Directed mutagenesis of the strongly conserved lysine 175 in the proposed nucleotide-binding domain of alpha-subunit from Escherichia coli F1-ATPase. (original) (raw)
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FEBS Letters, 1988
Binding of about 1 mol of adenosine triphosphopyridoxal to Escherichia coli F,-ATPase resulted in the nearly complete inactivation of the enzyme [(1987) J. Biol. Chem. 262, 768676921. About two thirds of the label was bound to the a-subunit, and the rest to the p-subunit. The present study revealed that LysZo' in the a-subunit and L~s'*~ in the glycinerich region of the B-subunit are the major sites labeled with this reagent. Thus, these two residues might be located close to the y-phosphate of the bound ATP.
Archives of Biochemistry and Biophysics, 1989
Cys) were isolated, and the kinetic properties of their F,-ATPases were studied. All the mutations so far identified are clustered in the two defined regions of the LY subunit. With F1 of strain KF114, as with F, of ~ux~4401 (Ser-373 + Phe; T. Noumi, M. Futai, and H. Kanazawa (1984) J. Biol. Ch.em 259, 10076-10079), the rate of multisite hydrolysis of ATP was 4 X lo-"-fold lower than that with wild-type Fi, suggesting that residues Ser-373 and Arg-376 or the regions in their vicinities are essential for positive catalytic cooperativity. With F1 from strain KFlOl, multisite hydrolysis was higher (about 40% of that of the wild type j, but the F, was unstable and showed defective interaction with the membrane sector (F,). The F1 from KF154 had lower multisite hydrolysis (about lo'% of that of the wild type) but could support slow growth by oxidative phosphorylation.
Biochemistry, 1992
Cysteine residues have been exchanged for serine residues at positions 10 and 108 in the e subunit of the Escherichia coli F1 ATPase by site-directed mutagenesis to create two mutants, e-S1OC and e-S108C. These two mutants and wild-type enzyme were reacted with [ 14C]N-ethylmaleimide (NEM) to examine the solvent accessibility of Cys residues and with novel photoactivated cross-linkers, tetrafluorophenyl azide-maleimides (TFPAM's), to examine near-neighbor relationships of subunits. In native wild-type F1 ATPase, N E M reacted with a subunits at a maximal level of 1 mol/mol of enzyme (1 mo1/3 a subunits) and with the 6 subunit at 1 mol/mol of enzyme; other subunits were not labeled by the reagent. In the mutants e-SlOC and e-S108C, Cyslo and cyslO& respectively, were also labeled by NEM, indicating that these are surface residues. Reaction of wild-type enzyme with TFPAM's gave cross-linking of the 6 subunit to both a and /3 subunits. Reaction of the mutants with TFPAM's also cross-linked 6 to a and (3 and in addition formed covalent links between Cyslo of the E subunit and the y subunit and between Cys108 of the e subunit and the a subunit. The yield of cross-linking between sites on e and other subunits depended on the nucleotide conditions used; this was not the case for 6-a or 6-/3 cross-linked products. In the presence of ATP + EDTA the yield of cross-linking between e-Cysl0 and y was high (close to 50%) while the yield of t-Cyslos and a was low (around 10%). In the presence of ATP + Mgz+ the yield of e-CyslO-y was lower than in ATP + EDTA (only 22%) and the yield of cross-linking between c-Cyslo8 and a was higher (now around 30%). These changes in cross-linking of E to near-neighbor subunits support previous work [Mendel-Hartvig, J., & Capaldi, R. A. (1991) Biochemistry 30, 1278-12841 in showing that there are ligand-dependent conformational changes and/or binding changes of the E subunit. Cross-linking of the e subunit to y had very little effect on ATPase activity, while cross-linking of the e subunit to an a subunit inhibited ATPase activity dramatically. 'This work was supported by NIH Grants HL24526 to R.A.C. and *Institute of Molecular Biology. GM27137 to J.F.W.K. Department of Chemistry. sites during ATP hydrolysis and ATP synthesis (Mendel-Hartvig & Capaldi, 1991a,b; Bragg & Hou, 1987; Richter & McCarty, 1987). During ATP hydrolysis these changes or shifts are related to Pi binding in the catalytic sites containing ADP + Mgz+ and are blocked by DCCD modification of Fo (Mendel-Hartvig & Capaldi, 199 1 b).
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 ⑀.