ε-Binding regions of the γ subunit of Escherichia coli ATP synthase (original) (raw)
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Archives of Biochemistry and Biophysics, 1987
The properties of two monoclonal antibodies which recognize the epsilon subunit of Escherichia coli F1-ATPase were studied in detail. The epsilon subunit is a tightly bound but dissociable inhibitor of the ATPase activity of soluble F1-ATPase. Antibody epsilon-1 binds free epsilon with a dissociation constant of 2.4 nM but cannot bind epsilon when it is associated with F1-ATPase. Likewise epsilon cannot associate with F1-ATPase in the presence of high concentrations of epsilon-1. Thus epsilon-1 activates F1-ATPase which contains the epsilon subunit, and prevents added epsilon from inhibiting the enzyme. Epsilon-1 cannot bind to membrane-bound F1-ATPase. The epsilon-4 antibody binds free epsilon with a dissociation constant of 26 nM. Epsilon-4 can bind to the F1-ATPase complex, but, like epsilon-1, it reverses the inhibition of F1-ATPase by the epsilon subunit. The epsilon subunit remains crosslinkable to both the beta and gamma subunits in the presence of epsilon-4, indicating that it is not grossly displaced from its normal position by the antibody. Presumably the activation arises from more subtle conformational effects. Antibodies epsilon-4 and delta-2, which recognizes the delta subunit, both bind to F1F0 in E. coli membrane vesicles, indicating that these subunits are substantially exposed in the membrane-bound complex. Epsilon-4 inhibits the ATPase activity of the membrane-bound enzyme by about 50%, and Fab prepared from epsilon-4 inhibits by about 40%. This inhibition is not associated with any substantial change in the major apparent Km for ATP. These results suggest that inhibition of membrane-bound F1-ATPase arises from steric effects of the antibody.
Journal of Biological Chemistry, 1998
An affinity resin for the F 1 sector of the Escherichia coli ATP synthase was prepared by coupling the b subunit to a solid support through a unique cysteine residue in the N-terminal leader. b 24-156 , a form of b lacking the N-terminal transmembrane domain, was able to compete with the affinity resin for binding of F 1. Truncated forms of b 24-156 , in which one or four residues from the C terminus were removed, competed poorly for F 1 binding, suggesting that these residues play an important role in b-F 1 interactions. Sedimentation velocity analytical ultracentrifugation revealed that removal of these C-terminal residues from b 24-156 resulted in a disruption of its association with the purified ␦ subunit of the enzyme. To determine whether these residues interact directly with ␦, cysteine residues were introduced at various C-terminal positions of b and modified with the heterobifunctional cross-linker benzophenone-4-maleimide. Cross-links between b and ␦ were obtained when the reagent was incorporated at positions 155 and 158 (two residues beyond the normal C terminus) in both the reconstituted b 24-156-F 1 complex and the membranebound F 1 F 0 complex. CNBr digestion followed by peptide sequencing showed the site of cross-linking within the 177-residue ␦ subunit to be C-terminal to residue 148, possibly at Met-158. These results indicate that the b and ␦ subunits interact via their C-terminal regions and that this interaction is instrumental in the binding of the F 1 sector to the b subunit of F 0 .
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).
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.
Nucleic acids research, 1981
The nucleotide sequence of the promoter distal region of the atp (or unc) operon of Escherichia coli has been determined. It encodes the gamma, beta and epsilon subunits of the ATP-synthase complex and includes a noncoding sequence in which transcription of the operon probably terminates. This work completes the nucleotide sequence of the operon which contains nine genes: eight encode structural proteins of the ATP-synthase complex; a ninth, the first in the operon, may be a pilot for assembly. The genes for the alpha and beta subunits have evolved from a common ancestor.
Archives of Biochemistry and Biophysics, 1986
Mutant genes for the y subunit of H+-translocating ATPase (H+-ATPase) were cloned from eight different strains of Eschmichiu coli isolated in this laboratory. Determination of their nucleotide sequences revealed that they are amber nonsense mutations: a Gln codon at position 15,158,227,262, and 270, respectively, was replaced by a termination codon in these strains. As terminal Met is missing in the y subunit, these results indicate that these strains are capable of synthesizing fragments of y subunits of 13,156, 225, 260, and 268 amino acid residues, respectively. Studies on the properties of membranes of these strains suggested the importance of the region between Gln 269 and the carboxyl terminus (residue 286) for forming a stable F1 complex with ATPase activity and the region between Gln 226 and Gln 261 for normal interaction of F1 with FO. The sequence from Gln 261 to Gln 269 also seemed to be important for stability of F1 assembly on the membranes. The high frequency of the nonsense mutations suggested that the number of essential residues is limited in this subunit. Comparison of the homologies of the amino acid sequences of the y subunits from four different sources confirmed this notion: 19% of amino acid residues are identically conserved in these four strains, and the conserved regions are the amino terminal and carboxyl terminal regions. o 19% Academic Press. Inc.