scholarly journals Interface mobility between monomers in dimeric bovine ATP synthase participates in the ultrastructure of inner mitochondrial membranes

2021 ◽  
Vol 118 (8) ◽  
pp. e2021012118
Author(s):  
Tobias E. Spikes ◽  
Martin G. Montgomery ◽  
John E. Walker
Keyword(s):  
Atp Synthase ◽  
Catalytic Mechanism ◽  
Membrane Domains ◽  
Membrane Domain ◽  
Mitochondrial Membranes ◽  
Angular Variation ◽  
Physiological Conditions ◽  
Catalytic Domains ◽  
Cryo Electron Microscopy ◽  
Lipid Structures

The ATP synthase complexes in mitochondria make the ATP required to sustain life by a rotary mechanism. Their membrane domains are embedded in the inner membranes of the organelle, and they dimerize via interactions between their membrane domains. The dimers form extensive chains along the tips of the cristae with the two rows of monomeric catalytic domains extending into the mitochondrial matrix at an angle to each other. Disruption of the interface between dimers by mutation affects the morphology of the cristae severely. By analysis of particles of purified dimeric bovine ATP synthase by cryo-electron microscopy, we have shown that the angle between the central rotatory axes of the monomeric complexes varies between ca. 76 and 95°. These particles represent active dimeric ATP synthase. Some angular variations arise directly from the catalytic mechanism of the enzyme, and others are independent of catalysis. The monomer–monomer interaction is mediated mainly by j subunits attached to the surface of wedge-shaped protein-lipid structures in the membrane domain of the complex, and the angular variation arises from rotational and translational changes in this interaction, and combinations of both. The structures also suggest how the dimeric ATP synthases might be interacting with each other to form the characteristic rows along the tips of the cristae via other interwedge contacts, molding themselves to the range of oligomeric arrangements observed by tomography of mitochondrial membranes, and at the same time allowing the ATP synthase to operate under the range of physiological conditions that influence the structure of the cristae.

scholarly journals The mobility of interfaces between monomers in dimeric bovine ATP synthase participates in the ultrastructure of inner mitochondrial membranes

2020 ◽  
Author(s):  
Tobias E. Spikes ◽  
Martin G. Montgomery ◽  
John E. Walker
Keyword(s):  
Atp Synthase ◽  
Catalytic Mechanism ◽  
Membrane Domains ◽  
Membrane Domain ◽  
Mitochondrial Membranes ◽  
Angular Variation ◽  
Physiological Conditions ◽  
Catalytic Domains ◽  
Cryo Electron Microscopy ◽  
Lipid Structures

SUMMARYThe ATP synthase complexes in mitochondria make the ATP required to sustain life by a rotary mechanism. Their membrane domains are embedded in the inner membranes of the organelle and they dimerize via interactions between their membrane domains. The dimers form extensive chains along the tips of the cristae with the two rows of monomeric catalytic domains extending into the mitochondrial matrix at an angle to each other. When the interaction between membrane domains is disrupted in living cells, the morphology of the cristae is affected severely. By analysis of particles of purified dimeric bovine ATP synthase by cryo-electron microscopy, we have shown that the angle between the central rotatory axes of the monomeric complexes varies between ca. 76° and ca. 95°. Some variations in this angle arise directly from the catalytic mechanism of the enzyme, and others are independent of catalysis. The monomer-monomer interaction is mediated mainly by j-subunits attached to the surface of wedge shaped protein-lipid structures in the membrane domain of the complex, and the angular variation arises from rotational and translational changes in this interaction, and combinations of both. The structures also suggest how the dimeric ATP synthases might be interacting with each other to form the characteristic rows along the tips of the cristae via other inter-wedge contacts, moulding themselves to the range of oligomeric arrangements observed by tomography of mitochondrial membranes, and at the same time allowing the ATP synthase to operate under the range of physiological conditions that influence the structure of the cristae.

scholarly journals F1F0-ATP synthase from bovine heart mitochondria: development of the purification of a monodisperse oligomycin-sensitive ATPase

1993 ◽  
Vol 295 (3) ◽  
pp. 799-806 ◽  
Author(s):  
R Lutter ◽  
M Saraste ◽  
H S van Walraven ◽  
M J Runswick ◽  
M Finel ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Atp Hydrolysis ◽  
Gel Filtration ◽  
Primary Objective ◽  
Membrane Domain ◽  
Mitochondrial Membranes ◽  
F1 Atpase ◽  
Ammonium Sulphate Precipitation ◽  
F1f0 Atp Synthase ◽  
Hydrolysis Activity

