A mechanism of proton translocation by F1F0 ATP synthases suggested by double mutants of the a subunit.

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

10.1101/023770 ◽

2015 ◽

Author(s):

Anna Zhou ◽

Alexis Rohou ◽

Daniel G Schep ◽

John V Bason ◽

Martin G Montgomery ◽

...

Keyword(s):

Atp Synthase ◽

Catalytic Mechanism ◽

Proton Translocation ◽

Atp Synthesis ◽

Bioinformatic Analysis ◽

Mitochondrial Atp Synthase ◽

A Subunit ◽

Classification Of Images ◽

Atp Synthases

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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S1.10 Keysteps during proton translocation by ATP synthases

Biochimica et Biophysica Acta (BBA) - Bioenergetics ◽

10.1016/j.bbabio.2008.05.048 ◽

2008 ◽

Vol 1777 ◽

pp. S11

Author(s):

Christoph von Ballmoos ◽

Judith Zingg Ebneter ◽

Alexander Wiedenmann ◽

Peter Dimroth

Keyword(s):

Proton Translocation ◽

Atp Synthases

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The V-ATPase a3 Subunit: Structure, Function and Therapeutic Potential of an Essential Biomolecule in Osteoclastic Bone Resorption

International Journal of Molecular Sciences ◽

10.3390/ijms22136934 ◽

2021 ◽

Vol 22 (13) ◽

pp. 6934

Author(s):

Anh Chu ◽

Ralph A. Zirngibl ◽

Morris F. Manolson

Keyword(s):

Bone Resorption ◽

Proton Translocation ◽

Critical Role ◽

Lipid Binding ◽

Bone Diseases ◽

Osteoclastic Bone Resorption ◽

Subunit Structure ◽

A Subunit ◽

A3 Subunit

This review focuses on one of the 16 proteins composing the V-ATPase complex responsible for resorbing bone: the a3 subunit. The rationale for focusing on this biomolecule is that mutations in this one protein account for over 50% of osteopetrosis cases, highlighting its critical role in bone physiology. Despite its essential role in bone remodeling and its involvement in bone diseases, little is known about the way in which this subunit is targeted and regulated within osteoclasts. To this end, this review is broadened to include the three other mammalian paralogues (a1, a2 and a4) and the two yeast orthologs (Vph1p and Stv1p). By examining the literature on all of the paralogues/orthologs of the V-ATPase a subunit, we hope to provide insight into the molecular mechanisms and future research directions specific to a3. This review starts with an overview on bone, highlighting the role of V-ATPases in osteoclastic bone resorption. We then cover V-ATPases in other location/functions, highlighting the roles which the four mammalian a subunit paralogues might play in differential targeting and/or regulation. We review the ways in which the energy of ATP hydrolysis is converted into proton translocation, and go in depth into the diverse role of the a subunit, not only in proton translocation but also in lipid binding, cell signaling and human diseases. Finally, the therapeutic implication of targeting a3 specifically for bone diseases and cancer is discussed, with concluding remarks on future directions.

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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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Essential arginine residue of the Fo-a subunit in FoF1-ATP synthase has a role to prevent the proton shortcut without c-ring rotation in the Fo proton channel

Biochemical Journal ◽

10.1042/bj20100621 ◽

2010 ◽

Vol 430 (1) ◽

pp. 171-177 ◽

Cited By ~ 43

Author(s):

Noriyo Mitome ◽

Sakurako Ono ◽

Hiroki Sato ◽

Toshiharu Suzuki ◽

Nobuhito Sone ◽

...

Keyword(s):

Atp Synthase ◽

Proton Translocation ◽

Critical Role ◽

Arginine Residue ◽

Proton Channel ◽

Ring Rotation ◽

A Subunit ◽

Coupled Reactions ◽

Single Polypeptide ◽

Fof1 Atp Synthase

In FoF1 (FoF1-ATP synthase), proton translocation through Fo drives rotation of the oligomer ring of Fo-c subunits (c-ring) relative to Fo-a. Previous reports have indicated that a conserved arginine residue in Fo-a plays a critical role in the proton transfer at the Fo-a/c-ring interface. Indeed, we show in the present study that thermophilic FoF1s with substitution of this arginine (aR169) to other residues cannot catalyse proton-coupled reactions. However, mutants with substitution of this arginine residue by a small (glycine, alanine, valine) or acidic (glutamate) residue mediate the passive proton translocation. This translocation requires an essential carboxy group of Fo-c (cE56) since the second mutation (cE56Q) blocks the translocation. Rotation of the c-ring is not necessary because the same arginine mutants of the ‘rotation-impossible’ (c10-a)FoF1, in which the c-ring and Fo-a are fused to a single polypeptide, also exhibits the passive proton translocation. The mutant (aR169G/Q217R), in which the arginine residue is transferred to putatively the same topological position in the Fo-a structure, can block the passive proton translocation. Thus the conserved arginine residue in Fo-a ensures proton-coupled c-ring rotation by preventing a futile proton shortcut.

