticity in cohesin-dockerin binding may facilitate cellulosome assembly in vivo or, alternatively, provide a conformational switch that promotes access of the tethered cellulosomal enzymes to their polysaccharide substrates.

Efficient degradation of cellulose by the anaerobic thermophilic bacterium, Clostridium thermocellum, is carried out by the multi-enzyme cellulosome complex. The enzymes on the complex are attached in a calcium-dependent manner via their dockerin (Doc) module to a cohesin (Coh) module of the cellulosomal scaffoldin subunit. In this study, we have optimized the Coh-Doc interaction for the purpose of protein affinity purification. A C. thermocellum Coh module was thus fused to a carbohydrate-binding module, and the resultant fusion protein was applied directly onto beaded cellulose, thereby serving as a non-covalent "activation" procedure. A complementary Doc module was then fused to a model protein target: xylanase T-6 from Geobacillus stearothermophilus. However, the binding to the immobilized Coh was only partially reversible upon treatment with EDTA, and only negligible amounts of the target protein were eluted from the affinity column. In order to improve protein elution, a series of truncated Docs were designed in which the calcium-coordinating function was impaired without appreciably affecting high-affinity binding to Coh. A shortened Doc of only 48 residues was sufficient to function as an effective affinity tag, and highly purified target protein was achieved directly from crude cell extracts in a single step with near-quantitative recovery of the target protein. Effective EDTA-mediated elution of the sequestered protein from the column was the key step of the procedure. The affinity column was reusable and maintained very high levels of capacity upon repeated rounds of loading and elution. Reusable Coh-Doc affinity columns thus provide an efficient and attractive approach for purifying proteins in high yield by modifying the calcium-binding loop of the Doc module.

The cellulosome is a large extracellular multi-enzyme complex that facilitates the efficient hydrolysis and degradation of crystalline cellulosic substrates. During the course of our studies on the cellulosome of the rumen bacterium Ruminococcus flavefaciens, we focused on the critical ScaA dockerin (ScaADoc), the unique dockerin that incorporates the primary enzyme-integrating ScaA scaffoldin into the cohesin-bearing ScaB adaptor scaffoldin. In the absence of a high-resolution structure of the ScaADoc module, we generated a computational model, and, upon its analysis, we were surprised to discover a putative stacking interaction between an N-terminal Trp and a C-terminal Pro, which we termed intramolecular clasp. In order to verify the existence of such an interaction, these residues were mutated to alanine. Circular dichroism spectroscopy, intrinsic tryptophan and ANS fluorescence, and NMR spectroscopy indicated that mutation of these residues has a destabilizing effect on the functional integrity of the Ca2+-bound form of ScaADoc. Analysis of recently determined dockerin structures from other species revealed the presence of other well-defined intramolecular clasps, which consist of different types of interactions between selected residues at the dockerin termini. We propose that this thematic interaction may represent a major distinctive structural feature of the dockerin module.