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The yeast mitochondrial ATPase has been genetically modified to include a His6 Ni-affinity tag on the amino end of the mature β-subunit. The modified β-subunit is imported into the mitochondrion, properly processed to the mature form, and assembled into a mature and fully active ATP synthase. The F1-ATPase has been purified from submitochondrial particles after release from the membrane with chloroform, followed by Ni-chelate-affinity and gel filtration chromatography. The final enzyme is a homogeneous preparation with full activity and no apparent degradation products. This enzyme preparation has been used to obtain crystals that diffract to better than 2.8Å resolution. [Copyright &y& Elsevier]

A genetically modified (His6-tagged) form of the mitochondrial F1 -ATPase (MW = 370 kDa) has been purified from the yeast Saccharomyces cerevisiae and crystallized in the presence of polythelene glycol (PEG) 6000 as a precipitant, 1 mM NiCl2, 1mM Mg AMP-PNP and 50 μM Mg ADP. X-ray diffraction data were obtained on three separate occasions using synchrotron radiation, with a progression in the quality of the diffraction data, which improved from 3.3 to 3.0 to 2.8 &Aring . On the second occasion, the diffraction was improved by a crystal-annealing procedure. The crystals belong to the monoclinic space group P21, with unit-cell parameters a = 110.6, b = 294.2, c = 190.4 Å, β = 101.6°. The asymmetric unit contains three molecules of yeast F1, with a corresponding volume per protein weight (VM) of 2.8 ų Da-1 and a solvent content of 55%. [ABSTRACT FROM AUTHOR]

Examines the complementation of deletion mutants in the genes encoding the F(sub 1)-ATPase. Expression of the corresponding bovine subunits in yeast Saccharomyces cerevisiae; Construction of the null mutants; Expression constructs generation.

Unisite ATPase kinetic constants were measured for wild-type yeast Saccharomyces cerevisiae F1-ATPase and F1-ATPase with the Thr197→Ser mutation in the β subunit. Under unisite conditions, the concentration of ATP is greater than that of the enzyme, ATP hydrolysis is slow and the affinity of the enzyme for ATP and ADP is high, The Thr197→Ser mutation in the yeast F1-ATPase increases the specific activity of ATP hydrolysis threefold and makes the enzyme much less sensitive to azide and oxyanions [Mueller, D. M. (1989) J. Biol. Chem. 264, 16552-16556]. A unifying hypothesis is that the affinity of F1-ATPase for ADP is altered by azide, oxyanions and the Thr197→Ser mutation. To address this hypothesis, kinetic and thermodynamic constants were measured for the wild-type and mutant enzymes in the absence and presence of azide and oxyanions. The results indicate that sulfite and azide do not significantly alter unisite thermodynamic binding constants of either enzyme for ADP at the catalytic site. The mutation Thr197→Ser has little effect on the binding constant for ADP, or on other unisite kinetic constants of the enzyme, in the presence or absence of azide or oxyanions. However, the binding of ADP to the enzyme was affected by oxyanions and the Thr197→Ser mutation as measured by determining the KiADP values for multi-site ATPase activity (saturating ATP). The Ki for ADP on ATPase activity was measured for the wild-type and mutant enzymes in the presence and absence of sulfite under mulfisite conditions. Sulfite increases the KiADP values for ATP hydrolysis under multisite conditions approximately threefold for the wildtype and mutant enzymes and the Thr197→Ser mutation increases KiADP ninefold. The effect of sulfite on KiADP is additive to the effect of the Thr197→Ser mutation, suggesting that these are distinct effects. These results indicate that the effects of azide, oxyanions, and the Thr197→Ser mutation on the biochemistry of F1-ATPase are limited primarily to multisite conditions. Both sulfite and the Thr197→Ser mutation decrease the affinity of the enzyme for ADP, as measured by the increase in the Ki values. Furthermore, the mechanisms of activation by sulfite and the Thr197→Ser mutations are different. This difference occurs despite their common biochemical consequences on the apparent affinity for ADP. [ABSTRACT FROM AUTHOR]

