Subunit F modulates ATP binding and migration in the nucleotide-binding subunit B of the A1AO ATP synthase of Methanosarcina mazei Gö1

The interaction of the nucleotide-binding subunit B with subunit F is essential in coupling of ion pumping and ATP synthesis in A₁A_O ATP synthases. Here we provide structural and thermodynamic insights on the nucleotide binding to the surface of subunits B and F of Methanosarcina mazei Gö1 A₁A_O ATP synthase, which initiated migration to its final binding pocket via two transitional intermediates on the surface of subunit B. NMR- and fluorescence spectroscopy as well as ITC data combined with molecular dynamics simulations of the nucleotide bound subunit B and nucleotide bound B-F complex in explicit solvent, suggests that subunit F is critical for the migration to and eventual occupancy of the final binding site by the nucleotide of subunit B. Rotation of the C-terminus and conformational changes in subunit B are initiated upon binding with subunit F causing a perturbation that leads to the migration of ATP from the transition site 1 through an intermediate transition site 2 to the final binding site 3. This mechanism is elucidated on the basis of change in binding affinity for the nucleotide at the specific sites on subunit B upon complexation with subunit F. The change in enthalpy is further explained based on the fluctuating local environment around the binding sites.     

Three-dimensional organization of the archaeal A1-ATPase from Methanosarcina mazei Gö1

A modified isolation procedure provides a homogeneous A(1)-ATPase from the archaeon Methanosarcina mazei G?1, containing the five subunits in stoichiometric amounts of A(3):B(3):C:D:F. A(1) obtained in this way was characterized by three-dimensional electron microscopy of single particles, resulting in the first three-dimensional reconstruction of an A(1)-ATPase at a resolution of 3.2 nm. The A(1) consists of a headpiece of 10.2 nm in diameter and 10.8 nm in height, formed by the six elongated subunits A(3) and B(3). At the bottom of the A(3)B(3) complex, a stalk of 3.0 nm in length can be seen. The A(3)B(3) domain surrounds a large cavity that extends throughout the length of the A(3)B(3) barrel. A part of the stalk penetrates inside this cavity and is displaced toward an A-B-A triplet. To investigate further the topology of the stalk subunits C-F in A(1), cross-linking has been carried out by using dithiobis[sulfosuccinimidylpropionate] (DSP) and 1-ethyl-3-(dimethylaminopropyl)-carbodiimide (EDC). In experiments where DSP was added the cross-linked products B-F, A(x)-D, A-B-D, and A(x)-B(x)-D were formed. Subunits B-F, A-D, A-B-D, and A-B-C-D could be cross-linked by EDC. The subunit-subunit interaction in the presence of DSP was also studied as a function of nucleotide binding, demonstrating movements of subunits C, D, and F during ATP cleavage. Finally, the three-dimensional organization of this A(1) complex is discussed in terms of the relationship to the F(1)- and V(1)-ATPases at a resolution of 3.2 nm.     

1H, 13C, and 15N resonance assignments of subunit F of the A1AO ATP synthase from Methanosarcina mazei Gö1

Energy coupling between the A₁ ATPase of archaea type A₁A_O ATP synthase and its integral membrane sub-complex A_O occurs via the stalk part, formed by the subunits C, D and F. To provide a molecular basis of the energy coupling, we performed NMR studies. Here, we report the assignment of the subunit F. Shovanlal Gayen and Subramanian Vivekanandan contributed equally to this work.     

Defined subcomplexes of the A1 ATPase from the archaeon Methanosarcina mazei Gö1: biochemical properties and redox regulation

The potential A(1) ATPase genes ahaA, ahaB, ahaC, ahaD, ahaE, ahaF, and ahaG from the anaerobic archaeon Methanosarcina mazei G?1 were overexpressed in Escherichia coli DK8 (pTL2). An A(1) complex was purified to apparent homogeneity and shown by Western blot and N-terminal sequence analyses to contain subunits A, B, C, D, and F but to be devoid of subunits E and G. Further removal of subunit C was without effect on ATPase activity. The enzyme was most active at pH 5.2 and required bisulfite and acetate for maximal activity. Kinetic studies confirmed three new inhibitors for A(1) ATPases (diethylstilbestrol and its derivatives hexestrol and dienestrol) and identified redox modulation as a new type of regulation of archaeal A(1) ATPases.     

