Structural snapshots of V/A-ATPase reveal the rotary catalytic mechanism of rotary ATPases

Abstract

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.

Structural transition of the ground-state structure to steady-state structures by sequential binding of ATP to V/A-ATPase

“…Together, these results suggest that 120° rotation does not occur immediately by the zippering motion of ABopen with ATP in the absence of ATP binding on ABsemi. This is consistent with the results of a previous study, in which both the zippering motion of AB open with ATP and hydrolysis of ATP in AB semi drive the unzipper motion of AB closed with the release of ADP and Pi, which are coupled simultaneously with the 120° rotation of the shaft[23].…”

“…Moreover, the structural snapshots indicate three catalytic processes simultaneously proceed; ⅰ) zipper movement in AB open with the ATP, ⅱ) ATP hydrolysis on AB semi , and iii) unzipper movement of AB closed with the release of ADP and Pi. In other words, simultaneous interlocking of the three catalytic processes and 120° rotation of the axis are necessary for continuous catalytic reactions [23]. AB closed should be changed to AB open with a 120° step of the rotor.…”

“…Single-particle cryo-electron microscopy (cryo-EM) analysis of the V/A-ATPase identified three rotational states (state 1–3) and an asymmetric structure of the three AB dimers, termed “open (AB open ),” “semi-closed (AB semi ),” and “closed (AB closed ) (Figure 1B). Even in the presence or absence of nucleotide binding, conformations of both AB semi and AB closed remained closed, which does not allow easy access of ATP to the catalytic sites [23, 24]. Therefore, ATP binding occurs in AB open where the catalytic site is fully open.…”

Direct Observation of Stepping Rotation of V-ATPase Reveals Rigid Coupling between Vo and V1 Motors

“…Single-molecule studies using fluorescent probes (8)(9)(10)(11), gold nanoparticle (AuNP) or nanorod probes (12)(13)(14)(15)(16)(17)(18)(19)(20), and Förster resonance energy transfer (FRET) (15,21,22) have revealed rotational dynamics of the rotary ATPases for both ATP hydrolysis/synthesis directions. Furthermore, recent cryo-electron microscopic (cryo-EM) single-particle analyses revealed entire architectures of the rotary ATPases with different structural states at atomic resolutions (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34). In particular, several studies have demonstrated elastic coupling of FoF1 due to large deformations of the peripheral stalk connecting Fo and F1 (24,28,34).…”

CryoEM Reveals the Complexity and Diversity of ATP Synthases

“…V/A-ATPases have been subjected to extensive structural analysis by x-ray crystallography, negative stain EM, and cryoEM (Boekema et al, 1999 ; Bernal and Stock, 2004 ; Murata et al, 2005 ; Numoto et al, 2009 ; Lau and Rubinstein, 2010 , 2012 ; Lee et al, 2010 ). Similar to F-type ATP synthase, cryoEM has allowed computational separation of rotational states and elucidation of the a subunit fold by evolutionary covariance analysis (Schep et al, 2016 ), with subsequent high-resolution structures revealing further details of the catalytic mechanism (Zhou and Sazanov, 2019 ; Kishikawa et al, 2022 ).…”