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At least 19 recordsLinked to original sources
PubMed2026

Genomic and structural insights into the atpB L173I substitution: modulation of the F₀ rotor architecture in Mycobacterium tuberculosis ATP synthase and altered Bedaquiline binding dynamics.

The F₀F₁ ATP synthase of Mycobacterium tuberculosis (M. tuberculosis) is an essential membrane-embedded rotary motor responsible for ATP synthesis and maintenance of the proton motive force in bacteria. The transmembrane F₀ domain comprises the c-subunit (atpE) and the a-subunit (atpB). Their coordinated interactions are needed for proton translocation and torque generation. Bedaquiline (BDQ), FDA-approved diarylquinoline for the treatment of multidrug-resistant tuberculosis (MDR-TB), targets the F₀ motor by binding at the a-c interface and inhibiting rotary catalysis. To the best of our knowledge, this study represents the first attempt to analyze the effects of mutations in the atpB protein on its structural stability in the F₀ domain, thereby highlighting the novelty of this work. In this study, we integrated Indian whole-genome sequencing (WGS) datasets (PRJNA37907) with long-timescale (1000 ns) membrane-embedded molecular dynamics (MD) simulations. Among 57 atpB mutations identified from WGS analysis, L173I was selected for structural and MD analysis. L173I is located at the atpB-atpE interface near the BDQ-binding region, despite V177L and S184A showing higher prevalence. Comparative MD simulations encompassed four systems: wild-type apo, wild-type with BDQ, L173I apo, and L173I with BDQ. Structural interrogation revealed that the L173I substitution induces subtle destabilization of the global fold of the atpB-atpE complex relative to the apo state, while more critically attenuating inter-subunit contacts between the a-subunit and the c-ring. These perturbations provide a mechanistic rationale for reduced BDQ susceptibility, arising from altered interfacial dynamics rather than complete abrogation of drug binding. This integrative genomic-structural framework advances our understanding of ATP synthase-mediated resistance in M. tuberculosis.

Diarylquinolines↗
PubMed2025

Dominant negative ATP5F1A variants disrupt oxidative phosphorylation causing neurological disorders.

ATP5F1A encodes the α-subunit of complex V of the respiratory chain, which is responsible for mitochondrial ATP synthesis. We describe 6 probands with heterozygous de novo missense ATP5F1A variants that presented with developmental delay, intellectual disability, and movement disorders. All variants were located at the contact points between the α- and β-subunits. Functional studies in C. elegans revealed that the variants were damaging via a dominant negative genetic mechanism. Biochemical and proteomics studies of proband-derived cells showed a marked reduction in complex V abundance and activity. Mitochondrial physiology studies revealed increased oxygen consumption, yet decreased mitochondrial membrane potential and ATP levels indicative of uncoupled oxidative phosphorylation as a pathophysiologic mechanism. Our findings contrast with the previously reported ATP5F1A variant, p.Arg207His, indicating a different pathological mechanism. This study expands the phenotypic and genotypic spectrum of ATP5F1A-associated conditions and highlights how functional studies can provide an understanding of the genetic, molecular, and cellular mechanisms of ATP5F1A variants of uncertain significance. With 12 heterozygous individuals now reported, ATP5F1A is the most frequent nuclear genome cause of complex V deficiency.

Humans↗
PubMed1992

Surface presentation of Shigella flexneri invasion plasmid antigens requires the products of the spa locus.

