Insights into the regulatory function of the ɛ subunit from bacterial F-type ATP synthases: a comparison of structural, biochemical and biophysical data (original) (raw)
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Journal of Biological Chemistry, 2013
Background: Bacterial ATP synthases are autoinhibited by the subunit ⑀ C-terminal domain. Results: Nucleotide hydrolysis is required to form the ⑀-inhibited state, which also responds dynamically to different ligand conditions. Conclusion: ⑀ inhibition initiates at the catalytic dwell angle, but reversible rotation over ϳ40°is probably involved in nucleotide effects on the inhibitory state of ⑀. Significance: ⑀ inhibition may provide a new target for antimicrobial discovery. F 1-ATPase is the catalytic complex of rotary nanomotor ATP synthases. Bacterial ATP synthases can be autoinhibited by the C-terminal domain of subunit ⑀, which partially inserts into the enzyme's central rotor cavity to block functional subunit rotation. Using a kinetic, optical assay of F 1 ⅐⑀ binding and dissociation, we show that formation of the extended, inhibitory conformation of ⑀ (⑀ X) initiates after ATP hydrolysis at the catalytic dwell step. Prehydrolysis conditions prevent formation of the ⑀ X state, and post-hydrolysis conditions stabilize it. We also show that ⑀ inhibition and ADP inhibition are distinct, competing processes that can follow the catalytic dwell. We show that the N-terminal domain of ⑀ is responsible for initial binding to F 1 and provides most of the binding energy. Without the C-terminal domain, partial inhibition by the ⑀ N-terminal domain is due to enhanced ADP inhibition. The rapid effects of catalytic site ligands on conformational changes of F 1-bound ⑀ suggest dynamic conformational and rotational mobility in F 1 that is paused near the catalytic dwell position. ATP synthases play a key role in energy metabolism in most living organisms and achieve energy coupling as dual engine rotary nanomotors (1-3). The F-type ATP synthase of Escherichia coli (Fig. 1), a bacterial prototype, is composed of core subunits that all have homologs in the ATP synthases of mitochondria and chloroplasts (4). The membrane-embedded F O complex (ab 2 c 10) acts like a turbine to transport protons across the membrane, and the external F 1 complex (␣ 3  3 ␥␦⑀) contains three cooperative catalytic sites for ATP synthesis or hydrolysis. The ring of c-subunits, with the critical proton transport sites, is the rotor complex of F O and connects to the central rotor stalk of F 1 , composed of ␥ and the N-terminal domain (NTD) 2 of ⑀. The three catalytic  subunits alternate with three ␣ subunits to surround the upper half of the asymmetric rotor stalk of ␥, and the ␦-b 2 connection forms a peripheral stator stalk anchoring ␣ 3  3 to the other stator subunit of F O , a. In vitro, F 1 from eukaryotes and bacteria can be dissociated from F O as a soluble, rotary motor ATPase, and these F 1-ATPases have been useful for both mechanistic studies and the determination of high resolution structures. Despite general conservation between bacterial and mitochondrial ATP synthases, it has been demonstrated that bacterial ATP synthase can be an effective target for antibacterial treatment. It is the target of a novel class of compounds that are bactericidal for actively replicating and dormant mycobacteria (5, 6) and that show promising effects against multidrug-resistant tuberculosis in phase II clinical trials (7). However, the lead compound is only effective against a narrow spectrum of mycobacteria, and, because it targets the H ϩ-transporting sites of F O , adapting this scaffold to target other pathogenic bacteria introduces a significant risk of cross-reaction with mitochondrial ATP synthase. Recently, our group determined the first crystal structure of a bacterial F 1-ATPase that is in an autoinhibited state mediated by the C-terminal domain (CTD) of its ⑀ subunit (8). Inhibition by ⑀ may serve regulatory roles in ATP synthases of bacteria (2, 9) and chloroplasts (10) but does not occur in mitochondrial ATP synthase, which has a distinct inhibitor protein (11). Recent studies confirmed that the bacterial ⑀CTD inhibits ATP synthesis as well as hydrolysis (12, 13), indicating that ⑀ inhibition may provide a new target for future development of antimicrobial drugs selective for bacteria. With that in mind, the current study focuses on improving our biochemical understanding of how the catalytic F 1 complex of E. coli ATP synthase is inhibited by ⑀.