Structural Insights into the A1 ATPase from the archaeon, Methanosarcina mazei Gö1

The low-resolution structure and overall dimensions of the A(3)B(3)CDF complex of the A(1) ATPase from Methanosarcina mazei G?1 in solution is analyzed by synchrotron X-ray small-angle scattering. The radius of gyration and the maximum size of the complex are 5.03 +/- 0.1 and 18.0 +/- 0.1 nm, respectively. The low-resolution shape of the protein determined by two independent ab initio approaches has a knob-and-stalk-like feature. Its headpiece is approximately 9.4 nm long and 9.2 nm wide. The stalk, which is known to connect the headpiece to its membrane-bound A(O) part, is approximately 8.4 nm long. Limited tryptic digestion of the A(3)B(3)CDF complex was used to probe the topology of the smaller subunits (C-F). Trypsin was found to cleave subunit C most rapidly at three sites, Lys(20), Lys(21), and Arg(209), followed by subunit F. In the A(3)B(3)CDF complex, subunit D remained protected from proteolysis.     

Identification of genes involved in salt adaptation in the archaeon Methanosarcina mazei Gö1 using genome-wide gene expression profiling

Methanosarcina mazei is a nonhalophilic methanogen that can adapt to 800 mM NaCl. Microarray studies have been used to examine the effect of elevated salinities on the regulation of gene expression in M. mazei. Eighty-four genes of different functional categories, such as solute transport and biosynthesis, Na(+) export, stress response, ion, protein and phosphate transport, metabolic enzymes, regulatory proteins, DNA-modification systems, and cell-surface modulators, were found to be stronger expressed at high salinities. Moreover, 10 genes encoding different metabolic functions including potassium uptake and ATP synthesis were reduced in expression under high salt. The overall expression profiles suggest that M. mazei is able to adapt to high salinities by multiple upregulation of many different cellular functions including protective pathways such as solute transport and biosynthesis, import of phosphate, export of Na(+), and upregulation of pathways for modification of DNA and cell surface architecture.     

Regulation of ATP hydrolysis by the ε subunit, ζ subunit and Mg-ADP in the ATP synthase of Paracoccus denitrificans

F₁F_O-ATP synthase is a crucial metabolic enzyme that uses the proton motive force from respiration to regenerate ATP. For maximum thermodynamic efficiency ATP synthesis should be fully reversible, but the enzyme from Paracoccus denitrificans catalyzes ATP hydrolysis at far lower rates than it catalyzes ATP synthesis, an effect often attributed to its unique ζ subunit. Recently, we showed that deleting ζ increases hydrolysis only marginally, indicating that other common inhibitory mechanisms such as inhibition by the C-terminal domain of the ε subunit (ε-CTD) or Mg-ADP may be more important. Here, we created mutants lacking the ε-CTD, and double mutants lacking both the ε-CTD and ζ subunit. No substantial activation of ATP hydrolysis was observed in any of these strains. Instead, hydrolysis in even the double mutant strains could only be activated by oxyanions, the detergent lauryldimethylamine oxide, or a proton motive force, which are all considered to release Mg-ADP inhibition. Our results establish that P. denitrificans ATP synthase is regulated by a combination of the ε and ζ subunits and Mg-ADP inhibition.     

Tissue-specific differences of the mitochondrial protein import machinery: in vitro import,processing and degradation of the pre-F1β subunit of the ATP synthase in spinach leaf and root mitochondria

In this study we report the first comparison of the mitochondrial protein import and processing events in two different tissues from the same organism. Both spinach leaf and root mitochondria were able to import and process the in vitro transcribed and translated Neurospora crassa F₁ subunit of ATP synthase to the mature size product. Temperature optimum for protein import, 20 °C, was considerably lower than that found in other systems. In spinach leaf mitochondria, the processing peptidase has been shown to constitute an integral part of the bc₁ complex of the respiratory chain. In accordance with these results, the majority of the processing activity in root mitochondria was also localized in the membrane. However, although the same amount of the processing peptidase was present per mg of membrane protein in both leaf and root mitochondria, as determined immunologically, the specific processing activity was several-fold higher in roots. Furthermore, in contrast to the processing enzyme in leaf, a portion of the processing activity could be disassociated from the root membrane with relatively weak salt treatment. The processing event in both the leaf and root membranes was always accompanied by a degradation of the F₁ precursor. The degradation activity was found to be several-fold higher in roots than in leaves and was also partially dissociated from the membrane after salt treatment. Both the processing and degradation activities were inhibited by orthophenanthroline, a known metalloprotease inhibitor. These results show tissue-specific differencies of the processing event catalyzed by the bc₁ complex and indicate the presence of two populations of the processing peptidase in root mitochondria.     

