The regulatory C-terminal domain of subunit ε of F₀F₁ ATP synthase is dispensable for growth and survival of Escherichia coli

The C-terminal domain of subunit ε of the bacterial F_oF₁ ATP synthase is reported to be an intrinsic inhibitor of ATP synthesis/hydrolysis activity in vitro, preventing wasteful hydrolysis of ATP under low-energy conditions. Mutants defective in this regulatory domain exhibited no significant difference in growth rate, molar growth yield, membrane potential, or intracellular ATP concentration under a wide range of growth conditions and stressors compared to wild-type cells, suggesting this inhibitory domain is dispensable for growth and survival of Escherichia coli.F_oF₁ ATP synthases are ubiquitous enzymes that synthesize ATP using a transmembrane electrochemical potential of protons or proton motive force (PMF) generated by the respiratory chain across the cytoplasmic membrane of bacteria, the thylakoid membrane of chloroplasts, or the mitochondrial inner membrane (4, 5, 37). The enzyme consists of two parts: membrane-embedded F_o subcomplex (a complex of subunits a, b, and c in bacteria) and hydrophilic F₁ subcomplex (composed of subunits α, β, γ, δ, and ε). The enzyme is also known as a molecular motor, which is composed of the stator subcomplex (α, β, δ, a, and b) and the rotor subcomplex (γ, ε, and c), and its rotation is coupled to ATP synthesis and proton flow across the membrane (20, 31, 52). The reaction of the enzyme is reversible; ATP is hydrolyzed into ADP and inorganic phosphate, the rotor subcomplex rotates in reverse, and protons are extruded to the periplasmic side, resulting in the generation of PMF. Although some bacteria utilize the reverse reaction under particular conditions, the primary function of F_oF₁ ATP synthase is generation of ATP from the PMF. Therefore, the direction of the activity of F_oF₁ ATP synthase is regulated to avoid wasteful ATP hydrolysis.Subunit ε in bacterial F_oF₁ has been known to be an intrinsic inhibitor of F₁ and F_oF₁ complex (18, 21, 23) and is proposed to have a regulatory function (10, 11, 42). Although the inhibitory effects of subunit ε vary among species, in general, ε inhibits ATP hydrolysis activity while repressing ATP synthesis activity to a lesser degree (14, 27). This regulatory function of the ε subunit is mediated almost exclusively by the C-terminal region of ε, which is comprised of two antiparallel α-helices (18, 49, 50). Biochemical and crystallographic studies have revealed that the C-terminal helices can adopt two different conformations (34, 46, 47, 48). In the retracted conformation, the α-helices form a hairpin-like structure and sit on the N-terminal β-sandwich domain of the ε subunit. When the ε subunit exhibits an inhibitory effect, it adopts a more extended conformation in which the C-terminal α-helices extend along the γ subunit, which composes the central stalk. It has also been shown that basic, positively charged residues on the second α-helix of the ε subunit interact with negatively charged residues in the DELSEED segment of subunit β to exert the inhibitory effect (12).Escherichia coli mutants deleted in the entire ε subunit exhibit a reduced growth rate and growth yield, and this effect is proposed to be a result of a deficiency in assembly of the F_o and F₁ complexes (21). The N-terminal β-sandwich domain of the ε subunit is responsible for the assembly of F_o and F₁ and is therefore important for efficient coupling between proton translocation through F_o and ATP synthesis/hydrolysis in F₁ (15, 39). Deletion of the ε subunit leads to dissociation of the F_oF₁ complex and wasteful ATP hydrolysis by free (cytoplasmic) F₁ and dissipation of PMF through free F_o (21, 22, 51).While the importance of the entire ε subunit in the whole-cell physiology of E. coli is fairly well established, the role of the regulatory C-terminal region of ε has received little attention and warrants investigation to determine if the regulatory functions (e.g., inhibition of ATP hydrolysis) observed in vitro are manifested in the physiology of E. coli under various growth conditions. To address this question, we constructed isogenic E. coli mutants that were deleted in the C-terminal region of ε subunit (ε^DC) and used these strains to compare physiological properties of wild-type versus ε^DC cells under a wide range of environmental conditions and stressors.     