An avirulent, invasion plasmid insertion mutant of Shigella flexneri 5 (pHS1059) was restored to the virulence phenotype by transformation with a partial HindIII library of the wild-type invasion plasmid constructed in pBR322. Western immunoblot analysis of pHS1059 whole-cell lysates revealed that the synthesis of the invasion plasmid antigens VirG, IpaA, IpaB, IpaC, and IpaD was similar to that seen in the corresponding isogenic S. flexneri 5 virulent strain, M90T. IpaB and IpaC, however, were not present on the surface of pHS1059 as was found in M90T, suggesting that the transport or presentation of the IpaB and IpaC proteins onto the bacterial surface was defective in the mutant. pHS1059 was complemented by pWR266, which carried contiguous 1.2- and 4.1-kb HindIII fragments of the invasion plasmid. pHS1059(pWR266) cells were positive in the HeLa cell invasion assay as well as colony immunoblot and enzyme-linked immunosorbent assays, using monoclonal antibodies to IpaB and IpaC. These studies established that the antigens were expressed on the surface of the transformed bacteria. In addition, water extraction of pHS1059 and pHS1059(pWR266) whole cells, which can be used to remove IpaB and IpaC antigens from the surface of wild-type M90T bacteria, yielded significant amounts of these antigens from pHS1059(pWR266) but not from pHS1059. Minicell and DNA sequence analysis indicated that several proteins were encoded by pWR266, comprising the spa loci, which were mapped to a region approximately 18 kb upstream of the ipaBCDAR gene cluster. Subcloning and deletion analysis revealed that more than one protein was involved in complementing the Spa- phenotype in pHS1059. One of these proteins, Spa47, showed striking homology to ORF4 of the Bacillus subtilis flaA locus and the fliI gene sequence of Salmonella typhimurium, both of which bear strong resemblance to the alpha and beta subunits of bacterial, mitochondrial, and chloroplast proton-translocating F0F1 ATPases.

Adenosine Triphosphate↗
PubMed1975

Stimulation of active uptake of nucleosides and amino acids by cyclic adenosine 3' :5'-monophosphate in the yeast Schizosaccharomyces pombe.

In conditions of glucose starvation, the maximum velocity of the mediated transport of nonmetabolized and metabolized amino acids, uridine, adenosine, and sucrose across the plasma membrane is stimulated by a factor of two by the addition of 1 mM adenosine 3':5'-monophosphate to Schizosaccharomyces pombe 972h- wild strain, to the glucose-super-repressed and derepressed mutants COB5 and COB6, and to Saccharomyces cerevisiae strain IL 216-IA. The mediated uptake of 2-D-deoxyglucose and the apparently nonmediated uptake of guanosine are not stimulated by the cyclic nucleotide. N6,O2'-Dibutyryl adenosine 3':5'-monophosphate is also efficient, whereas theophylline, guanosine 3':5'-monophosphate, 5'-AMP, ATP, and adenosine are ineffective. The cellular ATP content of glycerol-grown S. pombe COB5 is about 10 nmol per mg of protein and is not decreased by further incubation in the starvation medium. The addition of 100 mM glucose markedly enhances transport without any increase of the cellular ATP content. The addition of antimycin A or Dio-9 decreases markedly both cellular ATP content and transport. The addition of 2.5 mM glucose to antimycin A-containing medium restores both transport is not necessarily of mitochondrial origin. The uptake of 2-D-deoxyglucose is unaffected by the respiratory inhibitors. Stimulation of uptake by cyclic adenosine 3':5'-monophosphate occurs only in glucose-deprived cells. The addition of 10 mM glucose elicits the disappearance of the stimulation and prevents the 30% decrease of the cellular adenosine 3':5'-monophosphate content produced by glucose starvation. Adenosine 3':5'-'monophosphate does not enhance the steady state ATP level but requires cellular ATP produced either by endogenous respiration or, in the absence of respiration blocked by antimycin A, by further addition of 2.5 mM glucose. Stimulation of active uptake by adenosine 3':5'-monophosphate does not require protein synthesis because the addition of cycloheximide or anisomycin does not prevent the stimulation of L-leucine uptake. In the absence of respiration, Dio-9, and ATPase inhibitor, suppresses instantaneously the cellular ejection of protons as well as the uptake of uridine and amino acids. It abolishes also the adenosine 3':5'-monophosphate-stimulated transport. In the presence of antimycin A, specific mitochondrial ATPase inhibitors such as venruricidin A do not inhibit metabolite uptakes and their stimulation by adenosine 3':5'-monophosphate. These results suggest that in these conditions, the target of Dio-9 is not the mitochondrial ATPase but a plasma membrane proton-translocating function generating an electrochemical gradient required for active transport. That adenosine 3':5'-monophosphate enhances the Dio-9-sensitive proton extrusion supports the view that the cyclic nucleotide might modulate the plasma membrane ATPase.