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Human H+ATPase a4 subunit mutations causing renal tubular acidosis reveal a role for interaction with phosphofructokinase-1

AJP Renal Physiology ◽

10.1152/ajprenal.90258.2008 ◽

2008 ◽

Vol 295 (4) ◽

pp. F950-F958 ◽

Cited By ~ 36

Author(s):

Ya Su ◽

Katherine G. Blake-Palmer ◽

Sara Sorrell ◽

Babak Javid ◽

Katherine Bowers ◽

...

Keyword(s):

Renal Tubular Acidosis ◽

Proton Translocation ◽

Point Mutations ◽

Glycolytic Enzyme ◽

Protein Levels ◽

Expression Studies ◽

A Subunit ◽

Renal Tubular ◽

And Function

The vacuolar-type ATPase (H+ATPase) is a ubiquitously expressed multisubunit pump whose regulation is poorly understood. Its membrane-integral a-subunit is involved in proton translocation and in humans has four forms, a1–a4. This study investigated two naturally occurring point mutations in a4's COOH terminus that cause recessive distal renal tubular acidosis (dRTA), R807Q and G820R. Both lie within a domain that binds the glycolytic enzyme phosphofructokinase-1 (PFK-1). We recreated these disease mutations in yeast to investigate effects on protein expression, H+ATPase assembly, targeting and activity, and performed in vitro PFK-1 binding and activity studies of mammalian proteins. Mammalian studies revealed complete loss of binding between the COOH terminus of a4 containing the G-to-R mutant and PFK-1, without affecting PFK-1's catalytic activity. In yeast expression studies, protein levels, H+ATPase assembly, and targeting of this mutant were all preserved. However, severe (78%) loss of proton transport but less decrease in ATPase activity (36%) were observed in mutant vacuoles, suggesting a requirement for the a-subunit/PFK-1 binding to couple these two functions. This role for PFK in H+ATPase function was supported by similar functional losses and uncoupling ratio between the two proton pump domains observed in vacuoles from a PFK-null strain, which was also unable to grow at alkaline pH. In contrast, the R-to-Q mutation dramatically reduced a-subunit production, abolishing H+ATPase function completely. Thus in the context of dRTA, stability and function of the metabolon composed of H+ATPase and glycolytic components can be compromised by either loss of required PFK-1 binding (G820R) or loss of pump protein (R807Q).

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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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Cardiolipin binds selectively but transiently to conserved lysine residues in the rotor of metazoan ATP synthases

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1608396113 ◽

2016 ◽

Vol 113 (31) ◽

pp. 8687-8692 ◽

Cited By ~ 64

Author(s):

Anna L. Duncan ◽

Alan J. Robinson ◽

John E. Walker

Keyword(s):

Proton Translocation ◽

Proton Channel ◽

Head Group ◽

Residence Times ◽

Functional Roles ◽

Equivalent Position ◽

Lysine Residues ◽

Lipid Head Group ◽

Α Helix ◽

Atp Synthases

The anionic lipid cardiolipin is an essential component of active ATP synthases. In metazoans, their rotors contain a ring of eight c-subunits consisting of inner and outer circles of N- and C-terminal α-helices, respectively. The beginning of the C-terminal α-helix contains a strictly conserved and fully trimethylated lysine residue in the lipid head-group region of the membrane. Larger rings of known structure, from c9-c15 in eubacteria and chloroplasts, conserve either a lysine or an arginine residue in the equivalent position. In computer simulations of hydrated membranes containing trimethylated or unmethylated bovine c8-rings and bacterial c10- or c11-rings, the head-groups of cardiolipin molecules became associated selectively with these modified and unmodified lysine residues and with adjacent polar amino acids and with a second conserved lysine on the opposite side of the membrane, whereas phosphatidyl lipids were attracted little to these sites. However, the residence times of cardiolipin molecules with the ring were brief and sufficient for the rotor to turn only a fraction of a degree in the active enzyme. With the demethylated c8-ring and with c10- and c11-rings, the density of bound cardiolipin molecules at this site increased, but residence times were not changed greatly. These highly specific but brief interactions with the rotating c-ring are consistent with functional roles for cardiolipin in stabilizing and lubricating the rotor, and, by interacting with the enzyme at the inlet and exit of the transmembrane proton channel, in participation in proton translocation through the membrane domain of the enzyme.

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