Subunit epsilon ofE. coliF₁F_oATP synthase attenuates enzyme activity by modulating central stalk flexibility

F1FoATP synthase functions as a biological rotary generator that makes a major contribution to cellular energy production. Proton flow through the Fomotor generates rotation of the central stalk, inducing conformational changes in the F1motor that catalyzes ATP production via flexible coupling. Here we present a range of cryo-EM structures ofE. coliATP 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 ε (εCTD) disassociating from the central stalk to adopt a condensed “down” conformation. The transition to the εCTD down state increases the torsional flexibility of the central stalk allowing its foot to rotate by ∼50°, with further flexing in the peripheral stalk enabling thec-ring to rotate by two sub-steps in the Fomotor. Truncation mutants lacking the second helix of the εCTD suggest that central stalk rotational flexibility is important for F1FoATP synthase function. Overall this study identifies the potential role played by torsional flexing within the rotor and how this could be influenced by the ε subunit.