Adenosine Diphosphate↗
PubMed1992

Mitochondrial ATP-synthase deficiency in a child with 3-methylglutaconic aciduria.

We report the finding of mitochondrial ATP-synthase deficiency in a child with persistent 3-methylglutaconic aciduria. The child presented in the neonatal period with severe lactic acidosis, which was controlled by Na-HCO3 and glucose infusions. During the 1st y of life, there were several episodes of lactic acidosis precipitated by infections or prolonged intervals between meals. The excretion of lactate in urine was variable, but there was a persistent high excretion of 3-methylglutaconic acid. The activity of 3-methylglutaconyl-CoA hydratase in fibroblasts was normal. The child had a hypertrophic cardiomyopathy and magnetic resonance images revealed hypoplasia of corpus callosum. The gross motor and mental development was retarded, but there were no other neurologic signs. Investigation of muscle mitochondrial function at 1 y of age revealed a severe mitochondrial ATP-synthase deficiency (oligomycin-sensitive, dinitrophenol-stimulated Mg2+ ATPase activity: 27 nmol x min-1 x (mg protein)-1, control range 223-673 nmol x min-1 x (mg protein)-1. The mitochondrial respiratory rate was low and tightly coupled. The respiratory rate was normalized by the addition of an uncoupler. Low Mg2+ ATPase activity was also demonstrated by histochemical methods. Morphologic examination revealed ultrastructural abnormalities of mitochondria. There was no deletion of mitochondrial DNA. The sequences of the ATP synthase subunit genes of mitochondrial DNA were in accordance with published normal sequences.

Acidosis, Lactic↗
PubMed1992

Structure/function analysis of yeast mitochondrial ATP synthase subunit 8.

Subunit 8 of yeast mitochondrial ATP synthase is a small hydrophobic component of the membrane-associated F0 sector. Structure/function relations in subunit 8 were studied by focusing on three structural domains: a highly conserved NH2-terminal region, a central hydrophobic region (previously suggested to be a transmembrane stem), and a COOH-terminal region bearing a conserved array of three positively charged residues. A combined approach was used, which encompasses site-directed mutagenesis, in vitro import and assembly tests, and an in vivo allotopic expression system (using host cells unable to synthesise subunit 8 in mitochondria). The results indicate that the NH2-terminal region of subunit 8 is involved functionally in the F0 sector. As the central hydrophobic region can functionally tolerate the introduction of multiple, positively charged residues (which abolishes the proteolipid solubility characteristics of the entire subunit), the role of this hydrophobic region as a transmembrane stem is brought into question. Each of the three positively charged residues toward the COOH-terminus of subunit 8 is required for the efficient assembly of this subunit into the F0 sector. Removal of the more proximal charged residues Arg37 or Arg42 has a more severe impact on subunit 8 assembly than does removal of the most distal residue Lys47 in terms of both in vitro import and assembly as well as the ability of the subunit 8 variant to function in mitochondrial ATP synthase in vivo.

Amino Acid Sequence↗
PubMed1992

The effect of lysophosphatidylcholine on the activity of various mitochondrial enzymes.

