Characterization of regulatory central stalk epitopes of mycobacterial- and acetogen F-ATP synthases

F-ATP synthases are multimeric membrane-embedded proteins which can synthesize/hydrolyze ATP depending on the energy requirements of the cell. The canonical composition includes F1 part comprised of α3:β3:γ:δ:ε and FO part composed of subunits a and c-ring attached to the dimeric peripheral stalk b2 (1). In mycobacterial cells, ATP synthesis occurs by utilizing the proton-motive force (PMF) to drive the catalytic events in the F-ATP synthase and form ATP from ADP and Pi, similarly to the mechanism found in other bacterial F-ATP synthases. In contrast, ATPase activity of Mtb enzyme is latent due to the action of a specific regulatory mechanism, allowing it to conserve ATP and thrive in the hypoxic environment (2). This trait gave rise to Mtb population establishment in energetically unfavorable conditions found in human lungs contributing to the latent TB infections (LTBI) and thus prompting a need for further structural and functional characterization of MtF-ATP synthase. Previously it was established that the genetically-fused extended C-terminal domain of mycobacterial α (Mtα) reduced the ATPase activity of the complex G. stearothermophilus α3:β3:γ indicating its involvement in the inhibition of the ATPase activity (3). In the present study, the synergistic effect of mycobacteria-specific epitopes (MtαCTD and Mtε) on inhibition of ATPase activity was revealed via the hybrid complex αchi3:β3:γ:Mtε used as a model system. The complex αchi3:β3:γ:Mtε was visualized for the first time, using the negative-stain electron microscopy, revealing the binding of the heterologous Mtε as well as the position of the Mtα-specific C-terminal epitope within the 3D reconstructed map (4). The interaction of these mycobacterial epitopes was described using mutagenesis and cysteine crosslinking experiments. The kinetic study of the ATPase activity advocates a strong dependence of the mycobacterial F-ATP synthase on Mtε for coupling and establishment of the fully-functional complex. The mutations in Mtε were designed to dissect the role of its C-terminal domain, hinge domain and N-terminal domain in the catalysis and stability of the reconstituted αchi3:β3:γ:Mtε(x) complexes. The mutations MtεE87A, MtεR62L, and Mtε1-120 described the coupling and the entrapment of the inhibitory Mg-ADP, while the mutant Mtε6-121 emphasized the role of MtεNTD for the protein stability in comparison to the other bacterial counterparts (4,5). In summary, the structural data obtained from NS-EM, together with the mutational and functional studies describe the importance of mycobacteria-specific epitopes, extended MtαCTD as well as Mtε in the regulation of the catalytic events in mycobacterial F-ATP synthases. On the other side of evolution, away from the Kingdom of Bacteriae, an archaeal species Acetobacterium woodii displays unique features of its F-ATP synthase, developed in response to the environmental energy availability. The evolutionary pressure to adapt had driven the specialization of this F-ATP synthase to accommodate several structural and regulatory mechanisms specific for a particular niche. As halophilic, thermophilic, acetogenic species, A. woodii was facing hypersalinity, oxygen-deprivation, high temperatures, and high content of organic acids which all created an environment whereby energy conservation is paramount for survival. To utilize the hypersalinity for the energy generation, A. woodii has invented a hybrid Na+-translocating c-ring that contains the V-ATPase-like subunit c1 and F-ATP synthase ¬c2/3, arranged in the c10 ring of the c1:c2/3 stoichiometry 1:9 (6,7). The Na+-motive force operates on a similar principle found in PMF-dependant F-ATP synthases. By transversing through the c-ring down the chemical gradient, Na+ ions affect its rotation, causing the rotation of the central stalk subunits γ-ε, which in turn imposes the structural changes in the nucleotide-binding α3β3 catalytic hexamer and drives the ATP synthesis. In this thesis, the entire A. woodii F1FO ATP synthase was visualized for the first time, using NS-EM and the 2D projections were obtained pointing towards canonical subunit composition with a stoichiometry α3:β3:γ:δ:ε:a:b2:(c2/3)9:c1. A unique loop (195TSGKEEKTEETKSK211) in the A. woodii subunit γ was unraveled, and its role was examined by generating the deletion mutant γΔ195-211 whose ATP synthesis activity was entirely abolished coupled with diminished Na+-translocation. T, the present thesis has established the structural basis of regulation of Na+-translocation, ATP synthesis, and revealed the regulatory role of the A. woodii unique γ-loop.Doctor of Philosoph