Molecular simulations reveal that a short helical loop regulates thermal stability of type I cohesin-dockerin complexes.

The cellulosome provides a fully worked out example of evolved radical nanotechnology. Improved understanding, and first steps toward re-engineering this biological nanomachine, is providing design rules for the formulation of advanced synthetic materials that can harness molecular flexibility and sticking interactions for applications in clean energy, environmental monitoring, and miniaturized devices. Computer simulations provide atomic scale insights into the mechanical stability of the component protein units, flexibility of short peptides that tether the units into scaffolds, and thermodynamic stability of protein-protein and protein-carbohydrate complexes, complementing and in some cases directing experiments. In the present work, a systematic computational study of cohesin-dockerin pairs, the strongly-bound protein complexes that glue the cellulosome nano-architecture in place, reveals that a short alpha-helix in the middle of the smaller dockerin protein becomes disordered at elevated temperatures and weakens cohesin-dockerin binding in mesophilic species. In thermophilic species, a more extensive and more thermally resistant H-bond network ensures the structure remains ordered at elevated temperatures of up to 400 K. The simulations predict that simply grafting the most crucial eight-residue peptide sequence into the mesophilic complex can, for one species and one of two possible binding modes, potentially create a new thermally resistant complex, providing leads for future experiments to re-engineer designer cellulosomes that can withstand elevated temperatures and so provide clean, renewable biocatalysts.

Steered molecular dynamics simulations reveal the role of Ca2+ in regulating mechanostability of cellulose-binding proteins.

The conversion of cellulosic biomass into biofuels requires degradation of the biomass into fermentable sugars. The most efficient natural cellulase system for carrying out this conversion is an extracellular multi-enzymatic complex named the cellulosome. In addition to temperature and pH stability, mechanical stability is important for functioning of cellulosome domains, and experimental techniques such as Single Molecule Force Spectroscopy (SMFS) have been used to measure the mechanical strength of several cellulosomal proteins. Molecular dynamics computer simulations provide complementary atomic-resolution quantitative maps of domain mechanical stability for identification of experimental leads for protein stabilization. In this study, we used multi-scale steered molecular dynamics computer simulations, benchmarked against new SMFS measurements, to measure the intermolecular contacts that confer high mechanical stability to a family 3 Carbohydrate Binding Module protein (CBM3) derived from the archetypal Clostridium thermocellum cellulosome. Our data predicts that electrostatic interactions in the calcium binding pocket modulate the mechanostability of the cellulose-binding module, which provides an additional design rule for the rational re-engineering of designer cellulosomes for biotechnology. Our data offers new molecular insights into the origins of mechanostability in cellulose binding domains and gives leads for synthesis of more robust cellulose-binding protein modules. On the other hand, simulations predict that insertion of a flexible strand can promote alternative unfolding pathways and dramatically reduce the mechanostability of the carbohydrate binding module, which gives routes to rational design of tailormade fingerprint complexes for force spectroscopy experiments.

On the distinct binding modes of expansin and carbohydrate-binding module proteins on crystalline and nanofibrous cellulose: implications for cellulose degradation by designer cellulosomes.

Transformation of cellulose into monosaccharides can be achieved by hydrolysis of the cellulose chains, carried out by a special group of enzymes known as cellulases. The enzymatic mechanism of cellulases is well described, but the role of non-enzymatic components of the cellulose-degradation machinery is still poorly understood, and difficult to measure using experiments alone. In this study, we use a comprehensive set of atomistic molecular dynamics simulations to probe the molecular details of binding of the family-3a carbohydrate-binding module (CBM3a) and the bacterial expansin protein (EXLX1) to a range of cellulose substrates. Our results suggest that CBM3a behaves in a similar way on both crystalline and amorphous cellulose, whereas binding of the dual-domain expansin protein depends on the substrate crystallinity, and we relate our computed binding modes to the experimentally measured features of CBM and expansin action on cellulose.

Non-local effects of point mutations on the stability of a protein module.

We combine experimental and theoretical methods to assess the effect of a set of point mutations on c7A, a highly mechanostable type I cohesin module from scaffoldin CipA from Clostridium thermocellum. We propose a novel robust and computationally expedient theoretical method to determine the effects of point mutations on protein structure and stability. We use all-atom simulations to predict structural shifts with respect to the native protein and then analyze the mutants using a coarse-grained model. We examine transitions in contacts between residues and find that changes in the contact map usually involve a non-local component that can extend up to 50 Å. We have identified mutations that may lead to a substantial increase in mechanical and thermodynamic stabilities by making systematic substitutions into alanine and phenylalanine in c7A. Experimental measurements of the mechanical stability and circular dichroism data agree qualitatively with the predictions provided the thermal stability is calculated using only the contacts within the secondary structures.

Nanoscale Engineering of Designer Cellulosomes.

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.