The influence of lysophosphatidylcholine (LPC) on H(+)-ATPase, cytochrome oxidase (COX), glycerolphosphate dehydrogenase (GPDH) and malate dehydrogenase (MDH) was followed. The activities of H(+)-ATPase and COX increased with increasing LPC concentration up to 0.5 mg/mg protein when maxima were achieved. This activatory effect is LPC-specific, because Lubrol-treated or frozen-thawed mitochondria showed lower activities of these enzymes. H(+)-ATPase was not influenced by higher concentration of LPC, while COX activity decreased with increasing amount of LPC. The activity of GPDH decreased at very low concentration of LPC and was not further modified at higher LPC concentration. In an attempt to find the concentration of LPC necessary for a complete permeabilization of inner mitochondrial membrane we followed the influence of lysolipid on the release of MDH activity from the mitochondrial matrix. The full activity of this enzyme was obtained with a concentration 0.75 mg LPC/mg protein indicating that mitochondria were completely broken. Our data indicate that LPC significantly affects activity of enzymes connected with mitochondrial membrane and can be useful for evaluation of the importance of phospholipid microenvironment for the enzyme function.

Adipose Tissue↗
PubMed1992

Study of the lipid-protein interaction of F ATPases.

Mg2+ may play a role in altering the lipid fluidity of the bilayers which would induce a change in conformation of the F0 portion of the H(+)-ATPase complex. This change could be transmitted to the soluble F1 portion, the conformation of which is in turn altered, resulting in higher enzymic activity. In addition to mitochondrial H(+)-ATPase, similar Mg2+ effects on the reconstitution of chloroplast H(+)-ATPase and other intrinsic membrane proteins have also been observed in our laboratory.

Animals↗
PubMed1978

Adenosine triphosphate in the bovine chromaffin granule.

1. pH and potential gradients are generated across the membranes of chromaffin granule 'ghost' by incubating them with MgATP: the inside of the 'ghosts' is positive and acid with respect to the incubation medium. 2. The pH gradient is partially dissipated by inclusion of a substrate for the catecholamine pump, or a mitochondrial uncoupling agent, but is enhanced by reserpine. 3. An imposed pH gradient leads to amine uptake by the 'ghosts': a potential gradient leads to ATP uptake. Studies with inhibitors confirm that amine accumulation by chromaffin granules is dependent on the former, and that ATP uptake results from ATPase-induced potential difference generation. 4. ATP has two known roles in chromaffin granule structure: the first is as a substrate for a membrane-bound proton-translocating ATPase; the second is as a component of the intragranular catecholamine storage complex.

Adenosine Triphosphate↗
PubMed1992

Identification of mitochondrial proteins in membrane preparations from Chlamydomonas reinhardtii.

Preparations enriched in Chlamydomonas reinhardtii thylakoids have proven useful in the study of photosynthesis. Many of their polypeptides however remain unidentified. We report here on three of those, h1 (34 kDa), h2 (11 kDa), and P3 (63 kDa). h1, h2, and P3 are present in all tested mutants of C. reinhardtii lacking either one or several of the photosynthetic chain complexes or depleted in thylakoid membranes. h2 is an ascorbate-reducible, soluble c550-type cytochrome encoded in the nucleus. It cross-reacts immunologically with mitochondrial cytochromes c from various sources and contains a hexapeptide encoded in C. reinhardtii cytochrome c cDNA. P3, a nuclear-encoded peripheral protein, cross-reacts with various ATP synthase beta subunits. Its N-terminal sequence is encoded in C. reinhardtii mitochondrial beta subunit cDNA. h1 behaves as an integral hemoprotein; it is absent in a mitochondrial mutant that carries a deletion in apocytochrome b gene. We conclude that C. reinhardtii mitochondrial membranes copurify with thylakoid membranes. h1 is part of the cytochrome bc1 complex, h2 is cytochrome c, and P3 is the beta subunit of mitochondrial ATP synthase.

Amino Acid Sequence↗
PubMed1992

The alpha 3 beta 3 and alpha 1 beta 1 complexes of ATP synthase.

