Research Article

Rotary substates of mitochondrial ATP synthase reveal the basis of flexible F1-Fo coupling

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Science  21 Jun 2019:
Vol. 364, Issue 6446, eaaw9128
DOI: 10.1126/science.aaw9128

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Flexible domains in a well-oiled machine

Motors convert one form of energy into another. For biological motors, adenosine triphosphate (ATP) serves as chemical energy and its hydrolysis is coupled to conformational changes that exert mechanical force. ATP synthases reverse this process in a multistep process: first converting an electrochemical gradient to rotational kinetic energy, and then coupling rotation to formation of high-energy phosphodiester bonds. Murphy et al. investigated these energy changes in the dimeric mitochondrial F1-Fo ATP synthase from Polytomella sp., a unicellular alga. They solved high-resolution cryo–electron microscopy structures of the ATP synthase complex, extracting 13 rotational substates. This collection of structures revealed that the rotation of the Fo ring and central stalk is coupled with partial rotations of the F1 head. This flexibility may enable the head to better couple continuous rotation with discrete ATP synthesis events.

Science, this issue p. eaaw9128

Structured Abstract

INTRODUCTION

Mitochondrial F1-Fo adenosine triphosphate (ATP) synthases are macromolecular turbines that couple proton translocation across a membrane to ATP synthesis. Protons are translocated through the Fo subcomplex in the lipid bilayer by a rotor composed of a defined number of c subunits, each with a proton-binding site, to generate ring rotation. A central stalk is firmly anchored to the c ring and conveys rotary motion to the catalytic F1 subcomplex in the mitochondrial matrix, where ATP is produced by rotary catalysis. A peripheral stalk connects the two subcomplexes to prevent idle rotation of F1 with Fo.

RATIONALE

Although ATP synthase complexes have been investigated for more than 50 years, several key questions remain. An enduring question is how the stoichiometrically mismatched c ring in Fo (composed of 8 to 17 c subunits) and the three-fold symmetric F1 head are efficiently coupled. Another open question is the exact pathway taken by protons through the membrane, which has been the least well characterized part of the mechanism.

RESULTS

We used single-particle cryo–electron microscopy (cryo-EM) to characterize the structure and dynamics of a complete and active dimeric mitochondrial ATP synthase from the chlorophyll-less unicellular alga Polytomella sp. Together with data obtained by genome sequencing and mass spectrometry, our 2.7- to 2.8-Å resolution map allowed us to build a full atomic model of the 1.6-MDa complex. The model includes the newly identified subunit ASA10, which interlinks the two ATP synthase monomers on the lumenal side of the membrane. Separation of 13 independent rotary states provides a detailed molecular description of the movements that accompany c-ring rotation. We find that the F1 head rotates together with the central stalk and c ring through approximately 30°, or one c subunit, at the beginning of each 120° step. Flexible coupling of the F1 head to the Fo motor is mediated primarily by a hinge at the interdomain link of the oligomycin sensitivity–conferring protein (OSCP) subunit that joins the F1 head to the peripheral stalk. The extended two-helix bundle of the central stalk γ subunit interacts with the catch-loop region of one β subunit of the F1 head. The resulting mechanism of flexible coupling is likely to be conserved in other F1-Fo ATP synthases. Our results provide much-needed context to a wealth of published data indicating that OSCP is a hub of metabolic control in the cell.

Our high-resolution map of the proton-translocating Fo complex has revealed a strong density, very likely a metal ion, ligated by two histidine residues. Recent cryo-EM studies of yeast and spinach chloroplast ATP synthase contain unannotated densities at the same position. Mutational experiments in Escherichia coli have shown that an equivalent residue is essential to proton translocation. By three-dimensional classification, we separated two different rotational positions of the c ring and showed that the coordination environment of the metal ion changes with c-ring position. This evidence points toward a role for the metal ion in synchronizing c-ring protonation with its rotation.

CONCLUSION

In ATP synthases, the F1 catalytic head can accompany the rotor through a rotation of ~30° at the beginning of each ~120° step. This movement allows flexible coupling of F1 and Fo. The interdomain hinge of OSCP facilitates flexible coupling and makes this subunit an apposite point for the regulation of ATP synthesis.

Cryo-EM structure of the Polytomella ATP synthase dimer.

The F1 head (green) is linked to the c-ring rotor (yellow) by the central stalk and the peripheral stalk. Insets (beginning at top right) show the flexible OSCP hinge (orange); F1 rotary substates with subunits β (green), γ (blue), and c (yellow); a coordinated metal ion in the proton access channel (light blue); and the dimer-forming subunit ASA10 (red).

Abstract

F1Fo–adenosine triphosphate (ATP) synthases make the energy of the proton-motive force available for energy-consuming processes in the cell. We determined the single-particle cryo–electron microscopy structure of active dimeric ATP synthase from mitochondria of Polytomella sp. at a resolution of 2.7 to 2.8 angstroms. Separation of 13 well-defined rotary substates by three-dimensional classification provides a detailed picture of the molecular motions that accompany c-ring rotation and result in ATP synthesis. Crucially, the F1 head rotates along with the central stalk and c-ring rotor for the first ~30° of each 120° primary rotary step to facilitate flexible coupling of the stoichiometrically mismatched F1 and Fo subcomplexes. Flexibility is mediated primarily by the interdomain hinge of the conserved OSCP subunit. A conserved metal ion in the proton access channel may synchronize c-ring protonation with rotation.

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