Role of the mitochondrial ATP synthase central stalk subunits γ and δ in the activity and assembly of the mammalian enzyme

The central stalk of mitochondrial ATP synthase consists of subunits γ, δ, and ε, and along with the membraneous subunit c oligomer constitutes the rotor domain of the enzyme. Our previous studies showed that mutation or deficiency of ε subunit markedly decreased the content of ATP synthase, which was otherwise functionaly and structuraly normal. Interestingly, it led to accumulation of subunit c aggregates, suggesting the role of the ε subunit in assembly of individual enzyme domains. In the present study we focused on the role of subunits γ and δ. Using shRNA knockdown in human HEK293 cells, the protein levels of γ and δ were decreased to 30% and 10% of control levels, respectively. The content of the assembled ATP synthase decreased in accordance with the levels of the silenced subunits, which was also the case for most structural subunits. In contrast, the hydrophobic c subunit was increased to 130% or 180%, respectively and most of it was detected as aggregates of 150–400?kDa by 2D PAGE. In addition the IF₁ protein was upregulated to 195% and 300% of control levels. Both γ and δ subunits silenced cells displayed decreased ATP synthase function - lowered rate of ADP-stimulated respiration, a two-fold increased sensitivity of respiration to inhibitor oligomycin, and impaired utilization of mitochondrial membrane potential for ADP phosphorylation. In summary, similar phenotype of γ, δ and ε subunit deficiencies suggest uniform requirement for assembled central stalk as driver of the c-oligomer attachment in the assembly process of mammalian ATP synthase.     

The salt-induced ABC transporter Ota of the methanogenic archaeon Methanosarcina mazei Gö1 is a glycine betaine transporter

The genes encoding the three subunits of the primary ABC transporter Ota of the methanogenic archaeon Methanosarcina mazei G?1 were cloned in an expression vector (pBAD24) and transformed into the glycine betaine transport-negative mutant Escherichia coli MKH13. Ota was produced as demonstrated by Western blotting. Uptake studies revealed that Ota catalyzed the transport of glycine betaine in E. coli MKH13(pBAD-Ota) with a K(m) of 10+/-5 microM and a maximal velocity of 1.5+/-0.5 nmol min(-1) mg protein(-1). Transport was ATP dependent. Ota was activated by salinity gradients, but only marginally by sugar gradients across the membrane. Glycine betaine transport was inhibited to a small extent by an excess of dimethylglycin or proline betaine, but not by sarcosine or glycine.     

Unique mechanistic features of post-translational regulation of glutamine synthetase activity in Methanosarcina mazei strain Gö1 in response to nitrogen availability

PII-like signal transduction proteins are found in all three domains of life and have been shown to play key roles in the control of bacterial nitrogen assimilation. This communication reports the first target protein of an archaeal PII-like protein, representing a novel PII receptor. The GlnK(1) protein of the methanogenic archaeon Methanosarcina mazei strain Go1 interacts and forms stable complexes with glutamine synthetase (GlnA(1)). Complex formation with GlnK(1) in the absence of metabolites inhibits the activity of GlnA(1). On the other hand, the activity of this enzyme is directly stimulated by the effector molecule 2-oxoglutarate. Moreover, 2-oxoglutarate antagonized the inhibitory effects of GlnK(1) on GlnA(1) activity but did not prevent GlnK(1)/GlnA(1) complex formation. On the basis of these findings, we hypothesize that besides the dominant effector molecule 2-oxoglutarate, the nitrogen sensor GlnK(1) allows finetuning control of the glutamine synthetase activity under changing nitrogen availabilities and propose the following model. (i) Under nitrogen limitation, increasing concentrations of 2-oxoglutarate stimulate maximal GlnA(1) activity and transform GlnA(1) into an activated conformation, which prevents inhibition by GlnK(1). (ii) Upon a shift to nitrogen sufficiency after a period of nitrogen limitation, GlnA(1) activity is reduced by decreasing internal 2-oxoglutarate concentrations through diminished direct activation and by GlnK(1) inhibition.     