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.     

What Is the Role of ε in the Escherichia coli ATP Synthase?

The ATP synthase from Escherichia coli is a prototype of the ATP synthases that are found in many bacteria, in the mitochondria of eukaryotes, and in the chloroplasts of plants. It contains eight different types of subunits that have traditionally been divided into F₁, a water-soluble catalytic sector, and F_o, a membrane-bound ion transporting sector. In the current rotary model for ATP synthesis, the subunits can be divided into rotor and stator subunits. Several lines of evidence indicate that is one of the three rotor subunits, which rotate through 360 degrees. The three-dimensional structure of is known and its interactions with other subunits have been explored by several approaches. In light of recent work by our group and that of others, the role of in the ATP synthase from E. coli is discussed.     

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.     

The regulatory subunit ε in Escherichia coli FOF1-ATP synthase

F-type ATP synthases are extraordinary multisubunit proteins that operate as nanomotors. The Escherichia coli (E. coli) enzyme uses the proton motive force (pmf) across the bacterial plasma membrane to drive rotation of the central rotor subunits within a stator subunit complex. Through this mechanical rotation, the rotor coordinates three nucleotide binding sites that sequentially catalyze the synthesis of ATP. Moreover, the enzyme can hydrolyze ATP to turn the rotor in the opposite direction and generate pmf. The direction of net catalysis, i.e. synthesis or hydrolysis of ATP, depends on the cell's bioenergetic conditions. Different control mechanisms have been found for ATP synthases in mitochondria, chloroplasts and bacteria. This review discusses the auto-inhibitory behavior of subunit ε found in F_OF₁-ATP synthases of many bacteria. We focus on E. coli F_OF₁-ATP synthase, with insights into the regulatory mechanism of subunit ε arising from structural and biochemical studies complemented by single-molecule microscopy experiments.     

Structure of theEscherichia coli ATP synthase and role of the γ and ε subunits in coupling catalytic site and proton channeling functions

The structure of theEscherichia coli ATP synthase has been studied by electron microscopy and a model developed in which the and subunits of the F₁ part are arranged hexagonally (in top view) alternating with one another and surrounding a central cavity of around 35 Å at its widest point. The and subunits are interdigitated in side view for around 60 Å of the 90 Å length of the molecule. The F₁ narrows and has three-fold symmetry at the end furthest from the F₀ part. The F₁ is linked to F₀ by a stalk approximately 45 Å long and 25–30 Å in diameter. The F₀ part is mostly buried in the lipid bilayer. The subunit provides a domain that extends into the central cavity of the F₁ part. The and subunits are in a different conformation when ATP+Mg²⁺ are present in catalytic sites than when ATP+EDTA are present. This is consistent with these two small subunits switching conformations as a function of whether or not phosphate is bound to the enzyme at the position of the phosphate of ATP. We suggest that this switching is the key to the coupling of catalytic site events with proton translocation in the F₀ part of the complex.     

Modulation of nucleotide binding to the catalytic sites of thermophilic F1-ATPase by the ε subunit: Implication for the role of the ε subunit in ATP synthesis

Effect of ε subunit on the nucleotide binding to the catalytic sites of F₁-ATPase from the thermophilic Bacillus PS3 (TF₁) has been tested by using α₃β₃γ and α₃β₃γε complexes of TF₁ containing βTyr341 to Trp substitution. The nucleotide binding was assessed with fluorescence quenching of the introduced Trp. The presence of the ε subunit weakened ADP binding to each catalytic site, especially to the highest affinity site. This effect was also observed when GDP or IDP was used. The ratio of the affinity of the lowest to the highest nucleotide binding sites had changed two orders of magnitude by the ε subunit. The differences may relate to the energy required for the binding change in the ATP synthesis reaction and contribute to the efficient ATP synthesis.     

