Cryo EM structure of intact rotary H+-ATPase/synthase from Thermus thermophilus

Abstract

Proton translocating rotary ATPases couple ATP hydrolysis/synthesis, which occurs in the soluble domain, with proton flow through the membrane domain via a rotation of the common central rotor complex against the surrounding peripheral stator apparatus. Here, we present a large data set of single particle cryo-electron micrograph images of the V/A type H+-rotary ATPase from the bacterium Thermus thermophilus, enabling the identification of three rotational states based on the orientation of the rotor subunit. Using masked refinement and classification with signal subtractions, we obtain homogeneous reconstructions for the whole complexes and soluble V1 domains. These reconstructions are of higher resolution than any EM map of intact rotary ATPase reported previously, providing a detailed molecular basis for how the rotary ATPase maintains structural integrity of the peripheral stator apparatus, and confirming the existence of a clear proton translocation path from both sides of the membrane.

The mechanical inhibition of the isolated Vofrom V-ATPase for proton conductance

“…For Tth V/A-ATPase, acquisition of micrographs was carried out using a Titan Krios equipped with a Falcon II direct electron detector. Cryo-EM micrographs of the complexes reconstituted into nanodiscs resulted in higher resolution EM maps compared with the LMNG solubilized preparation previous reported ( 17 ). The strategy of single particle analysis for Tth V/A-ATPase is summarized in Figure S2A.…”

“…Structural analysis using cryogenic microscopy (cryoEM) of the holo V/A-ATPase, including our recent study, revealed several rotational states of the entire holo complex ( 17, 27 ). However, understanding of the inhibition mechanism of the isolated Tth V o is currently limited due to a lack of a high resolution structure.…”

“…The density for the scaffold proteins and lipids of the nanodiscs is clearly visible surrounding the membrane domain of the isolated V o . Following 3D classification of the V o , only one major class was identified indicating that the isolated V o is very structurally homogenous, in contrast to the Tth V/A-ATPase which is clearly visible in three different rotational states ( 17 ). Our 3D reconstruction map of the isolated V o was obtained with an overall resolution of 3.9 Å.…”

The Peripheral Stalk of Rotary ATPases

“…(B) Models that correspond to three rotational states of one of the peripheral stalks of the V-ATPase of S. cerevisiae ; PDBs 3J9T, 3J9U, 3J9V from Zhao et al (2015) . (C) Models that correspond to three rotational states of one of the peripheral stalks of the A-ATPase of T. thermophilus ; PDBs 5Y5X, 5Y5Z, 5Y60 from Nakanishi et al (2018) . The black horizontal lines indicate the mitochondrial inner membrane.…”

“…The authors argue that this can be due to the fact that this enzyme has a larger rotational asymmetry (3:12) compared to the one of the yeast V-ATPase (3:10) and bovine F-ATPase (3:8), which may cause it to adopt an energetically favorable rotational state in which most of the images are obtained, resulting in an apparent lack of flexibility. Recently, a larger data set of single particle images obtained by Nakanishi et al (2018) allowed the identification of the missing third rotational state of the enzyme of T. thermophilus ; taken together, the structures show there is a dynamic rearrangement of the peripheral stalks in the transitions between each rotational state ( Nakanishi et al, 2018 ; Figure 7C ).…”