A new procedure for the isolation of ATP synthase from bovine mitochondria has been developed, with the primary objective of producing enzyme suitable for crystallization trials. Proteins were extracted from mitochondrial membranes with dodecyl-beta-D-maltoside, and the ATP synthase was purified from the extract in the presence of the same detergent by a combination of ion-exchange and gel-filtration chromatography and ammonium sulphate precipitation. This simple and rapid procedure yields 20-30 mg of highly pure and monodisperse enzyme, evidently consisting of 14 different subunits, amongst them, in apparently stoichiometric amounts with the established subunits, subunit e, a recently discovered subunit of unknown function. The enzyme preparation has an oligomycin-sensitive ATP hydrolysis activity, and so the F1 domain is functionally associated with the membrane domain, F0. In contrast with the N-termini of some of the subunits of bovine mitochondrial F1-ATPase, those of the F1F0-ATP synthase are not degraded by proteolysis during the isolation procedure. This preparation therefore satisfies prerequisites for crystallization trials.

scholarly journals Structure of the dimeric ATP synthase from bovine mitochondria

2020 ◽  
Vol 117 (38) ◽  
pp. 23519-23526 ◽  
Author(s):  
Tobias E. Spikes ◽  
Martin G. Montgomery ◽  
John E. Walker
Keyword(s):  
Atp Synthase ◽  
Enzyme Complex ◽  
Membrane Domains ◽  
Mitochondrial Matrix ◽  
Membrane Domain ◽  
Rotational States ◽  
Peripheral Stalk ◽  
B Subunit ◽  
Proton Uptake ◽  
Grotthus Mechanism

The structure of the dimeric ATP synthase from bovine mitochondria determined in three rotational states by electron cryo-microscopy provides evidence that the proton uptake from the mitochondrial matrix via the proton inlet half channel proceeds via a Grotthus mechanism, and a similar mechanism may operate in the exit half channel. The structure has given information about the architecture and mechanical constitution and properties of the peripheral stalk, part of the membrane extrinsic region of the stator, and how the action of the peripheral stalk damps the side-to-side rocking motions that occur in the enzyme complex during the catalytic cycle. It also describes wedge structures in the membrane domains of each monomer, where the skeleton of each wedge is provided by three α-helices in the membrane domains of the b-subunit to which the supernumerary subunits e, f, and g and the membrane domain of subunit A6L are bound. Protein voids in the wedge are filled by three specifically bound cardiolipin molecules and two other phospholipids. The external surfaces of the wedges link the monomeric complexes together into the dimeric structures and provide a pivot to allow the monomer–monomer interfaces to change during catalysis and to accommodate other changes not related directly to catalysis in the monomer–monomer interface that occur in mitochondrial cristae. The structure of the bovine dimer also demonstrates that the structures of dimeric ATP synthases in a tetrameric porcine enzyme have been seriously misinterpreted in the membrane domains.

scholarly journals Crystallographic insight into the catalytic mechanism of subunit A of the A-ATP synthase and the P-loop switch in evolution

2010 ◽  
Vol 1797 ◽  
pp. 32-33
Author(s):  
Anil Kumar ◽  
Malathy Sony Subramanian Manimekalai ◽  
Asha Manikkoth Balakrishna ◽  
Gerhard Grüber
Keyword(s):  
Atp Synthase ◽  
Catalytic Mechanism ◽  
Subunit A ◽  
Insight Into

scholarly journals Cryo-EM structure of phosphodiesterase 6 reveals insights into the allosteric regulation of type I phosphodiesterases

2019 ◽  
Vol 5 (2) ◽  
pp. eaav4322 ◽  
Author(s):  
Sahil Gulati ◽  
Krzysztof Palczewski ◽  
Andreas Engel ◽  
Henning Stahlberg ◽  
Lubomir Kovacik
Keyword(s):  
Conformational Changes ◽  
Second Messengers ◽  
Structural Features ◽  
Full Length ◽  
Type I ◽  
Regulatory Domains ◽  
Catalytic Domains ◽  
Cryo Electron Microscopy ◽  
Density Map ◽  
G Protein Coupled

Cyclic nucleotide phosphodiesterases (PDEs) work in conjunction with adenylate/guanylate cyclases to regulate the key second messengers of G protein–coupled receptor signaling. Previous attempts to determine the full-length structure of PDE family members at high-resolution have been hindered by structural flexibility, especially in their linker regions and N- and C-terminal ends. Therefore, most structure-activity relationship studies have so far focused on truncated and conserved catalytic domains rather than the regulatory domains that allosterically govern the activity of most PDEs. Here, we used single-particle cryo–electron microscopy to determine the structure of the full-length PDE6αβ2γ complex. The final density map resolved at 3.4 Å reveals several previously unseen structural features, including a coiled N-terminal domain and the interface of PDE6γ subunits with the PDE6αβ heterodimer. Comparison of the PDE6αβ2γ complex with the closed state of PDE2A sheds light on the conformational changes associated with the allosteric activation of type I PDEs.

scholarly journals Role of the Two Catalytic Domains of DSR-E Dextransucrase and Their Involvement in the Formation of Highly α-1,2 Branched Dextran

