Mechanisms of the type III secretion system ATPase complex

Molecular motors play a vital role in cell dynamics and are a ubiquitous feature in molecular biology. Bacteria possess a vast array of secretion systems such as the type three secretion system (T3SS), a complex membrane-spanning structure used to infect eukaryotic cells (vT3SS) and synthesise flagella (fT3SS) that is markedly similar to F1/V1 ATPases. The Flil6FliJ assembly of the fT3SS docks to membrane embedded export proteins FlhA and FlhB, supported by peripheral FliH spoke proteins, to ensure efficient coupling of ATP hydrolysis with protein export and proton influx. Protein unfolding and export gate activation are required for effective protein transport although the process is not fully elucidated at present An integrated approach was taken in this thesis to rationalise molecular processes associated with vT3SS and fT3SS ATPase complex in Campylobacter jejuni. Computational methods were employed to examine the rotation mechanism and ligand binding of the T3SS ATPase complex: protein structure prediction algorithms, using homology modelling and molecular dynamics (MD) simulations. Single nucleotide polymorphisms (SNPs) of C. jejuni fT3SS fliF and fliH genes resulted in pseudoreversion and the recovery of motility in a non-motile strain to a wild-type level. Phenotypic assays combined with molecular modeling and MD simulations were applied to explain pseudoreversions of C. jejuni by a SNP of the fliH gene. The rotation dynamics of F1/V1 ATPases were extrapolated to probe conserved rotation protein dynamics of the T3SS. Experimentally derived structures of the T3SS were incorporated into in silico assembly models and MD simulations were used to apply rotation potentials to decipher aspects of the mechanisms of the T3SS ATPase complex. The results indicate a specific rotation profile of the vT3SS ATPase complex with a more energetically favourable axial protein rotation in a clockwise direction. Furthermore, they suggested that a rotation mechanism paired with a predicted conserved tilted conformation of the rotor protein results in a mechanistically similar but conformationally distinct system to the F1/V1 ATPases. Application of experimental and computational analyses of pseudorevertant mutations indicated mutations of FliH protein binding domains result in the recovery of the motile phenotypes but this will need to be further investigated in the future to fully explain the molecular intricacies of C. jejuni pseudoreversion.