Molecular dynamics modelling of the cellulosome multi-enzyme complex

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. One way to enhance the efficiency of the cellulosome for biomass conversion is to improve the stability and well as the binding affinity of its constituent domains so that they are compatible with industrial processing conditions. In this thesis, we investigate the mechanical, thermal stabilities as well as the binding affinity of cellulosomal proteins, using molecular dynamics simulations. Firstly, steered molecular dynamics computer simulations was used 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 simulations identified candidates for site-directed mutagenesis experiments in the calcium binding pocket, providing molecular insights into the origins of mechanostability in cellulose binding domains and leads for synthesis of more robust cellulose-binding protein modules. Given that elevated hydrolysis temperatures >50o C significantly enhance industrial scale lignocellulose degradation, high thermal stability is important for native functioning of cellulosome domains. Atomic resolution results from MD simulations of three cohesin dockerin systems (one thermophilic and two mesophilic) provide insight on the substantial flexibility of a linker region between alpha-helices H1 and H3 in mesophilic dockerins, at high temperatures. Consequently, weaker cohesin-dockerin binding energies were calculated at higher temperatures of 350K and 400K, in mesophilic systems. Binding affinity of four different, six-monomer cellulose glucan chains and ZgGH5 enzyme, found in the bacteria Zobellia galactanivorans were tested in simulations. Results illustrates how the ‘S-shaped’ binding pocket better fits the natural conformation of the substrate with β-1,3 linkage between sub-units +1 and -1, with the most favourable binding energy value. Finally, we present a complete all-atomistic model (approx. 5 million atoms) in order to study the assembly of a trivalent designer cellulosome (DC) structure. From the simulations, it is clear that the flexible nature and modularity of the scaffoldin influences the conformation of the DC. After 50 ns simulations, bending of scaffoldin linkers as well as some domain-domain hydrogen bond interaction results in a compacted structural state of the DC. Our data shed light into the mechanisms driving the physical, mechanical and thermal properties of the cellulosome components and therefore provide insight into the rational re-engineering of complex biological nanomachines for biotechnology.