• The ATP1A2 spectrum should include early onset severe epileptic encephalopathy. • The phenotype consists of hemiplegia and seizures starting by 2.5 years of age. • It also includes developmental delay and recurrent encephalopathy with regression. • NMDAR antagonist therapy was associated with apparent improvement in all 5 patients. ATP1A2 mutations cause hemiplegic migraine with or without epilepsy or acute reversible encephalopathy. Typical onset is in adulthood or older childhood without subsequent severe long-term developmental impairments. We aimed to describe the manifestations of early onset severe ATP1A2 -related epileptic encephalopathy and its underlying mutations in a cohort of seven patients. A retrospective chart review of a cohort of seven patients was conducted. Response to open-label memantine therapy, used off-label due to its NMDA receptor antagonist effects, was assessed by the Global Rating Scale of Change (GRSC) and Clinical Global Impression Scale of Improvement (CGI-I) methodologies. Molecular modeling was performed using PyMol program. Patients (age 2.5–20 years) had symptom onset at an early age (6 days–1 year). Seizures were either focal or generalized. Common features were: drug resistance, recurrent status epilepticus, etc., severe developmental delay with episodes of acute severe encephalopathy often with headaches, dystonias, hemiplegias, seizures, and developmental regression. All had variants predicted to be disease causing (p.Ile293Met, p.Glu1000Lys, c.1017+5G>A, p.Leu809Arg, and 3 patients with p.Met813Lys). Modeling revealed that mutations interfered with ATP1A2 ion binding and translocation sites. Memantine, given to five, was tolerated in all (mean treatment: 2.3 years, range 6 weeks–4.8 years) with some improvements reported in all five. Our observations describe a distinctive clinical profile of seven unrelated probands with early onset severe ATP1A2 -related epileptic encephalopathy, provide insights into structure–function relationships of ATP1A2 mutations, and support further studies of NMDAR antagonist therapy in ATP1A2- encephalopathy. [ABSTRACT FROM AUTHOR]

The ATP synthase is under a number of mechanisms of regulation. The chloroplast ATPase has a unique mode of regulation in which activity is controlled by the redox state in the organelle. This mode of regulation is determined by a small unique region within the γ-subunit and this region contains two cysteine residues. Introduction of this region within the yeast γ-subunit causes a defect in oxidative phosphorylation. Oxidative phosphorylation is restored if the cysteine residues are replaced with serine. Biochemical analysis of the chimeric mitochondrial ATPase indicates that the ATP synthase is not largely altered with the cysteine residues in either the oxidized or reduced states. However, the level and activity of cytochrome c oxidase are decreased by about 90%, whereas that of NADH dehydrogenase and cytochrome c reductase are unchanged as compared with the wild-type enzymes. The level and activity of cytochrome c oxidase are restored with replacement of the cysteine residues with serine in the regulatory region. These results indicate that the chimeric ATP synthase containing cysteine, but not serine, decreases the expression or assembly of cytochrome c oxidase with little effect on the activity of the ATP synthase. [ABSTRACT FROM AUTHOR]

The mitochondrial ATP synthase is a molecular motor, which couples the flow of protons with phosphorylation of ADP. Rotation of the central stalk within the core of ATP synthase effects conformational changes in the active sites driving the synthesis of ATP. Mitochondrial genome integrity (mgi) mutations have been previously identified in the α-, β-, and γ-subunits of ATP synthase in yeast Kluyveromyces lactis and trypanosome Trypanosoma brucei. These mutations reverse the lethality of the loss of mitochondrial DNA in petite negative strains. Introduction of the homologous mutations in Saccharomyces cerevisiae results in yeast strains that lose mitochondrial DNA at a high rate and accompanied decreases in the coupling of the ATP synthase. The structure of yeast F1-ATPase reveals that the mgi residues cluster around the γ-subunit and selectively around the collar region of F1. These results indicate that residues within the mgi complementation group are necessary for efficient coupling of ATP synthase, possibly acting as a support to fix the axis of rotation of the central stalk. [ABSTRACT FROM AUTHOR]

The F1-ATPase is a multimeric enzyme (α3β3γδε) primarily responsible for the synthesis of ATP under aerobic conditions. The entire coding region of each of the genes was deleted separately in yeast, providing five null mutant strains. Strains with a deletion in the genes encoding α-, β-, γ- or δ-subunits were unable to grow, while the strain with a null mutation in ε was able to grow slowly on medium containing glycerol as the carbon source. In addition, strains with a null mutation in γ or δ became 100% ρ0/ρ­ and the strain with the null mutation in γ grew much more slowly on medium containing glucose. These additional phenotypes were not observed in strains with the double mutations: ΔαΔγ, ΔβΔγ, Δatp11Δγ, ΔαΔδ, ΔβΔγ or Δatp11Δδ. These results indicate that ε is not an essential component of the ATP synthase and that mutations in the genes encoding the α- and β-subunits and in ATP11 are epistatic to null mutations in the genes encoding the γ- and δ-subunits. These data suggest that the propensity to form ρ0/ρ­ mutations in the γ and δ null deletion mutant stains and the slow growing phenotypes of the null γ mutant strain are due to the assembly of F1 deficient in the corresponding subunit. These results have profound implications for the physiology of normal cells. [ABSTRACT FROM AUTHOR]

