V/A-ATPase is a motor protein that shares a common rotary catalytic mechanism with FoF1 ATP synthase. When powered by ATP hydrolysis, the V1 domain rotates the central rotor against the A3B3 hexamer, composed of three catalytic AB dimers adopting different conformations (ABopen, ABsemi, and ABclosed). Here, we report the atomic models of 18 catalytic intermediates of the V1 domain of V/A-ATPase under different reaction conditions, determined by single particle cryo-EM. The models reveal that the rotor does not rotate immediately after binding of ATP to the V1. Instead, three events proceed simultaneously with the 120˚ rotation of the shaft: hydrolysis of ATP in ABsemi, zipper movement in ABopen by the binding ATP, and unzipper movement in ABclosed with release of both ADP and Pi. This indicates the unidirectional rotation of V/A-ATPase by a ratchet-like mechanism owing to ATP hydrolysis in ABsemi, rather than the power stroke model proposed previously for F1-ATPase.

ATP synthases (FoF1-ATPases) are crucial for all aerobic organisms. F1, a water-soluble domain, can catalyze both the synthesis and hydrolysis of ATP with the rotation of the central γε rotor inside a cylinder made of α3β3 in three different conformations (referred to as βE, βTP, and βDP). In this study, we determined multiple cryo-electron microscopy structures of bacterial FoF1 exposed to different reaction conditions. The structures of nucleotide-depleted FoF1 indicate that the ε subunit directly forces βTP to adopt a closed form independent of the nucleotide binding to βTP. The structure of FoF1 under conditions that permit only a single catalytic β subunit per enzyme to bind ATP is referred to as unisite catalysis and reveals that ATP hydrolysis unexpectedly occurs on βTP instead of βDP, where ATP hydrolysis proceeds in the steady-state catalysis of FoF1. This indicates that the unisite catalysis of bacterial FoF1 significantly differs from the kinetics of steady-state turnover with continuous rotation of the shaft.

F1 domain of ATP synthase is a rotary ATPase complex in which rotation of central γ-subunit proceeds in 120° steps against a surrounding α3β3 fueled by ATP hydrolysis. How the ATP hydrolysis reactions occurring in three catalytic αβ dimers are coupled to mechanical rotation is a key outstanding question. Here we describe catalytic intermediates of the F1 domain in FoF1 synthase from Bacillus PS3 sp. during ATP mediated rotation captured using cryo-EM. The structures reveal that three catalytic events and the first 80° rotation occur simultaneously in F1 domain when nucleotides are bound at all the three catalytic αβ dimers. The remaining 40° rotation of the complete 120° step is driven by completion of ATP hydrolysis at αDβD, and proceeds through three sub-steps (83°, 91°, 101°, and 120°) with three associated conformational intermediates. All sub-steps except for one between 91° and 101° associated with phosphate release, occur independently of the chemical cycle, suggesting that the 40° rotation is largely driven by release of intramolecular strain accumulated by the 80° rotation. Together with our previous results, these findings provide the molecular basis of ATP driven rotation of ATP synthases.

V/A-ATPase is a motor protein that shares a common rotary catalytic mechanism with FoF1 ATP synthase. When powered by ATP hydrolysis, the V1 moiety rotates the central rotor against the A3B3 hexamer, composed of three catalytic AB dimers adopting different conformations (ABopen, ABsemi, and ABclosed). Here we have determined the atomic models of 18 catalytic intermediates of the V1 moiety of V/A-ATPase under different reaction conditions by single particle Cryo-EM, which revealed that the rotor does not rotate immediately after binding of ATP to the V1. Instead, three events proceed simultaneously with the 120˚ rotation of the shaft: hydrolysis of ATP in ABsemi, zipper movement in ABopen by the binding ATP, and unzipper movement in ABclosed with release of both ADP and Pi. This indicates the unidirectional rotation of V/A-ATPase by a ratchet-like mechanism owing to ATP hydrolysis in ABsemi, rather than the power stroke model proposed previously for F1-ATPase.

F1 domain of ATP synthase is a rotary ATPase complex in which rotation of central gamma-subunit proceeds in 120 degree steps against a surrounding alpha3beta3 fueled by ATP hydrolysis. How the ATP hydrolysis reactions occurring in three catalytic alphabeta dimers are coupled to mechanical rotation is a key outstanding question. Here we describe catalytic intermediates of the F1 domain during ATP mediated rotation captured using cryo-EM. The structures reveal that three catalytic events and the first 80 degree rotation occur simultaneously in F1 domain when nucleotides are bound at all the three catalytic alphabeta dimers. The remaining 40 degree rotation of the complete 120 degree step is driven by completion of ATP hydrolysis at alphaDbetaD, and proceeds through three sub-steps (83 degree, 91 degree, 101 degree, and 120 degree) with three associated conformational intermediates. All sub-steps except for one between 91 degree and 101 degree associated with phosphate release, occur independently of the chemical cycle, suggesting that the 40 degree rotation is largely driven by release of intramolecular strain accumulated by the 80 degree rotation. Together with our previous results, these findings provide the molecular basis of ATP driven rotation of ATP synthases.