Browse
Submit Data
Databases
API
Help

Dataset Information

26 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

The N-terminal region of the ? subunit from cyanobacterial ATP synthase alone can inhibit ATPase activity.

ABSTRACT: ATP hydrolysis activity catalyzed by chloroplast and proteobacterial ATP synthase is inhibited by their ? subunits. To clarify the function of the ? subunit from phototrophs, here we analyzed the ? subunit-mediated inhibition (?-inhibition) of cyanobacterial F1-ATPase, a subcomplex of ATP synthase obtained from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. We generated three C-terminal ?-helix null ?-mutants; one lacked the C-terminal ?-helices, and in the other two, the C-terminal conformation could be locked by a disulfide bond formed between two ?-helices or an ?-helix and a ?-sandwich structure. All of these ?-mutants maintained ATPase-inhibiting competency. We then used single-molecule observation techniques to analyze the rotary motion of F1-ATPase in the presence of these ?-mutants. The stop angular position of the ? subunit in the presence of the ?-mutant was identical to that in the presence of the WT ?. Using magnetic tweezers, we examined recovery from the inhibited rotation and observed restoration of rotation by 80° forcing of the ? subunit in the case of the ADP-inhibited form, but not when the rotation was inhibited by the ?-mutants or by the WT ? subunit. These results imply that the C-terminal ?-helix domain of the ? subunit of cyanobacterial enzyme does not directly inhibit ATP hydrolysis and that its N-terminal domain alone can inhibit the hydrolysis activity. Notably, this property differed from that of the proteobacterial ?, which could not tightly inhibit rotation. We conclude that phototrophs and heterotrophs differ in the ? subunit-mediated regulation of ATP synthase.

SUBMITTER: Inabe K

PROVIDER: S-EPMC6664179 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

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

Project description: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.

| S-EPMC8698299 | biostudies-literature

Characterization of mutants deficient in N-terminal phosphorylation of the chloroplast ATP synthase subunit β.

Project description:Understanding the regulation of photosynthetic light harvesting and electron transfer is of great importance to efforts to improve the ability of the electron transport chain to supply downstream metabolism. A central regulator of the electron transport chain is ATP synthase, the molecular motor that harnesses the chemiosmotic potential generated from proton-coupled electron transport to synthesize ATP. ATP synthase is regulated both thermodynamically and post-translationally, with proposed phosphorylation sites on multiple subunits. In this study we focused on two N-terminal serines on the catalytic subunit β in tobacco (Nicotiana tabacum), previously proposed to be important for dark inactivation of the complex to avoid ATP hydrolysis at night. Here we show that there is no clear role for phosphorylation in the dark inactivation of ATP synthase. Instead, mutation of one of the two phosphorylated serine residues to aspartate to mimic constitutive phosphorylation strongly decreased ATP synthase abundance. We propose that the loss of N-terminal phosphorylation of ATPβ may be involved in proper ATP synthase accumulation during complex assembly.

| S-EPMC10022623 | biostudies-literature

Analysis of an N-terminal deletion in subunit a of the Escherichia coli ATP synthase.

Project description:Subunit a is a membrane-bound stator subunit of the ATP synthase and is essential for proton translocation. The N-terminus of subunit a in E. coli is localized to the periplasm, and contains a sequence motif that is conserved among some bacteria. Previous work has identified mutations in this region that impair enzyme activity. Here, an internal deletion was constructed in subunit a in which residues 6-20 were replaced by a single lysine residue, and this mutant was unable to grow on succinate minimal medium. Membrane vesicles prepared from this mutant lacked ATP synthesis and ATP-driven proton translocation, even though immunoblots showed a significant level of subunit a. Similar results were obtained after purification and reconstitution of the mutant ATP synthase into liposomes. The location of subunit a with respect to its neighboring subunits b and c was probed by introducing cysteine substitutions that were known to promote cross-linking: a_L207C + c_I55C, a_L121C + b_N4C, and a_T107C + b_V18C. The last pair was unable to form cross-links in the background of the deletion mutant. The results indicate that loss of the N-terminal region of subunit a does not generally disrupt its structure, but does alter interactions with subunit b.

| S-EPMC5376380 | biostudies-literature

The Unique C-Terminal Extension of Mycobacterial F-ATP Synthase Subunit α Is the Major Contributor to Its Latent ATP Hydrolysis Activity.