Two catalytic structures of H(+)-motive ATP synthase (Fig. 1), the alpha 3 beta 3 oligomer (M(r) = 319,581) and alpha 1 beta 1 promoter (M(r) = 106,527) (Fig. 2), were isolated using high pressure liquid chromatography (Fig. 3) and polyacrylamide gel electrophoresis (Figs. 4 and 5). These were reconstituted from the alpha and beta subunits of thermophilic F1 (TF1), and the alpha 3 beta 3 oligomer was also crystallized. Common to both F1 and the alpha 3 beta 3 oligomer were the nucleotide specificity, the two Km values, the presence of protomer-oligomer activities, and the one-hit--one-kill phenomenon. A synchrotron experiment on the ATP hydrolysis cycle revealed the dynamic shrinkage and expansion of F1(44) that correspond, respectively, to the ATP-induced association and ADP-induced dissociation of the alpha 3 beta 3 oligomer. The oligomer, like mitochondrial F1 and TF1, exhibited two kinds of ATPase activity: one was cooperative and was inhibited by only one inhibitor per hexamer, and the other was inhibited by three inhibitors per hexamer.

Adenosine Triphosphate↗
PubMed1979

Mitochondrial ATPase.

Considerable progress has been made in recent years in our understanding of the phosphorylating apparatus in mitochondria, chloroplasts, and bacteria. It has become clear that the structure and the function of the ATP synthesizing apparatus in these widely divergent organisms is similar if not virtually identical. The subunit composition of F1, its molecular architecture, the location and function of substrate binding sites, as well as putative control sites, understanding of the component parts of the oligomycin-sensitive ATPase complex, and the role of these components in the function of the complex all are under active investigation in many laboratories. The developing information and the new insights provided have begun to permit experimental approaches, at the molecular level, to the mode of action of the ATPase in electron-transport-coupled ATP synthesis.

Adenosine Triphosphatases↗
PubMed1992

Mitochondrial ATP synthase regulation in heart: defects in hypertension are restored after treatment with captopril.

Control of mitochondrial ATP synthase capacity was investigated in cultured cardiomyocytes from normotensive (Wistar-Kyoto) and spontaneously hypertensive rats. Cells from spontaneously hypertensive rats have a higher basal ATP synthase capacity than those from normotensives, but lack the normal up-regulation in response to an increased energy demand. After treatment of spontaneously hypertensive rats with captopril (60 mg/kg per day for 12 weeks), cellular hypertrophy characteristic of the hypertensives was abolished and the cardiomyocytes showed a normal ATP synthase capacity. Normal up-regulation of this enzyme was also restored. All cells showed a normal down-regulation of the synthase in response to cyanide. Experiments with the calcium antagonists, verapamil and ruthenium red, suggest that abnormal ATP synthase regulation observed in the untreated spontaneously hypertensive rats results from an alteration of Ca2+ handling in cardiac cells under chronic high workload, which is reversed by captopril treatment.

Animals↗
PubMed1992

Does pyrophosphate bind to the catalytic sites of mitochondrial F1-ATPase?

The interactions between the pyrophosphate (PPi) binding sites and the nucleotide binding sites on mitochondrial F1-ATPase have been investigated, using F1 preparations containing different numbers of catalytic and noncatalytic nucleotide-binding sites occupied by ligands. In all cases, the total number of moles of bound nucleotides and PPi per mole of F1 was less than or equal to six. F1 preparations containing either three or two filled noncatalytic sites and no filled catalytic sites (referred as F1[3,0] and F1[2,0]) were found to bind 3 mol of PPi/mol of F1. Tight binding of ADP-fluoroberyllate complexes to two of the catalytic sites of F1 converted the three heterogeneous PPi-binding sites into three homogeneous binding sites, each exhibiting the same affinity for PPi. The addition of PPi at saturating concentrations to F1 containing GDP bound to two catalytic sites (F1[2,2]) resulted in the release of 1 mol of GDP. Furthermore, the addition of PPi to F1 filled with ADP-fluoroberyllate at the catalytic sites resulted in the release of 1 mol of tightly bound ADP/mol of F1. Taken together, these results indicate that PPi binds to specific sites that interact with both the catalytic and the noncatalytic nucleotide-binding sites of F1.

Adenosine Diphosphate↗