Structure and conformational states of the bovine mitochondrial ATP synthase by cryo-EM

Adenosine triphosphate (ATP), the chemical energy currency of biology, is synthesized in eukaryotic cells primarily by the mitochondrial ATP synthase. ATP synthases operate by a rotary catalytic mechanism where proton translocation through the membrane-inserted FO region is coupled to ATP synthesis in the catalytic F1 region via rotation of a central rotor subcomplex. We report here single particle electron cryomicroscopy (cryo-EM) analysis of the bovine mitochondrial ATP synthase. Combining cryo-EM data with bioinformatic analysis allowed us to determine the fold of the a subunit, suggesting a proton translocation path through the FO region that involves both the a and b subunits. 3D classification of images revealed seven distinct states of the enzyme that show different modes of bending and twisting in the intact ATP synthase. Rotational fluctuations of the c8-ring within the FO region support a Brownian ratchet mechanism for proton-translocation driven rotation in ATP synthases.

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Structure of a bacterial ATP synthase

10.1101/463968 ◽

2018 ◽

Cited By ~ 1

Author(s):

Hui Guo ◽

Toshiharu Suzuki ◽

John L. Rubinstein

Keyword(s):

Atp Synthase ◽

Biochemical Analysis ◽

Atp Hydrolysis ◽

Genetic Manipulation ◽

Proton Translocation ◽

Atp Synthesis ◽

Mitochondrial Complex ◽

Rotational States ◽

Membrane Region ◽

Atp Synthases

AbstractATP synthases produce ATP from ADP and inorganic phosphate with energy from a transmembrane proton motive force. Bacterial ATP synthases have been studied extensively because they are the simplest form of the enzyme and because of the relative ease of genetic manipulation of these complexes. We expressed theBacillusPS3 ATP synthase inEschericia coli, purified it, and imaged it by cryo-EM, allowing us to build atomic models of the complex in three rotational states. The position of subunitεshows how it is able to inhibit ATP hydrolysis while allowing ATP synthesis. The architecture of the membrane region shows how the simple bacterial ATP synthase is able to perform the same core functions as the equivalent, but more complicated, mitochondrial complex. The structures reveal the path of transmembrane proton translocation and provide a model for understanding decades of biochemical analysis interrogating the roles of specific residues in the enzyme.

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Structure of a bacterial ATP synthase

eLife ◽

10.7554/elife.43128 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 46

Author(s):

Hui Guo ◽

Toshiharu Suzuki ◽

John L Rubinstein

Keyword(s):

Atp Synthase ◽

Biochemical Analysis ◽

Atp Hydrolysis ◽

Genetic Manipulation ◽

Proton Translocation ◽

Atp Synthesis ◽

Mitochondrial Complex ◽

Rotational States ◽

Membrane Region ◽

Atp Synthases

ATP synthases produce ATP from ADP and inorganic phosphate with energy from a transmembrane proton motive force. Bacterial ATP synthases have been studied extensively because they are the simplest form of the enzyme and because of the relative ease of genetic manipulation of these complexes. We expressed the Bacillus PS3 ATP synthase in Eschericia coli, purified it, and imaged it by cryo-EM, allowing us to build atomic models of the complex in three rotational states. The position of subunit ε shows how it is able to inhibit ATP hydrolysis while allowing ATP synthesis. The architecture of the membrane region shows how the simple bacterial ATP synthase is able to perform the same core functions as the equivalent, but more complicated, mitochondrial complex. The structures reveal the path of transmembrane proton translocation and provide a model for understanding decades of biochemical analysis interrogating the roles of specific residues in the enzyme.

