Subunit epsilon of E. coli F1Fo ATP synthase attenuates enzyme activity by modulating central stalk flexibility

F1Fo ATP synthase functions as a biological rotary generator that makes a major contribution to cellular energy production. Proton flow through the Fo motor generates rotation of the central stalk, inducing conformational changes in the F1 motor that catalyzes ATP production via flexible coupling. Here we present a range of cryo-EM structures of E. coli ATP synthase in different rotational and inhibited states observed following a 45 second incubation with 10 mM MgATP. The structures generated describe multiple changes that occur following addition of MgATP, with the inhibitory C-terminal domain of subunit {varepsilon} ({varepsilon}CTD) disassociating from the central stalk to adopt a condensed "down" conformation. The transition to the {varepsilon}CTD down state increases the torsional flexibility of the central stalk allowing its foot to rotate by [~]50{degrees}, with further flexing in the peripheral stalk enabling the c-ring to rotate by two sub-steps in the Fo motor. Truncation mutants lacking the second helix of the {varepsilon}CTD suggest that central stalk rotational flexibility is important for F1Fo ATP synthase function. Overall this study identifies the potential role played by torsional flexing within the rotor and how this could be influenced by the {varepsilon} subunit.