Effects of site-directed mutation on the function of the chloroplast ATP synthase ε subunit

The previous work in our lab showed that the spinach chloroplast ATP synthase ε mutant with 3 amino acid residues deleted from the N-terminus had much lower ability to inhibit ATP hydrolysis and block proton leakage in comparison to a mutant with 1 or 2 residues deleted from the N-terminus. The present study aimed at determining whether there is special importance in the structure and function of the N-terminal third residue of the chloroplast ε subunit. The leucine residue at the N-terminal third site (Leu3) of the spinach chloroplast ε subunit was replaced with Ile, Phe, Thr, Arg, Glu or Pro by site-directed mutagenesis, forming mutants εL3I, εL3F, εL3T, εL3R, εL3E and εL3P, respectively. These ε variants all showed lower abilities to inhibit ATP hydrolysis and to block proton leakage, as compared to the wild type ε subunit (εWT). The abilities of mutants εL3I and εL3F to restore the ATP synthesis activity of reconstituted membranes were higher than those of εWT, but the abilities of the other ε variants were lower than that of εWT. These results indicate that the hydrophobic and neutral characteristics of Leu3 of the chloroplast ε subunit are very important for its ability to inhibit ATP hydrolysis and block proton leakage, and for the ATP synthesis ability of ATP synthase.     

Stabilization of the Escherichia coli DNA polymerase III ε subunit by the θ subunit favors in vivo assembly of the Pol III catalytic core

Escherichia coli DNA polymerase III holoenzyme (HE) contains a core polymerase consisting of three subunits: α (polymerase), ε (3'-5' exonuclease), and θ. Genetic experiments suggested that θ subunit stabilizes the intrinsically labile ε subunit and, furthermore, that θ might affect the cellular amounts of Pol III core and HE. Here, we provide biochemical evidence supporting this model by analyzing the amounts of the relevant proteins. First, we show that a ΔholE strain (lacking θ subunit) displays reduced amounts of free ε. We also demonstrate the existence of a dimer of ε, which may be involved in the stabilization of the protein. Second, θ, when overexpressed, dissociates the ε dimer and significantly increases the amount of Pol III core. The stability of ε also depends on cellular chaperones, including DnaK. Here, we report that: (i) temperature shift-up of ΔdnaK strains leads to rapid depletion of ε, and (ii) overproduction of θ overcomes both the depletion of ε and the temperature sensitivity of the strain. Overall, our data suggest that ε is a critical factor in the assembly of Pol III core, and that this is role is strongly influenced by the θ subunit through its prevention of ε degradation.     

Conformational change of the α subunit of Escherichia coli F1 ATPase: ATP changes the trypsin sensitivity of the subunit

Conformational change in the α subunit of Escherichia coli proton-translocating ATPase was studied using trypsin. The subunit was cleaved with a small amount of trypsin (1 μg/mg subunit) to peptides of less than 8000 daltons. On the other hand, the subunit was cleaved to two main polypeptides (30,000 and 25,000 daltons) in the presence of sufficient ATP (1 mm-0.5 μm) to saturate the high-affinity site of the subunit. Analysis of digests of the subunit combined with fluorescent maleimide suggested that the subunit was digested in the middle of the polypeptide chain in the presence of the nucleotide. ADP and adenylyl imidodiphosphate had the same effect as ATP. These results suggest that the conformation of the subunit changed to form two trypsin-resistant domains upon binding of ATP to the high-affinity site.     

F-ATP-ase of Escherichia coli membranes: The ubiquitous MgADP-inhibited state and the inhibited state induced by the ε–subunit's C-terminal domain are mutually exclusive