In the past two decades, 7 coronaviruses have infected the human population, with two major outbreaks caused by SARS-CoV and MERS-CoV in the year 2002 and 2012, respectively. Currently, the entire world is facing a pandemic of another coronavirus, SARS-CoV-2, with a high fatality rate. The spike glycoprotein of SARS-CoV-2 mediates entry of virus into the host cell and is one of the most important antigenic determinants, making it a potential candidate for a vaccine. In this study, we have computationally designed a multi-epitope vaccine using spike glycoprotein of SARS-CoV-2. The overall quality of the candidate vaccine was validated in silico and Molecular Dynamics Simulation confirmed the stability of the designed vaccine. Docking studies revealed stable interactions of the vaccine with Toll-Like Receptors and MHC Receptors. The in silico cloning and codon optimization supported the proficient expression of the designed vaccine in E. coli expression system. The efficiency of the candidate vaccine to trigger an effective immune response was assessed by an in silico immune simulation. The computational analyses suggest that the designed multi-epitope vaccine is structurally stable which can induce specific immune responses and thus, can be a potential vaccine candidate against SARS-CoV-2. [ABSTRACT FROM AUTHOR]

In the past two decades, 7 coronaviruses have infected the human population, with two major outbreaks caused by SARS-CoV and MERS-CoV in the year 2002 and 2012, respectively. Currently, the entire world is facing a pandemic of another coronavirus, SARS-CoV-2, with a high fatality rate. The spike glycoprotein of SARS-CoV-2 mediates entry of virus into the host cell and is one of the most important antigenic determinants, making it a potential candidate for a vaccine. In this study, we have computationally designed a multi-epitope vaccine using spike glycoprotein of SARS-CoV-2. The overall quality of the candidate vaccine was validated in silico and Molecular Dynamics Simulation confirmed the stability of the designed vaccine. Docking studies revealed stable interactions of the vaccine with Toll-Like Receptors and MHC Receptors. The in silico cloning and codon optimization supported the proficient expression of the designed vaccine in E. coli expression system. The efficiency of the candidate vaccine to trigger an effective immune response was assessed by an in silico immune simulation. The computational analyses suggest that the designed multi-epitope vaccine is structurally stable which can induce specific immune responses and thus, can be a potential vaccine candidate against SARS-CoV-2. [ABSTRACT FROM AUTHOR]

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel composed of 1480 amino acids. The major mutation responsible for cystic fibrosis results in loss of amino acid residue, F508 (F508del). Loss of F508 in CFTR alters the folding pathway resulting in endoplasmic-reticulum-associated degradation. This study investigates the role of synonymous codon in the expression of CFTR and CFTR F508del in human HEK293 cells. DNA encoding the open reading frame (ORF) for CFTR containing synonymous codon replacements was expressed using a heterologous vector integrated into the genome. The results indicate that the codon usage greatly affects the expression of CFTR. While the promoter strength driving expression of the ORFs was largely unchanged and the mRNA half-lives were unchanged, the steady-state levels of the mRNA varied by as much as 30-fold. Experiments support that this apparent inconsistency is attributed to nonsense mediated decay independent of exon junction complex. The ratio of CFTR/mRNA indicates that mRNA containing native codons was more efficient in expressing mature CFTR as compared to mRNA containing synonymous high-expression codons. However, when F508del CFTR was expressed after codon optimization, a greater percentage of the protein escaped endoplasmic-reticulum-associated degradation resulting in considerable levels of mature F508del CFTR on the plasma membrane, which showed channel activity. These results indicate that codon usage has an effect on mRNA levels and protein expression, for CFTR, and likely on chaperone-assisted folding pathway, for F508del CFTR. [ABSTRACT FROM AUTHOR]

We report the high-resolution (1.9 Å) crystal structure of oligomycin bound to the subunit c10 ring of the yeast mitochondrial ATP synthase. Oligomycin binds to the surface of the c10 ring making contact with two neighboring molecules at a position that explains the inhibitory effect on ATP synthesis. The carboxyl side chain of Glu59, which is essential for proton translocation, forms an H-bond with oligomycin via a bridging water molecule but is otherwise shielded from the aqueous environment. The remaining contacts between oligomycin and subunit c are primarily hydrophobic. The amino acid residues that form the oligomycin-binding site are 100% conserved between human and yeast but are widely different from those in bacterial homologs, thus explaining the differential sensitivity to oligomycin. Prior genetics studies suggest that the oligomycin-binding site overlaps with the binding site of other antibiotics, including those effective against Mycobacterium tuberculosis, and thereby frames a common "drug-binding site." We anticipate that this drug-binding site will serve as an effective target for new antibiotics developed by rational design. [ABSTRACT FROM AUTHOR]

