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

J Kishikawa,A Furuta,T Kato,S Saeki,A Nakano,A Nakanishi,K Yokoyama,K Mistuoka

doi:10.1038/s41467-022-28832-5

J Kishikawa, A Furuta + Show 6 more

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https://doi.org/10.1038/s41467-022-28832-5

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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.

Highlights

V/A-ATPase is a motor protein that shares a common rotary catalytic mechanism with FoF1 ATP synthase
The chemical reaction (ATP hydrolysis/synthesis) in V1/F1 is tightly associated with proton movement through Vo/Fo using a rotary catalytic mechanism, where both reactions are coupled by rotation of the central rotor complex relative to the surrounding stator apparatus, which includes the ATPase active hexamer[6,10,11] (Fig. 1B)
When using 40 nm gold beads with almost negligible viscous resistance, V1 pauses every 120° even at an ATP concentration around Km without a sign of substeps[19]. These single-molecule experiments on V1 suggest that both catalytic events, ATP hydrolysis and product (ADP and Pi) release occur at an individual ATP binding position, and imply the presence of chemo-mechanically stable catalytic intermediates (Fig. 1C and Supplementary Fig. 1)

Summary

Introduction

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). 1234567890():,; The proton translocation ATPase/synthase family includes F-type enzymes found in eubacteria, mitochondria, and chloroplasts, and the V/A type enzymes found in archaea and some eubacteria[1–5] (Fig. 1A) These ATPases produce the majority of cytosolic ATP from ADP and Pi using energy derived from the transmembrane proton motive force generated by cellular respiration[6]. The V1 domain of Tth V/A-ATPase (A3B3D1F1) is an ATP-driven rotary motor where the central DF shaft rotates inside the hexameric A3B3 containing three catalytic sites, each composed of an AB dimer. These single-molecule experiments on V1 suggest that both catalytic events, ATP hydrolysis and product (ADP and Pi) release occur at an individual ATP binding position, and imply the presence of chemo-mechanically stable catalytic intermediates (Fig. 1C and Supplementary Fig. 1)

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