2005 ◽  
Vol 187 (1) ◽  
pp. 296-303 ◽  
Author(s):  
Emeline Fabre ◽  
Sophie Bozonnet ◽  
Audrey Arcache ◽  
René-Marc Willemot ◽  
Michel Vignon ◽  
...  
Keyword(s):  
High Performance ◽  
Leuconostoc Mesenteroides ◽  
Catalytic Mechanism ◽  
Small Scale ◽  
Gene Products ◽  
Large Protein ◽  
Catalytic Domains ◽  
Hydrolase Family ◽  
Glucan Binding Domain

ABSTRACT The dsrE gene from Leuconostoc mesenteroides NRRL B-1299 was shown to encode a very large protein with two potentially active catalytic domains (CD1 and CD2) separated by a glucan binding domain (GBD). From sequence analysis, DSR-E was classified in glucoside hydrolase family 70, where it is the only enzyme to have two catalytic domains. The recombinant protein DSR-E synthesizes both α-1,6 and α-1,2 glucosidic linkages in transglucosylation reactions using sucrose as the donor and maltose as the acceptor. To investigate the specific roles of CD1 and CD2 in the catalytic mechanism, truncated forms of dsrE were cloned and expressed in Escherichia coli. Gene products were then small-scale purified to isolate the various corresponding enzymes. Dextran and oligosaccharide syntheses were performed. Structural characterization by 13C nuclear magnetic resonance and/or high-performance liquid chromatography showed that enzymes devoid of CD2 synthesized products containing only α-1,6 linkages. On the other hand, enzymes devoid of CD1 modified α-1,6 linear oligosaccharides and dextran acceptors through the formation of α-1,2 linkages. Therefore, each domain is highly regiospecific, CD1 being specific for the synthesis of α-1,6 glucosidic bonds and CD2 only catalyzing the formation of α-1,2 linkages. This finding permitted us to elucidate the mechanism of α-1,2 branching formation and to engineer a novel transglucosidase specific for the formation of α-1,2 linkages. This enzyme will be very useful to control the rate of α-1,2 linkage synthesis in dextran or oligosaccharide production.

scholarly journals Glutathione-dependent activities of Trypanosoma cruzi p52 makes it a new member of the thiol:disulphide oxidoreductase family

1997 ◽  
Vol 322 (1) ◽  
pp. 43-48 ◽  
Author(s):  
Mireille MOUTIEZ ◽  
Eric QUÉMÉNEUR ◽  
Christian SERGHERAERT ◽  
Valérie LUCAS ◽  
André TARTAR ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Reductase Activity ◽  
Redox Balance ◽  
Ribonuclease A ◽  
Physiological Conditions ◽  
Active Site Motif ◽  
Key Enzyme ◽  
Low Rate

Trypanothione:glutathione disulphide thioltransferase of Trypanosoma cruzi (p52) is a key enzyme in the regulation of the intracellular thiolŐdisulphide redox balance by reducing glutathione disulphide. Here we show that p52, like other disulphide oxidoreductases possessing the CXXC active site motif, catalyses the reduction of low-molecular-mass disulphides (hydroxyethyldisulphide) as well as protein disulphides (insulin). However, p52 seems to be a poor oxidase under physiological conditions as evidenced by its very low rate for oxidative renaturation of reduced ribonuclease A. Like thioltransferase and protein disulphide isomerase, p52 was found to possess a glutathione-dependent dehydroascorbate reductase activity. The kinetic parameters were in the same range as those determined for mammalian dehydroascorbate reductases. A catalytic mechanism taking into account both trypanothione- and glutathione-dependent reduction reactions was proposed. This newly characterized enzyme is specific for the parasite and provides a new target for specific chemotherapy.

scholarly journals Cholesterol-dependent partitioning of PtdIns(4,5)P2 into membrane domains by the N-terminal fragment of NAP-22 (neuronal axonal myristoylated membrane protein of 22 kDa)

2004 ◽  
Vol 379 (3) ◽  
pp. 527-532 ◽  
Author(s):  
Richard M. EPAND ◽  
Phan VUONG ◽  
Christopher M. YIP ◽  
Shohei MAEKAWA ◽  
Raquel F. EPAND
Keyword(s):  
Membrane Protein ◽  
Fluorescence Microscopy ◽  
Fluorescence Spectroscopy ◽  
Membrane Domains ◽  
Domain Formation ◽  
Membrane Domain ◽  
Terminal Fragment ◽  
N Terminus ◽  
Total Internal Reflectance ◽  
Internal Reflectance

A myristoylated peptide corresponding to the N-terminus of NAP-22 (neuronal axonal myristoylated membrane protein of 22 kDa) causes the quenching of the fluorescence of BODIPY®-TMR-labelled PtdIns(4,5)P2 in bilayers of 1-palmitoyl-2-oleoyl phosphatidylcholine containing 40 mol% cholesterol and 0.1 mol% BODIPY®–PtdIns(4,5)2. Both fluorescence spectroscopy and total internal reflectance fluorescence microscopy revealed the cholesterol-dependent nature of PtdIns(4,5)P2-enriched membrane-domain formation.

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