ATP synthases are important energy-coupling, rotary motor enzymes in all kingdoms of life. In all F-type ATP synthases, the central rotor of the catalytic F₁ complex is composed of the γ subunit and the N-terminal domain (NTD) of the ε subunit. In the enzymes of diverse bacteria, the C-terminal domain of ε (εCTD) can undergo a dramatic conformational change to trap the enzyme in a transiently inactive state. This inhibitory mechanism is absent in the mitochondrial enzyme, so the εCTD could provide a means to selectively target ATP synthases of pathogenic bacteria for antibiotic development. For Escherichia coli and other bacterial model systems, it has been difficult to dissect the relationship between ε inhibition and a MgADP-inhibited state that is ubiquitous for F_OF₁ from bacteria and eukaryotes. A prior study with the isolated catalytic complex from E. coli, EcF₁, showed that these two modes of inhibition are mutually exclusive, but it has long been known that interactions of F₁ with the membrane-embedded F_O complex modulate inhibition by the εCTD. Here, we study membranes containing EcF_OF₁ with wild-type ε, ε lacking the full εCTD, or ε with a small deletion at the C-terminus. By using compounds with distinct activating effects on F-ATP-ase activity, we confirm that εCTD inhibition and ubiquitous MgADP inhibition are mutually exclusive for membrane-bound E. coli F-ATP-ase. We determine that most of the enzyme complexes in wild-type membranes are in the ε-inhibited state (>50%) or in the MgADP-inhibited state (30%).     

Multiple phosphorylation sites in the β subunit of thylakoid ATP synthase

Proteomic analyses of the β subunit of the plastid ATP synthase of barley (Hordeum vulgare L.) revealed that mature protein was not carboxy terminus processed and suggested the correction of the 274 codon (GAT to AAT) in the data bank that was confirmed by DNA sequencing. Six isoforms of the ATP synthase β subunit with pI ranging from 4.95 to 5.14 were resolved by two-dimensional electrophoresis (2-DE). Mass spectrometry analyses indicated that the six isoforms differ in their phosphorylation degree, which was confirmed by the disappearance of more acidic forms after incubation with the protein phosphatase calcineurin. Six Ser and/or Thr were detected as phosphorylated, among them the conserved Thr-179 that is also phosphorylated in the β subunit of human mitochondria. The results are discussed in relation with the proposed regulation of the ATP synthase by phosphorylation and 14-3-3 proteins.     

ATP synthase: Subunit–subunit interactions in the stator stalk

In ATP synthase, proton translocation through the F_o subcomplex and ATP synthesis/hydrolysis in the F₁ subcomplex are coupled by subunit rotation. The static, non-rotating portions of F₁ and F_o are attached to each other via the peripheral “stator stalk”, which has to withstand elastic strain during subunit rotation. In Escherichia coli, the stator stalk consists of subunits b₂δ; in other organisms, it has three or four different subunits. Recent advances in this area include affinity measurements between individual components of the stator stalk as well as a detailed analysis of the interaction between subunit δ (or its mitochondrial counterpart, the oligomycin-sensitivity conferring protein, OSCP) and F₁. The current status of our knowledge of the structure of the stator stalk and of the interactions between its subunits will be discussed in this review.     

ATP binding to the ϵ subunit of thermophilic ATP synthase is crucial for efficient coupling of ATPase and H+ pump activities

ATP binding to the ? subunit of F1-ATPase, a soluble subcomplex of TFoF1 (FoF1-ATPase synthase from the thermophilic Bacillus strain PS3), affects the regulation of F1-ATPase activity by stabilizing the compact, ATPase-active, form of the ? subunit [Kato, S., Yoshida, M. and Kato-Yamada, Y. (2007) J. Biol. Chem. 282, 37618-37623]. In the present study, we report how ATP binding to the ? subunit affects ATPase and H+ pumping activities in the holoenzyme TFoF1. Wild-type TFoF1 showed significant H+ pumping activity when ATP was used as the substrate. However, GTP, which bound poorly to the ? subunit, did not support efficient H+ pumping. Addition of small amounts of ATP to the GTP substrate restored coupling between GTPase and H+ pumping activities. Similar uncoupling was observed when TFoF1 contained an ATP-binding-deficient ? subunit, even with ATP as a substrate. Further analysis suggested that the compact conformation of the ? subunit induced by ATP binding was required to couple ATPase and H+ pumping activities in TFoF1 unless the ? subunit was in its extended-state conformation. The present study reveals a novel role of the ? subunit as an ATP-sensitive regulator of the coupling of ATPase and H+ pumping activities of TFoF1.     