Background: Bacterial ATP synthases are autoinhibited by the subunit ⑀ C-terminal domain. Results: Nucleotide hydrolysis is required to form the ⑀-inhibited state, which also responds dynamically to different ligand conditions. Conclusion: ⑀ inhibition initiates at the catalytic dwell angle, but reversible rotation over ϳ40°is probably involved in nucleotide effects on the inhibitory state of ⑀. Significance: ⑀ inhibition may provide a new target for antimicrobial discovery. F 1-ATPase is the catalytic complex of rotary nanomotor ATP synthases. Bacterial ATP synthases can be autoinhibited by the C-terminal domain of subunit ⑀, which partially inserts into the enzyme's central rotor cavity to block functional subunit rotation. Using a kinetic, optical assay of F 1 ⅐⑀ binding and dissociation, we show that formation of the extended, inhibitory conformation of ⑀ (⑀ X) initiates after ATP hydrolysis at the catalytic dwell step. Prehydrolysis conditions prevent formation of the ⑀ X state, and post-hydrolysis conditions stabilize it. We also show that ⑀ inhibition and ADP inhibition are distinct, competing processes that can follow the catalytic dwell. We show that the N-terminal domain of ⑀ is responsible for initial binding to F 1 and provides most of the binding energy. Without the C-terminal domain, partial inhibition by the ⑀ N-terminal domain is due to enhanced ADP inhibition. The rapid effects of catalytic site ligands on conformational changes of F 1-bound ⑀ suggest dynamic conformational and rotational mobility in F 1 that is paused near the catalytic dwell position. ATP synthases play a key role in energy metabolism in most living organisms and achieve energy coupling as dual engine rotary nanomotors (1-3). The F-type ATP synthase of Escherichia coli (Fig. 1), a bacterial prototype, is composed of core subunits that all have homologs in the ATP synthases of mitochondria and chloroplasts (4). The membrane-embedded F O complex (ab 2 c 10) acts like a turbine to transport protons across the membrane, and the external F 1 complex (␣ 3  3 ␥␦⑀) contains three cooperative catalytic sites for ATP synthesis or hydrolysis. The ring of c-subunits, with the critical proton transport sites, is the rotor complex of F O and connects to the central rotor stalk of F 1 , composed of ␥ and the N-terminal domain (NTD) 2 of ⑀. The three catalytic  subunits alternate with three ␣ subunits to surround the upper half of the asymmetric rotor stalk of ␥, and the ␦-b 2 connection forms a peripheral stator stalk anchoring ␣ 3  3 to the other stator subunit of F O , a. In vitro, F 1 from eukaryotes and bacteria can be dissociated from F O as a soluble, rotary motor ATPase, and these F 1-ATPases have been useful for both mechanistic studies and the determination of high resolution structures. Despite general conservation between bacterial and mitochondrial ATP synthases, it has been demonstrated that bacterial ATP synthase can be an effective target for antibacterial treatment. It is the target of a novel class of compounds that are bactericidal for actively replicating and dormant mycobacteria (5, 6) and that show promising effects against multidrug-resistant tuberculosis in phase II clinical trials (7). However, the lead compound is only effective against a narrow spectrum of mycobacteria, and, because it targets the H ϩ-transporting sites of F O , adapting this scaffold to target other pathogenic bacteria introduces a significant risk of cross-reaction with mitochondrial ATP synthase. Recently, our group determined the first crystal structure of a bacterial F 1-ATPase that is in an autoinhibited state mediated by the C-terminal domain (CTD) of its ⑀ subunit (8). Inhibition by ⑀ may serve regulatory roles in ATP synthases of bacteria (2, 9) and chloroplasts (10) but does not occur in mitochondrial ATP synthase, which has a distinct inhibitor protein (11). Recent studies confirmed that the bacterial ⑀CTD inhibits ATP synthesis as well as hydrolysis (12, 13), indicating that ⑀ inhibition may provide a new target for future development of antimicrobial drugs selective for bacteria. With that in mind, the current study focuses on improving our biochemical understanding of how the catalytic F 1 complex of E. coli ATP synthase is inhibited by ⑀.
