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The structure of the peripheral stalk of Thermus thermophilus H+-ATPase/synthase

Abstract

Proton-translocating ATPases are ubiquitous protein complexes that couple ATP catalysis with proton translocation via a rotary catalytic mechanism. The peripheral stalks are essential components that counteract torque generated from proton translocation during ATP synthesis or from ATP hydrolysis during proton pumping. Despite their essential role, the peripheral stalks are the least conserved component of the complexes, differing substantially between subtypes in composition and stoichiometry. We have determined the crystal structure of the peripheral stalk of the A-type ATPase/synthase from Thermus thermophilus consisting of subunits E and G. The structure contains a heterodimeric right-handed coiled coil, a protein fold never observed before. We have fitted this structure into the 23 Å resolution EM density of the intact A-ATPase complex, revealing the precise location of the peripheral stalk and new implications for the function and assembly of proton-translocating ATPases.

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Figure 1: Architecture and nomenclature of the intact T. thermophilus A-ATPase/synthase.
Figure 2: Structure of the E–G peripheral stalk complex.
Figure 3: Hydrophobic repeats forming the RHCC interface between subunits E and G.
Figure 4: Superposition of the P. horikoshii subunit E C-terminal domain and T. thermophilus subunit E.
Figure 5: Docking of the E–G peripheral stalk complex into the 23 Å EM density of the intact T. thermophilus A-ATPase/synthase.

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Protein Data Bank

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Acknowledgements

We thank the staff at beamline ID14-4 at the European Synchrotron Research Facility, Grenoble, France, the staff at beamline 14-ID (BioCARS) and 23-ID (General Medicine and Cancer Institutes Collaborative Access Team), the staff at the Advanced Photon Source, Chicago, USA, and the staff at beamline 3-BM1 at the Australian Synchrotron, Victoria, for support; and we acknowledge K. Miki and colleagues for providing coordinates of the T. thermophilus A1 structure before release from the Protein Data Bank. Use of the Advanced Photon Source was supported by the US Department of Energy, Basic Energy Sciences, Office of Science (contract DE-AC02-06CH11357), and use of the BioCARS Sector 14 was supported by the National Institutes of Health, National Center for Research Resources (grant RR007707). The General Medicine and Cancer Institutes Collaborative Access Team has been funded in whole or in part with federal funds from the National Cancer Institute (Y1-CO-1020) and the National Institute of General Medical Science (Y1-GM-1104). This work was supported by the Australian Synchrotron Research Program of the Australian Nuclear Science Technology Organization and Australian National Health & Medical Research Council (grant DP573712).

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L.K.L. collected X-ray diffraction data, solved, built and refined the crystal structure, performed the Fourier analysis of coiled coils and wrote the manuscript; A.G.S. expressed, purified and crystallized the E–G complex, solved, built and refined the crystal structure and wrote the manuscript; M.D. cloned, expressed, purified and crystallized the E–G complex; R.A.B. docked subunits into EM density; and D.S. conceived the project, collected X-ray diffraction data, built and refined the structure and wrote the manuscript.

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Correspondence to Daniela Stock.

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Lee, L., Stewart, A., Donohoe, M. et al. The structure of the peripheral stalk of Thermus thermophilus H+-ATPase/synthase. Nat Struct Mol Biol 17, 373–378 (2010). https://doi.org/10.1038/nsmb.1761

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