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Atomic model for the dimeric FO region of mitochondrial ATP synthase

Science ◽

10.1126/science.aao4815 ◽

2017 ◽

Vol 358 (6365) ◽

pp. 936-940 ◽

Cited By ~ 102

Author(s):

Hui Guo ◽

Stephanie A. Bueler ◽

John L. Rubinstein

Keyword(s):

Electron Microscopy ◽

Saccharomyces Cerevisiae ◽

Crystal Structures ◽

Atp Synthase ◽

Proton Translocation ◽

Atp Synthesis ◽

Atomic Model ◽

Eukaryotic Cells ◽

Cryo Electron Microscopy ◽

Mitochondrial Atp Synthase

Mitochondrial adenosine triphosphate (ATP) synthase produces the majority of ATP in eukaryotic cells, and its dimerization is necessary to create the inner membrane folds, or cristae, characteristic of mitochondria. Proton translocation through the membrane-embedded FO region turns the rotor that drives ATP synthesis in the soluble F1 region. Although crystal structures of the F1 region have illustrated how this rotation leads to ATP synthesis, understanding how proton translocation produces the rotation has been impeded by the lack of an experimental atomic model for the FO region. Using cryo–electron microscopy, we determined the structure of the dimeric FO complex from Saccharomyces cerevisiae at a resolution of 3.6 angstroms. The structure clarifies how the protons travel through the complex, how the complex dimerizes, and how the dimers bend the membrane to produce cristae.

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Structural basis of proton translocation and force generation in mitochondrial ATP synthase

eLife ◽

10.7554/elife.33274 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 33

Author(s):

Niklas Klusch ◽

Bonnie J Murphy ◽

Deryck J Mills ◽

Özkan Yildiz ◽

Werner Kühlbrandt

Keyword(s):

Atp Synthase ◽

Proton Translocation ◽

Potential Gradient ◽

Structural Basis ◽

Ring Rotation ◽

Mitochondrial Atp Synthase ◽

The Matrix ◽

A Subunit ◽

Proton Leakage ◽

Rotary Catalysis

ATP synthases produce ATP by rotary catalysis, powered by the electrochemical proton gradient across the membrane. Understanding this fundamental process requires an atomic model of the proton pathway. We determined the structure of an intact mitochondrial ATP synthase dimer by electron cryo-microscopy at near-atomic resolution. Charged and polar residues of the a-subunit stator define two aqueous channels, each spanning one half of the membrane. Passing through a conserved membrane-intrinsic helix hairpin, the lumenal channel protonates an acidic glutamate in the c-ring rotor. Upon ring rotation, the protonated glutamate encounters the matrix channel and deprotonates. An arginine between the two channels prevents proton leakage. The steep potential gradient over the sub-nm inter-channel distance exerts a force on the deprotonated glutamate, resulting in net directional rotation.

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In situ structure of trypanosomal ATP synthase dimer reveals a unique arrangement of catalytic subunits

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1612386114 ◽

2017 ◽

Vol 114 (5) ◽

pp. 992-997 ◽

Cited By ~ 25

Author(s):

Alexander W. Mühleip ◽

Caroline E. Dewar ◽

Achim Schnaufer ◽

Werner Kühlbrandt ◽

Karen M. Davies

Keyword(s):

Atp Synthase ◽

Conformational Changes ◽

Atp Synthesis ◽

Α Subunit ◽

Catalytic Subunits ◽

Mitochondrial Atp Synthase ◽

The Individual ◽

Electron Cryotomography ◽

Atp Synthases

We used electron cryotomography and subtomogram averaging to determine the in situ structures of mitochondrial ATP synthase dimers from two organisms belonging to the phylum euglenozoa:Trypanosoma brucei, a lethal human parasite, andEuglena gracilis,a photosynthetic protist. At a resolution of 32.5 Å and 27.5 Å, respectively, the two structures clearly exhibit a noncanonical F1head, in which the catalytic (αβ)3assembly forms a triangular pyramid rather than the pseudo-sixfold ring arrangement typical of all other ATP synthases investigated so far. Fitting of known X-ray structures reveals that this unusual geometry results from a phylum-specific cleavage of the α subunit, in which the C-terminal αCfragments are displaced by ∼20 Å and rotated by ∼30° from their expected positions. In this location, the αCfragment is unable to form the conserved catalytic interface that was thought to be essential for ATP synthesis, and cannot convert γ-subunit rotation into the conformational changes implicit in rotary catalysis. The new arrangement of catalytic subunits suggests that the mechanism of ATP generation by rotary ATPases is less strictly conserved than has been generally assumed. The ATP synthases of these organisms present a unique model system for discerning the individual contributions of the α and β subunits to the fundamental process of ATP synthesis.

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