Frontiers in Molecular Biosciences, 2023
It is a conjecture that the ε subunit regulates ATP hydrolytic function of the F 1 F o ATP synthase in bacteria. This has been proposed by the ε subunit taking an extended conformation, with a terminal helix probing into the central architecture of the hexameric catalytic domain, preventing ATP hydrolysis. The ε subunit takes a contracted conformation when bound to ATP, thus would not interfere with catalysis. A recent crystallographic study has disputed this; the Caldalkalibacillus thermarum TA2.A1 F 1 F o ATP synthase cannot natively hydrolyse ATP, yet studies have demonstrated that the loss of the ε subunit terminal helix results in an ATP synthase capable of ATP hydrolysis, supporting ε subunit function. Analysis of sequence and crystallographic data of the C. thermarum F 1 F o ATP synthase revealed two unique histidine residues. Molecular dynamics simulations suggested that the protonation state of these residues may influence ATP binding site stability. Yet these residues lie outside the ATP/Mg 2+ binding site of the ε subunit. We then probed the effect of pH on the ATP binding affinity of the ε subunit from the C. thermarum F 1 F o ATP synthase at various physiologically relevant pH values. We show that binding affinity changes 5.9 fold between pH 7.0, where binding is weakest, to pH 8.5 where it is strongest. Since the C. thermarum cytoplasm is pH 8.0 when it grows optimally, this correlates to the ε subunit being down due to ATP/Mg 2+ affinity, and not being involved in blocking ATP hydrolysis. Here, we have experimentally correlated that the pH of the bacterial cytoplasm is of critical importance for ε subunit ATP affinity regulated by secondshell residues thus the function of the ε subunit changes with growth conditions.
Escherichia coli ATP synthase (F-ATPase): catalytic site and regulation of H+ translocation
The Journal of experimental biology, 1992
We discuss our recent results on the Escherichia coli F-ATPase, in particular its catalytic site in the beta subunit and regulation of H+ transport by the gamma subunit. Affinity labelling experiments suggest that beta Lys-155 in the glycine-rich sequence is near the gamma-phosphate moiety of ATP bound at the catalytic site. The enzyme loses activity upon introduction of missense mutations in beta Lys-155 or beta Thr-156 and changes catalytic properties upon introduction of other mutations. By analysis of mutations and their pseudo revertants, residues beta Ser-174, beta Glu-192 and beta Val-198 were found to be located near the glycine-rich sequence. The combined approaches of chemical labelling and genetics have been fruitful in visualizing the structure of the catalytic site. Analysis of mutations in the gamma subunit suggests that this subunit has an essential role in coupling catalysis with proton translocation.
Structure of the Cytosolic Part of the Subunit b-Dimer of Escherichia coli F0F1-ATP Synthase
Biophysical Journal, 2008
The structure of the external stalk and its function in the catalytic mechanism of the F 0 F 1 -ATP synthase remains one of the important questions in bioenergetics. The external stalk has been proposed to be either a rigid stator that binds F 1 or an elastic structural element that transmits energy from the small rotational steps of subunits c to the F 1 sector during catalysis. We employed proteomics, sequence-based structure prediction, molecular modeling, and electron spin resonance spectroscopy using site-directed spin labeling to understand the structure and interfacial packing of the Escherichia coli b -subunit homodimer external stalk. Comparisons of bacterial, cyanobacterial, and plant b-subunits demonstrated little sequence similarity. Supersecondary structure predictions, however, show that all compared b-sequences have extensive heptad repeats, suggesting that the proteins all are capable of packing as left-handed coiled-coils. Molecular modeling subsequently indicated that b 2 from the E. coli ATP synthase could pack into stable left-handed coiled-coils. Thirty-eight substitutions to cysteine in soluble b-constructs allowed the introduction of spin labels and the determination of intersubunit distances by ESR. These distances correlated well with molecular modeling results and strongly suggest that the E. coli subunit b-dimer can stably exist as a left-handed coiled-coil.