The carboxyl-terminal helical domain of the ATP synthase γ subunit is involved in ε subunit conformation and energy coupling

The γ subunit located at the center of ATP synthase (F_OF₁) plays critical roles in catalysis. Escherichia coli mutant with Pro substitution of the γ subunit residue γLeu218, which are located the rotor shaft near the c subunit ring, decreased NADH-driven ATP synthesis activity and ATP hydrolysis-dependent H⁺ transport of membranes to ~60% and ~40% of the wild type, respectively, without affecting F_OF₁ assembly. Consistently, the mutant was defective in growth by oxidative phosphorylation, indicating that energy coupling is impaired by the mutation. The ε subunit conformations in the γLeu218Pro mutant enzyme were investigated by cross-linking between cysteine residues introduced into both the ε subunit (εCys118 and εCys134, in the second helix and the hook segment, respectively) and the γ subunit (γCys99 and γCys260, located in the globular domain and the carboxyl-terminal helix, respectively). In the presence of ADP, the two γ260 and ε134 cysteine residues formed a disulfide bond in both the γLeu218Pro mutant and the wild type, indicating that the hook segment of ε subunit penetrates into the α₃β₃-ring along with the γ subunits in both enzymes. However, γ260/ε134 cross-linking in the γLeu218Pro mutant decreased significantly in the presence of ATP, whereas this effect was small in the wild type. These results suggested that the γ subunit carboxyl-terminal helix containing γLeu218 is involved in the conformation of the ε subunit hook region during ATP hydrolysis and, therefore, is required for energy coupling in F_OF₁.     

Modulation of coupling in the Escherichia coli ATP synthase by ADP and Pi: Role of the ε subunit C-terminal domain

The ε-subunit of ATP-synthase is an endogenous inhibitor of the hydrolysis activity of the complex and its α-helical C-terminal domain (εCTD) undergoes drastic changes among at least two different conformations. Even though this domain is not essential for ATP synthesis activity, there is evidence for its involvement in the coupling mechanism of the pump. Recently, it was proposed that coupling of the ATP synthase can vary as a function of ADP and P_i concentration. In the present work, we have explored the possible role of the εCTD in this ADP- and P_i-dependent coupling, by examining an εCTD-lacking mutant of Escherichia coli. We show that the loss of P_i-dependent coupling can be observed also in the εCTD-less mutant, but the effects of P_i on both proton pumping and ATP hydrolysis were much weaker in the mutant than in the wild-type. We also show that the εCTD strongly influences the binding of ADP to a very tight binding site (half-maximal effect ≈ 1 nM); binding at this site induces higher coupling in EF_OF₁ and increases responses to P_i. It is proposed that one physiological role of the εCTD is to regulate the kinetics and affinity of ADP/P_i binding, promoting ADP/P_i-dependent coupling.     

The effect of NBD-Cl in nucleotide-binding of the major subunit α and B of the motor proteins F<Subscript>1</Subscript>F<Subscript>O</Subscript> ATP synthase and A<Subscript>1</Subscript>A<Subscript>O</Subscript> ATP synthase

Subunit α of the Escherichia coli F₁F_O ATP synthase has been produced, and its low-resolution structure has been determined. The monodispersity of α allowed the studies of nucleotide-binding and inhibitory effect of 4-Chloro-7-nitrobenzofurazan (NBD-Cl) to ATP/ADP-binding. Binding constants (K _d ) of 1.6 μM of bound MgATP-ATTO-647N and 2.9 μM of MgADP-ATTO-647N have been determined from fluorescence correlation spectroscopy data. A concentration of 51 μM and 55 μM of NBD-Cl dropped the MgATP-ATTO-647N and MgADP-ATTO-647N binding capacity to 50% (IC₅₀), respectively. In contrast, no effect was observed in the presence of N,N′-dicyclohexylcarbodiimide. As subunit α is the homologue of subunit B of the A₁A_O ATP synthase, the interaction of NBD-Cl with B of the A-ATP synthase from Methanosarcina mazei Gö1 has also been shown. The data reveal a reduction of nucleotide-binding of B due to NBD-Cl, resulting in IC₅₀ values of 41 μM and 42 μM for MgATP-ATTO-647N and MgADP-ATTO-647N, respectively.