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

2017 ◽  
Vol 114 (5) ◽  
pp. 992-997 ◽  
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.

scholarly journals Helical arrays of U-shaped ATP synthase dimers form tubular cristae in ciliate mitochondria

2016 ◽  
Vol 113 (30) ◽  
pp. 8442-8447 ◽  
Author(s):  
Alexander W. Mühleip ◽  
Friederike Joos ◽  
Christoph Wigge ◽  
Achilleas S. Frangakis ◽  
Werner Kühlbrandt ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Protein Complexes ◽  
Paramecium Tetraurelia ◽  
Mitochondrial Atp Synthase ◽  
Structural Differences ◽  
Membrane Protein Complexes ◽  
Subtomogram Averaging ◽  
Electron Cryotomography ◽  
Energy Converting

F1Fo-ATP synthases are universal energy-converting membrane protein complexes that synthesize ATP from ADP and inorganic phosphate. In mitochondria of yeast and mammals, the ATP synthase forms V-shaped dimers, which assemble into rows along the highly curved ridges of lamellar cristae. Using electron cryotomography and subtomogram averaging, we have determined the in situ structure and organization of the mitochondrial ATP synthase dimer of the ciliate Paramecium tetraurelia. The ATP synthase forms U-shaped dimers with parallel monomers. Each complex has a prominent intracrista domain, which links the c-ring of one monomer to the peripheral stalk of the other. Close interaction of intracrista domains in adjacent dimers results in the formation of helical ATP synthase dimer arrays, which differ from the loose dimer rows in all other organisms observed so far. The parameters of the helical arrays match those of the cristae tubes, suggesting the unique features of the P. tetraurelia ATP synthase are directly responsible for generating the helical tubular cristae. We conclude that despite major structural differences between ATP synthase dimers of ciliates and other eukaryotes, the formation of ATP synthase dimer rows is a universal feature of mitochondria and a fundamental determinant of cristae morphology.

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

eLife ◽  
2015 ◽  
Vol 4 ◽  
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.

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

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.

scholarly journals Structure of a bacterial ATP synthase

2018 ◽  
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.

scholarly journals Targeting Mycobacterial F-ATP Synthase C-Terminal α Subunit Interaction Motif on Rotary Subunit γ

Antibiotics ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1456
Author(s):  
Amaravadhi Harikishore ◽  
Chui-Fann Wong ◽  
Priya Ragunathan ◽  
Dennis Litty ◽  
Volker Müller ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Atp Hydrolysis ◽  
Atp Synthesis ◽  
Membrane Vesicles ◽  
Α Subunit ◽  
Rotational States ◽  
C Terminus ◽  
Inverted Membrane Vesicles ◽  
Hydrolysis Of ◽  
Micromolar Range

Mycobacteria regulate their energy (ATP) levels to sustain their survival even in stringent living conditions. Recent studies have shown that mycobacteria not only slow down their respiratory rate but also block ATP hydrolysis of the F-ATP synthase (α3:β3:γ:δ:ε:a:b:b’:c9) to maintain ATP homeostasis in situations not amenable for growth. The mycobacteria-specific α C-terminus (α533-545) has unraveled to be the major regulative of latent ATP hydrolysis. Its deletion stimulates ATPase activity while reducing ATP synthesis. In one of the six rotational states of F-ATP synthase, α533-545 has been visualized to dock deep into subunit γ, thereby blocking rotation of γ within the engine. The functional role(s) of this C-terminus in the other rotational states are not clarified yet and are being still pursued in structural studies. Based on the interaction pattern of the docked α533-545 region with subunit γ, we attempted to study the druggability of the α533-545 motif. In this direction, our computational work has led to the development of an eight-featured α533-545 peptide pharmacophore, followed by database screening, molecular docking, and pose selection, resulting in eleven hit molecules. ATP synthesis inhibition assays using recombinant ATP synthase as well as mycobacterial inverted membrane vesicles show that one of the hits, AlMF1, inhibited the mycobacterial F-ATP synthase in a micromolar range. The successful targeting of the α533-545-γ interaction motif demonstrates the potential to develop inhibitors targeting the α site to interrupt rotary coupling with ATP synthesis.

scholarly journals A critical appraisal of evidence for localized energy coupling. Kinetic studies on liposomes containing bacteriorhodopsin and ATP synthase

1985 ◽  
Vol 230 (2) ◽  
pp. 543-549 ◽  
Author(s):  
R L Van der Bend ◽  
J Petersen ◽  
J A Berden ◽  
K Van Dam ◽  
H V Westerhoff
Keyword(s):  
Electron Transfer ◽  
Atp Synthase ◽  
Critical Appraisal ◽  
Atp Synthesis ◽  
Kinetic Studies ◽  
Energy Coupling ◽  
Proton Pumps ◽  
Specific Interactions ◽  
Mitochondrial Atp Synthase ◽  
Different Levels