Journal of Bacteriology, 2009
Subunit b, the peripheral stalk of bacterial F 1 F o ATP synthases, is composed of a membrane-spanning and a soluble part. The soluble part is divided into tether, dimerization, and ␦-binding domains. The first solution structure of b30-82, including the tether region and part of the dimerization domain, has been solved by nuclear magnetic resonance, revealing an ␣-helix between residues 39 and 72. In the solution structure, b30-82 has a length of 48.07 Å. The surface charge distribution of b30-82 shows one side with a hydrophobic surface pattern, formed by alanine residues. Alanine residues 61, 68, 70, and 72 were replaced by single cysteines in the soluble part of subunit b, b22-156. The cysteines at positions 61, 68, and 72 showed disulfide formation. In contrast, no cross-link could be formed for the A70C mutant. The patterns of disulfide bonding, together with the circular dichroism spectroscopy data, are indicative of an adjacent arrangement of residues 61, 68, and 72 in both ␣-helices in b22-156. ATP synthesis by oxidative phosphorylation or photophosphorylation is a multistep membrane-located process that provides the bulk of cellular energy in eukaryotes and many prokaryotes. The majority of ATP synthesis is accomplished by the enzyme ATP synthase (EC 3.6.1.34), also called F 1 F o ATP synthase, which, in its simplest form, as in bacteria, is composed of eight different subunits (␣ 3 ,  3 , ␥, ␦, ε, a, b 2 , and c 9-12). This multisubunit complex is divided into the F 1 headpiece, ␣ 3 : 3 , and a membrane-embedded ion-translocating part known as F o , to which F 1 is attached by a central and a peripheral stalk (1, 5, 25). ATP is synthesized or hydrolyzed on the ␣ 3 : 3 hexamer, and the energy provided for or released during that process is transmitted to the membrane-bound F o sector, consisting of subunits a and c and part of subunit b (30, 31). The energy coupling between the two active domains occurs via the stalk part(s) (6). The central stalk is made of subunits ␥ and ε, and the peripheral stalk is formed by subunits ␦ and b. The peripheral stalk, which lies at the edge of the multisubunit assembly of the F 1 F o ATP synthase, acts as a stator to counter the tendency of the ␣ 3 : 3 hexamer to follow the rotation of the central stalk and the attached c-ring, and to anchor the membrane-embedded a subunit (17, 36). In Escherichia coli, subunit b with its 156 residues extends with its soluble part (b sol ; b21-156) from the top of the F 1 sector down, into, and across the membrane, where it is associated with subunit a (2, 15, 32, 34). The 156-residue b subunit has been divided into four functional domains (28). They are, in order from the N to the C terminus; the membrane domain, the tether region, the dimerization domain, and the ␦-binding
Nature Structural and Molecular Biology, 2011
Part of the Medical Biochemistry Commons, and the Medical Molecular Biology Commons This Article is brought to you for free and open access by the Jefferson Digital Commons. The Jefferson Digital Commons is a service of Thomas Jefferson University's Center for Teaching and Learning (CTL). The Commons is a showcase for Jefferson books and journals, peer-reviewed scholarly publications, unique historical collections from the University archives, and teaching tools. The Jefferson Digital Commons allows researchers and interested readers anywhere in the world to learn about and keep up to date with Jefferson scholarship. This article has been accepted for inclusion in Department of Biochemistry and Molecular Biology Faculty Papers by an authorized administrator of the Jefferson Digital Commons. For more information, please contact: JeffersonDigitalCommons@jefferson.edu. Recommended Citation Cingolani, Gino and Duncan, Thomas M, "Structure of the ATP synthase catalytic complex (F(1)) from Escherichia coli in an autoinhibited conformation." (2011).