The proton pore of the F1Fo ATP synthase consists of a ring of c subunits, which rotates, driven by downhill proton diffusion across the membrane. An essential carboxylate side chain in each subunit provides a proton-binding site. In all the structures of c-rings reported to date, these sites are in a closed, ion-locked state. Structures are here presented of the c10 ring from Saccharomyces cerevisiae determined at pH 8.3, 6.1 and 5.5, at resolutions of 2.0 Å, 2.5 Å and 2.0 Å, respectively. The overall structure of this mitochondrial c-ring is similar to known homologs, except that the essential carboxylate, Glu59, adopts an open extended conformation. Molecular dynamics simulations reveal that opening of the essential carboxylate is a consequence of the amphiphilic nature of the crystallization buffer. We propose that this new structure represents the functionally open form of the c subunit, which facilitates proton loading and release. [ABSTRACT FROM AUTHOR]

The mitochondrial ATP synthase couples the flow of protons with the phosphorylation of ADP. A class of mutations, the mitochondrial genome integrity (mgi) mutations, has been shown to uncouple this process in the yeast mitochondrial ATP synthase. Four mutant forms of the yeast F1 ATPase with mgi mutations were crystallized; the structures were solved and analyzed. The analysis identifies two mechanisms of structural uncoupling: one in which the empty catalytic site is altered and in doing so, apparently disrupts substrate (phosphate) binding, and a second where the steric hindrance predicted between γLeu83 and βDP residues, Leu-391 and Glu-395, located in Catch 2 region, is reduced allowing rotation of the γ-subunit with less impedance. Overall, the structures provide key insights into the critical interactions in the yeast ATP synthase involved in the coupling process. [ABSTRACT FROM AUTHOR]

The crystal structure of nucleotide-free yeast F1 ATPase has been determined at a resolution of 3.6 Å. The overall structure is very similar to that of the ground state enzyme, In particular, the βDP and βTP subunits both adopt the closed conformation found in the ground state structure despite the absence of bound nucleotides. This implies that interactions between the γ and β subunits are as important as nucleotide occupancy in determining the conformational state of the β subunits. Furthermore, this result suggests that for the mitochondrial enzyme, there is no state of nucleotide occupancy that would result in more than one of the β subunits adopting the open conformation. The adenine-binding pocket of the βTP subunit is disrupted in the apo-enzyme, suggesting that the βDP subunit is responsible for unisite catalytic activity. [ABSTRACT FROM AUTHOR]

The crystal structure of yeast mitochondrial F1 ATPase contains three independent copies of the complex, two of which have similar conformations while the third differs in the position of the central stalk relative to the α3β3 sub-assembly. All three copies display very similar asymmetric features to those observed for the bovine enzyme, but the yeast F1 ATPase structures provide novel information. In particular, the active site that binds ADP in bovine F1 ATPase has an ATP analog bound and therefore this structure does not represent the ADP-inhibited form. In addition, one of the complexes binds phosphate in the nucleotide-free catalytic site, and comparison with other structures provides a picture of the movement of the phosphate group during initial binding and subsequent catalysis. The shifts in position of the central stalk between two of the three copies of yeast F1 ATPase and when these structures are compared to those of the bovine enzyme give new insight into the conformational changes that take place during rotational catalysis. [ABSTRACT FROM AUTHOR]

The mitochondrial F1F0-ATP synthase is a multimeric enzyme complex composed of at least 16 unique peptides with an overall molecular mass of ∼600 kDa. F1-ATPase is composed of α3β3γδε with an overall molecular mass of 370 kDa. The genes encoding bovine F1-ATPase have been expressed in a quintuple yeast Saccharomyces cerevisiae deletion mutant (ΔαΔβΔγΔδΔε). This strain expressing bovine F1 is unable to grow on medium containing a non-fermentable carbon source (YPG), indicating that the enzyme is non-functional. However, daughter strains were easily selected for growth on YPG medium and these were evolved for improved growth on YPG medium. The evolution of the strains was presumably due to mutations, but mutations in the genes encoding the subunits of the bovine F1-ATPase were not required for the ability of the cell to grow on YPG medium. The bovine enzyme expressed in yeast was partially purified to a specific activity of about half of that of the enzyme purified from bovine heart mitochondria. These results indicate that the molecular machinery required for the assembly of the mitochondrial ATP synthase is conserved from bovine and yeast and suggest that yeast may be useful for the expression, mutagenesis, and analysis of the mammalian F1- or F1F0- ATP synthase. [ABSTRACT FROM AUTHOR]