Project description:Mycobacterial F1Fo-ATP synthases (α3:β3:γ:δ:ε:a:b:b':c9 ) are incapable of ATP-driven proton translocation due to their latent ATPase activity. This prevents wasting of ATP and altering of the proton motive force, whose dissipation is lethal to mycobacteria. We demonstrate that the mycobacterial C-terminal extension of nucleotide-binding subunit α contributes mainly to the suppression of ATPase activity in the recombinant mycobacterial F1-ATPase. Using C-terminal deletion mutants, the regions responsible for the enzyme's latency were mapped, providing a new compound epitope.

| S-EPMC7674025 | biostudies-literature

Mechanism of inhibition by C-terminal alpha-helices of the epsilon subunit of Escherichia coli FoF1-ATP synthase.

Project description:The epsilon subunit of bacterial FoF1-ATP synthase (FoF1), a rotary motor protein, is known to inhibit the ATP hydrolysis reaction of this enzyme. The inhibitory effect is modulated by the conformation of the C-terminal alpha-helices of epsilon, and the "extended" but not "hairpin-folded" state is responsible for inhibition. Although the inhibition of ATP hydrolysis by the C-terminal domain of epsilon has been extensively studied, the effect on ATP synthesis is not fully understood. In this study, we generated an Escherichia coli FoF1 (EFoF1) mutant in which the epsilon subunit lacked the C-terminal domain (FoF1epsilonDeltaC), and ATP synthesis driven by acid-base transition (DeltapH) and the K+-valinomycin diffusion potential (DeltaPsi) was compared in detail with that of the wild-type enzyme (FoF1epsilonWT). The turnover numbers (kcat) of FoF1epsilonWT were severalfold lower than those of FoF1epsilonDeltaC. FoF1epsilonWT showed higher Michaelis constants (Km). The dependence of the activities of FoF1epsilonWT and FoF1epsilonDeltaC on various combinations of DeltapH and DeltaPsi was similar, suggesting that the rate-limiting step in ATP synthesis was unaltered by the C-terminal domain of epsilon. Solubilized FoF1epsilonWT also showed lower kcat and higher Km values for ATP hydrolysis than the corresponding values of FoF1epsilonDeltaC. These results suggest that the C-terminal domain of the epsilon subunit of EFoF1 slows multiple elementary steps in both the ATP synthesis/hydrolysis reactions by restricting the rotation of the gamma subunit.

| S-EPMC2719386 | biostudies-literature

Inverse regulation of F1-ATPase activity by a mutation at the regulatory region on the gamma subunit of chloroplast ATP synthase.

Project description:Chloroplast ATP synthase is a thiol-modulated enzyme whose DeltamuH(+)-linked activation is strongly influenced by reduction and the formation of a disulphide bridge between Cys(199) and Cys(205) on the gamma subunit. In solubilized chloroplast coupling factor 1 (CF(1)), reduction of the disulphide bond elicits the latent ATP-hydrolysing activity. To assess the regulatory importance of the amino acid residues around these cysteine residues, we focused on the three negatively charged residues Glu(210)-Asp-Glu(212) close to the two cysteine residues and also on the following region from Leu(213) to Ile(230), and investigated the modulation of ATPase activity by chloroplast thioredoxins. The mutant gamma subunits were reconstituted with the alpha and beta subunits from F(1) of the thermophilic bacterium Bacillus PS3; the active ATPase complexes obtained were purified by gel-filtration chromatography. The complex formed with a mutant gamma subunit in which Glu(210) to Glu(212) had been deleted was inactivated rather than activated by reduction of the disulphide bridge by reduced thioredoxin, indicating inverse regulation. This complex was insensitive to the inhibitory CF(1)-epsilon subunit when the mutant gamma subunit was oxidized. In contrast, the deletion of Glu(212) to Ile(230) converted the complex from a modulated state into a highly active state.

| S-EPMC1221517 | biostudies-other

Structures of the thermophilic F1-ATPase epsilon subunit suggesting ATP-regulated arm motion of its C-terminal domain in F1.

