Subunit epsilon ofE. coliF1FoATP synthase attenuates enzyme activity by modulating central stalk flexibility

ABSTRACT F 1 F o ATP synthase functions as a biological rotary generator that makes a major contribution to cellular energy production. Proton flow through the F o motor generates rotation of the central stalk, inducing conformational changes in the F 1 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 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 the c -ring to rotate by two sub-steps in the F o motor. Truncation mutants lacking the second helix of the εCTD suggest that central stalk rotational flexibility is important for F 1 F o 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 ε subunit.