The mechanism of inhibition of yeast mitochondrial F1-ATPase by its natural regulatory peptide, IF1, was investigated by correlating the rate of inhibition by IF1 with the nucleotide occupancy of the catalytic sites. Nucleotide occupancy of the catalytic sites was probed by fluorescence quenching of a tryptophan, which was engineered in the catalytic site (β-Y345W). Fluorescence quenching of a β-Trp345 indicates that the binding of MgADP to F1 can be described as 3 binding sites with dissociation constants of Kd1 = 10 ± 2 nM, Kd2 = 0.22 ± 0.03 μM, and Kd3 = 16.3 ± 0.2 μM. In addition, the ATPase activity of the β-Trp345 enzyme followed simple Michaelis-Menten kinetics with a corresponding Km of 55 μM. Values for the Kd for MgATP were estimated and indicate that the Km (55 μM) for ATP hydrolysis corresponds to filling the third catalytic site on F1. IF1 binds very slowly to F1-ATPase depleted of nucleotides and under unisite conditions. The rate of inhibition by IF1 increased with increasing concentration of MgATP to about 50 μM, but decreased thereafter. The rate of inhibition was half-maximal at 5 μM MgATP, which is 10-fold lower than the Km for ATPase. The variations of the rate of IF1 binding are related to changes in the conformation of the IF1 binding site during the catalytic reaction cycle of ATP hydrolysis. A model is proposed that suggests that IF1 binds rapidly, but loosely to F1 with two or three catalytic sites filled, and is then locked in the enzyme during catalytic hydrolysis of ATP. [ABSTRACT FROM AUTHOR]

The inner membrane of the mitochondrion folds inwards, forming the cristae. This folding allows a greater amount of membrane to be packed into the mitochondrion. The data in this study demonstrate that subunits e and g of the mitochondrial ATP synthase are involved in generating mitochondrial cristae morphology. These two subunits are non-essential components of ATP synthase and are required for the dimerization and oligomerization of ATP synthase. Mitochondria of yeast cells deficient in either subunits e or g were found to have numerous digitations and onion-like structures that correspond to an uncontrolled biogenesis and/or folding of the inner mitochondrial membrane. The present data show that there is a link between dimerization of the mitochondrial ATP synthase and cristae morphology. A model is proposed of the assembly of ATP synthase dimers, taking into account the oligomerization of the yeast enzyme and earlier data on the ultrastructure of mitochondrial cristae, which suggests that the association of ATP synthase dimers is involved in the control of the biogenesis of the inner mitochondrial membrane. [ABSTRACT FROM AUTHOR]

Bedaquiline (BDQ, Sirturo) has been approved to treat multidrug resistant forms of Mycobacterium tuberculosis. Prior studies suggested that BDQ was a selective inhibitor of the ATP synthase from M. tuberculosis. However, Sirturo treatment leads to an increased risk of cardiac arrhythmias and death, raising the concern that this adverse effect results from inhibition at a secondary site. Here we show that BDQ is a potent inhibitor of the yeast and human mitochondrial ATP synthases. Single-particle cryo-EM reveals that the site of BDQ inhibition partially overlaps with that of the inhibitor oligomycin. Molecular dynamics simulations indicate that the binding mode of BDQ to this site is similar to that previously seen for a mycobacterial enzyme, explaining the observed lack of selectivity. We propose that derivatives of BDQ ought to be made to increase its specificity toward the mycobacterial enzyme and thereby reduce the side effects for patients that are treated with Sirturo. Luo, Zhou et al. show that Bedaquiline (BDQ, Sirturo), approved to treat multi-drug-resistant tuberculosis, inhibits the yeast and human mitochondrial ATP synthases in addition to its intended target, the Mycobacterium tuberculosis ATP synthase. The structure of the mitochondrial ATP synthase bound to BDQ suggests a means to modify this inhibitor to increase its specificity for the M. tuberculosis enzyme, thereby reducing its side effects for patients. [ABSTRACT FROM AUTHOR]

The article presents a study on high-resolution cryo-electron microscopy (cryo-EM) analysis of the yeast adenosine triphosphate (ATP) synthase in a lipid membrane. Topics include a high-resolution structure of the complete monomeric form of the mitochondrial ATP synthase which provides an understanding of the mechanism of inhibition by ogligomycin.