Interaction between γC87 and γR242 residues participates in energy coupling between catalysis and proton translocation in Escherichia coli ATP synthase

Functioning as a nanomotor, ATP synthase plays a vital role in the cellular energy metabolism. Interactions at the rotor and stator interface are critical to the energy transmission in ATP synthase. From mutational studies, we found that the γC87K mutation impairs energy coupling between proton translocation and nucleotide synthesis/hydrolysis. An additional glutamine mutation at γR242 (γR242Q) can restore efficient energy coupling to the γC87K mutant. Arrhenius plots and molecular dynamics simulations suggest that an extra hydrogen bond could form between the side chains of γC87K and β_TPE381 in the γC87K mutant, thus impeding the free rotation of the rotor complex. In the enzyme with γC87K/γR242Q double mutations, the polar moiety of γR242Q side chain can form a hydrogen bond with γC87K, so that the amine group in the side chain of γC87K will not hydrogen-bond with βE381. As a conclusion, the intra-subunit interaction between positions γC87 and γR242 modulates the energy transmission in ATP synthase. This study should provide more information of residue interactions at the rotor and stator interface in order to further elucidate the energetic mechanism of ATP synthase.     

Roles of the β subunit hinge domain in ATP synthase F1 sector: Hydrophobic network formed by introduced βPhe174 inhibits subunit rotation

The ATP synthase β subunit hinge domain (βPhe148 ∼ βGly186, P-loop/α-helixB/loop/β-sheet4, Escherichia coli residue numbering) dramatically changes in conformation upon nucleotide binding. We previously reported that F₁ with the βSer174 to Phe mutation in the domain lowered the γ subunit rotation speed, and thus decreased the ATPase activity [M. Nakanishi-Matsui, S. Kashiwagi, T. Ubukata, A. Iwamoto-Kihara, Y. Wada, M. Futai, Rotational catalysis of Escherichia coli ATP synthase F₁ sector. Stochastic fluctuation and a key domain of the β subunit, J. Biol. Chem. 282 (2007) 20698-20704.]. Homology modeling indicates that the amino acid replacement induces a hydrophobic network, in which the βMet159, βIle163, and βAla167 residues of the β subunit are involved together with the mutant βPhe174. The network is expected to stabilize the conformation of β_DP (nucleotide-bound form of the β subunit), resulting in increased activation energy for transition to β_E (empty β subunit). The modeling further predicts that replacement of βMet159 with Ala or Ile weakens the hydrophobic network. As expected, these two mutations experimentally suppressed the ATPase activities as well as subunit rotation of βS174F. Furthermore, the rotation rate decreased with the increase of the strength in the hydrophobic network. These results indicate that the smooth conformational change of the β subunit hinge domain is pertinent for the rotational catalysis.     

The ∈ subunit of the chloroplast ATP synthase of Pisum sativum

In contrast to the well-characterized spinach ( Spinacea oleracea) chloroplast ATP synthase (CF1–CFo), the properties of the chloroplast ATP synthase from pea (Pisum sativum ) have not been as intensively studied. Preliminary data suggested that the regulatory properties of the two enzymes differ. In the absence of activating treatments the ATPase activity of pea thylakoids in the dark was higher than that in spinach thylakoids. When assayed in the presence of sulfite, the MgATPase activity of pea thylakoids was inhibited to a maximum of 67% by tentoxin, indicating that the dark ATPase activity is in part catalyzed by CF1–CFo. The ATPase activity of purified pea CF1 was also higher than that of spinach CF1 in the absence of activating treatments. These differences could result from the different regulatory properties of the pea or subunit or both. The pea subunit was less effective in binding to or inhibiting the ATPase activity of pea o r spinach CF1 deficient in (CF1-). Spinach inhibited the ATPase activity of pea CF1- at lower concentrations than pea . The gene encoding the pea subunit was cloned and over-expressed. Recombinant pea did not restore low proton permeability to spinach thylakoid membranes reconstitituted with spinach CF1-, although pea was effective when tested with pea thylakoids reconstitituted with pea CF1-. These results confirm earlier suggestions that the C-terminal region of is important in -CF1 and -CFo interactions.