In intact systems (chloroplasts, mitochondria and bacteria) many experiments have been reported which are indicative of localized coupling between ATP synthase and electron transfer complexes. We have carried out similar experiments with a system in which we may assume that specific interactions between the proton pumps are absent: reconstituted vesicles containing bacteriorhodopsin and yeast mitochondrial ATP synthase. The only experiment that gives results which differ from those previously published for intact systems concerns the effect of uncouplers on the rate of ATP synthesis at different levels of inhibition of the ATP synthase. We propose that this type of experiment may discriminate between localized and delocalized coupling.

Phosphate exchange and ATP synthesis by DMSO-pretreated purified bovine mitochondrial ATP synthase

2001 ◽  
Vol 353 (2) ◽  
pp. 215-222
Author(s):  
Seelochan BEHARRY ◽  
Philip D. BRAGG
Keyword(s):  
Exchange Reaction ◽  
Atp Synthase ◽  
Atp Synthesis ◽  
Catalytic Site ◽  
Phosphate Removal ◽  
Subsequent Removal ◽  
F1fo Atp Synthase ◽  
Mitochondrial Atp Synthase ◽  
Phosphate Exchange

Purified soluble bovine mitochondrial F1Fo-ATP synthase contained 2mol of ATP, 2mol of ADP and 6mol of Pi/mol. Incubation of this enzyme with 1mM [32P]Pi caused the exchange of 2mol of Pi/mol of F1Fo-ATP synthase. The labelled phosphates were not displaced by ATP. Transfer of F1Fo-ATP synthase to a buffer containing 30% (v/v) DMSO and 1mM [32P]Pi resulted in the loss of bound nucleotides with the retention of 1mol of ATP/mol of F1Fo-ATP synthase. Six molecules of [32P]Pi were incorporated by exchange with the existing bound phosphate. Removal of the DMSO by passage of the enzyme through a centrifuged column of Sephadex G-50 resulted in the exchange of one molecule of bound [32P]Pi into the bound ATP. Azide did not prevent this [32P]Pi ↔ ATP exchange reaction. The bound labelled ATP could be displaced from the enzyme by exogenous ATP. Addition of ADP to the DMSO-pretreated F1Fo-ATP synthase in the original DMSO-free buffer resulted in the formation of an additional molecule of bound ATP. It was concluded that following pretreatment with and subsequent removal of DMSO the F1Fo-ATP synthase contained one molecule of ATP at a catalytic site which was competent to carry out a phosphateŐATP exchange reaction using enzyme-bound inorganic radiolabelled phosphate. In the presence of ADP an additional molecule of labelled ATP was formed from enzyme-bound Pi at a second catalytic site. The bound phosphateŐATP exchange reaction is not readily accommodated by current mechanisms for the ATP synthase.

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

2019 ◽  
Author(s):  
Bonnie J. Murphy ◽  
Niklas Klusch ◽  
Julian D. Langer ◽  
Deryck J. Mills ◽  
Özkan Yildiz ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Metal Ion ◽  
De Novo ◽  
Atp Synthesis ◽  
Joint Movement ◽  
Access Channel ◽  
Subunit Interface ◽  
Ring Rotation ◽  
Mitochondrial Atp Synthase ◽  
Ordered Water

F1Fo-ATP synthases play a central role in cellular metabolism, making the energy of the proton-motive force across a membrane available for a large number of energy-consuming processes. We determined the single-particle cryo-EM structure of active dimeric ATP synthase from mitochondria of Polytomella sp. at 2.7- 2.8 Å resolution. Separation of 13 well-defined rotary substates by 3D 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. The joint movement facilitates flexible coupling of the stoichiometrically mismatched F1 and Fo subcomplexes. Flexibility is mediated primarily by the interdomain hinge of the conserved OSCP subunit, a well-established target of physiologically important inhibitors. Our maps provide atomic detail of the c-ring/a-subunit interface in the membrane, where protonation and deprotonation of c-ring cGlu111 drives rotary catalysis. An essential histidine residue in the lumenal proton access channel binds a strong non-peptide density assigned to a metal ion that may facilitate c-ring protonation, as its coordination geometry changes with c-ring rotation. We resolve ordered water molecules in the proton access and release channels and at the gating aArg239 that is critical in all rotary ATPases. We identify the previously unknown ASA10 subunit and present complete de novo atomic models of subunits ASA1-10, which make up the two interlinked peripheral stalks that stabilize the Polytomella ATP synthase dimer.

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