Mechanistic insights into Escherichia coli alkyl hydroperoxide reductase complex formation
Date of Issue2017-03-30
School of Biological Sciences
Oxidative stress, an imbalance between the amount of Reactive Oxygen Species (ROS) and antioxidant defences, is detrimental to the survival of both benign and pathogenic bacteria . To keep ROS in balance bacteria have evolved various antioxidant enzymes, among them the alkyl hydroperoxide reductase (AhpR), consisting of 21 kDa AhpC and 57 kDa AhpF, which serve as a primary hydroperoxide scavenger that reduces hydroperoxide to water and its corresponding alcohol, and preventing thereby damages like protein oxidation, lipid peroxidation and DNA damage [2-5]. AhpC and -F represent one of the most efficient enzyme systems responsible for redox homeostasis in bacteria  To do that, the Escherichia coli AhpC (EcAhpC) directly donates electrons to peroxides reducing them to less harmful products. The oxidized AhpC is reduced by AhpF via the disulphide center (-CxxC-) in its N-terminal domain (NTD), with a catalytic turnover rate measured to be 4.0 x 10^7 M^-1 s^-1 in Salmonella typhimurium . By employing biophysical approaches, this thesis set out to understand the mechanistic basis for this efficiency. The C-terminal tail of AhpC has been implicated as a hydroperoxide sensor in eukaryotes but not in most bacteria . By generating C-terminally truncated mutants, EcAhpC_1-177, EcAhpC_1-182, EcAhpC_1-185, together with site-specific mutations EcAhpC_KJ86A and EcAhpC_1187G, the critical role of the C-terminus in oligomerization under reducing conditions has been examined. Since oligomerization has been associated with catalytic efficiency , NADH-dependent assays have been used to associate the C-terminal mutations to catalytic efficiency. In an attempt to gain structural insight into the C-terminal tail, the EcAhpC_1-182 crystal structure has been solved in its oxidised state that revealed five molecules in an asymmetric unit. The ring shape decamer conformation was generated by applying a symmetry calculation with the asymmetric unit cell. The absence of the C-terminal tail in the crystal structure indicates a flexibility of this region. Furthermore, the first interaction studies between the EcAhpC and EcAhpF have been performed. By generating constructs of the N- and C-terminal domains of EcAhpF, it has been confirmed that EcAhpF interacts entropically via its NTD with EcAhpC. The affinity for the EcAhpF_NTD-EcAhpC interaction was calculated to be 3.2 μM. The effect of the redox-state of EcAhpC has been found to be important to this interaction as only the oxidized AhpC interacts with the NTD. In comparison, reduction abolished the interaction altogether. Importantly, by using NMR spectroscopy, it has been revealed that the interaction of the full-length EcAhpC and the NTD is long-lived, whereas the C-terminal free AhpC (EcAhpC_1-172) interacts very dynamically with the NTD. This dynamic nature of the EcAhpC_1-172-EcAhpF_1-212 interaction enabled the observation of chemical shift perturbations of residues of the isotopically labelled NTD. Information obtained have been used to drive a HADDOCK docking of the complex and subsequently a binding epitope of the Cterminal tail of EcAhpC which it is proposed to wrap around the backside of EcAhpF_NTD. Taken together, this thesis has revealed a novel binding mechanism, where the C-terminus of AhpC wraps around the NTD to slow the dissociation rate upon complex formation for an efficient electron transfer process to occur, and a release mechanism mediated by a well-described conformational change of the Cterminus of EcAhpC upon reduction. In addition, by employing a high ionic buffer system, solution structures of the full-length oxidized EcAhpC have been solved using small angle x-ray scattering (SAXS) throwing light on the flexibility of the C-terminus and its importance to the EcAhpC-EcAhpF complex formation. In addition, the orientation of the full-length proteins in the complex in solution agrees very well with models obtained by NMR.