Project description:The epsilon subunit of bacterial and chloroplast F(o)F(1)-ATP synthases modulates their ATP hydrolysis activity. Here, we report the crystal structure of the ATP-bound epsilon subunit from a thermophilic Bacillus PS3 at 1.9-A resolution. The C-terminal two alpha-helices were folded into a hairpin, sitting on the beta sandwich structure, as reported for Escherichia coli. A previously undescribed ATP binding motif, I(L)DXXRA, recognizes ATP together with three arginine and one glutamate residues. The E. coli epsilon subunit binds ATP in a similar manner, as judged on NMR. We also determined solution structures of the C-terminal domain of the PS3 epsilon subunit and relaxation parameters of the whole molecule by NMR. The two helices fold into a hairpin in the presence of ATP but extend in the absence of ATP. The latter structure has more helical regions and is much more flexible than the former. These results suggest that the epsilon C-terminal domain can undergo an arm-like motion in response to an ATP concentration change and thereby contribute to regulation of F(o)F(1)-ATP synthase.

| S-EPMC2040882 | biostudies-literature

Fo-driven Rotation in the ATP Synthase Direction against the Force of F1 ATPase in the FoF1 ATP Synthase.

Project description:Living organisms rely on the FoF1 ATP synthase to maintain the non-equilibrium chemical gradient of ATP to ADP and phosphate that provides the primary energy source for cellular processes. How the Fo motor uses a transmembrane electrochemical ion gradient to create clockwise torque that overcomes F1 ATPase-driven counterclockwise torque at high ATP is a major unresolved question. Using single FoF1 molecules embedded in lipid bilayer nanodiscs, we now report the observation of Fo-dependent rotation of the c10 ring in the ATP synthase (clockwise) direction against the counterclockwise force of ATPase-driven rotation that occurs upon formation of a leash with Fo stator subunit a. Mutational studies indicate that the leash is important for ATP synthase activity and support a mechanism in which residues aGlu-196 and cArg-50 participate in the cytoplasmic proton half-channel to promote leash formation.

| S-EPMC4409238 | biostudies-literature

Entrapment of water by subunit c of ATP synthase.

Project description:We consider an ancient protein, and water as a smooth surface, and show that the interaction of the two allows the protein to change its hydrogen bonding to encapsulate the water. This property could have made a three-dimensional microenvironment, 3-4 Gyr ago, for the evolution of subsequent complex water-based chemistry. Proteolipid, subunit c of ATP synthase, when presented with a water surface, changes its hydrogen bonding from an alpha-helix to beta-sheet-like configuration and moves away from its previous association with lipid to interact with water surface molecules. Protein sheets with an intra-sheet backbone spacing of 3.7A and inter-sheet spacing of 6.0 A hydrogen bond into long ribbons or continuous surfaces to completely encapsulate a water droplet. The resulting morphology is a spherical vesicle or a hexagonal crystal of water ice, encased by a skin of subunit c. Electron diffraction shows the crystals to be highly ordered and compressed and the protein skin to resemble beta-sheets. The protein skin can retain the entrapped water over a temperature rise from 123 to 223 K at 1 x 10(-4) Pa, whereas free water starts to sublime significantly at 153 K.

| S-EPMC2500151 | biostudies-literature

A conformational change of the ? subunit indirectly regulates the activity of cyanobacterial F1-ATPase.

Project description:The central shaft of the catalytic core of ATP synthase, the ? subunit consists of a coiled-coil structure of N- and C-terminal ?-helices, and a globular domain. The ? subunit of cyanobacterial and chloroplast ATP synthase has a unique 30-40-amino acid insertion within the globular domain. We recently prepared the insertion-removed ?(3)?(3)? complex of cyanobacterial ATP synthase (Sunamura, E., Konno, H., Imashimizu-Kobayashi, M., and Hisabori, T. (2010) Plant Cell Physiol. 51, 855-865). Although the insertion is thought to be located in the periphery of the complex and far from catalytic sites, the mutant complex shows a remarkable increase in ATP hydrolysis activity due to a reduced tendency to lapse into ADP inhibition. We postulated that removal of the insertion affects the activity via a conformational change of two central ?-helices in ?. To examine this hypothesis, we prepared a mutant complex that can lock the relative position of two central ?-helices to each other by way of a disulfide bond formation. The mutant obtained showed a significant change in ATP hydrolysis activity caused by this restriction. The highly active locked complex was insensitive to N-dimethyldodecylamine-N-oxide, suggesting that the complex is resistant to ADP inhibition. In addition, the lock affected ? inhibition. In contrast, the change in activity caused by removal of the ? insertion was independent from the conformational restriction of the central axis component. These results imply that the global conformational change of the ? subunit indirectly regulates complex activity by changing both ADP inhibition and ? inhibition.

| S-EPMC3493913 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data