Nucleotide-dependent Movement of the ε Subunit between α and β Subunits in the Escherichia coli F1F0-type ATPase

Abstract

Mutants of ECF 1 -ATPase were generated, containing cysteine residues in one or more of the following positions: ␣Ser-411, ␤Glu-381, and ⑀Ser-108, after which disulfide bridges could be created by CuCl 2 induced oxidation in high yield between ␣ and ⑀, ␤ and ⑀, ␣ and ␥, ␤ and ␥ (endogenous Cys-87), and ␣ and ␤. All of these cross-links lead to inhibition of ATP hydrolysis activity. In the two double mutants, containing a cysteine in ⑀Ser-108 along with either the DELSEED region of ␤ (Glu-381) or the homologous region in ␣ (Ser-411), there was a clear nucleotide dependence of the cross-link formation with the ⑀ subunit. In ␤E381C/⑀S108C the ␤-⑀ cross-link was obtained preferentially when Mg 2؉ and ADP ؉ P i (addition of MgCl 2 ؉ ATP) was present, while the ␣-⑀ cross-link product was strongly favored in the ␣S411C/⑀S108C mutant in the Mg 2؉ ATP state (addition of MgCl 2 ؉ 5-adenylyl-␤,␥-imidodiphosphate). In the triple mutant ␣S411C/␤E381C/⑀S108C, the ⑀ subunit bound to the ␤ subunit in Mg 2؉ -ADP and to the ␣ subunit in Mg There has been significant recent progress in determining the structure of the F 1 part of the F 1 F 0 -type ATP synthase, a key enzyme in oxidative phosphorylation and photo-phosphorylation (Senior 1988(Senior , 1990Boyer, 1993). The F 1 , which can be detached from the F 0 and studied separately, is a complex of five different types of subunits ␣, ␤, ␥, ␦, and ⑀ in the molar ratio 3:3:1:1:1. Electron microscopy has shown that the ␣ and ␤ subunits are hexagonally arranged, and alternate around a central cavity in which the ␥ subunit is located (Gogol et al. 1989a(Gogol et al. , 1989bBoekema and Böttcher, 1992). Biochemical studies place the ␦ and ⑀ subunits (nomenclature for the Escherichia coli enzyme) at the bottom of the ␣ 3 ␤ 3 ␥ core complex (Beckers et al., 1992;Dallmann et al., 1992;Capaldi et al., 1994Capaldi et al., , 1995 in the stalk region which is a 40 -45-Å long structure that links the F 1 to the F 0 part (Gogol et al., 1987;Lü cken et al., 1990). The recently published high resolution structure of a major part of the F 1 molecule confirms the above described arrangement of the ␣, ␤, and ␥ subunits relative to one another and adds important details (Abrahams et al., 1994). The ␣ and ␤ subunits have a similar overall fold, each made up of three domains: an NH 2 -terminal, predominantly ␤-sheet domain, a nucleotide-binding domain of 9 ␤ strands and 9 ␣ helices, and a COOH-terminal, predominantly ␣-helical domain, that provides the binding site for the ⑀ subunit, probably the ␦ subunit, as well as the b subunit of the F 0 . The part of the ␥ subunit within the ring of ␣ and ␤ subunits is arranged as two ␣ helices: a long COOH-terminal helix extending from the top NH 2 -terminal domain of the ␣ and ␤ subunits into the stalk region, and a short NH 2 -terminal helix running from the catalytic site domain into the stalk. These two ␣ helices form a coiled coil. A third short ␣-helix of the ␥ subunit (residues 82-99 in the E. coli enzyme) is inclined at about 45°to the two larger helices...

Rotor/Stator Interactions of the ϵ Subunit in Escherichia coli ATP Synthase and Implications for Enzyme Regulation

“…An earlier study with chloroplast thylakoids indicated that it undergoes a rapid conformational change (Ͻ30 s) in response to membrane energizing (60). In contrast, previous cross-linking studies with ⑀S108C in EcF 0 F 1 (43,61) suggested that different nucleotide conditions trap the C-terminal domain of ⑀ in relatively static alternate states, because selective cross-linking was achieved through slow, Cu 2ϩ -catalyzed oxidation (15 min to Ͼ1 h). In the present study, ␤D380C-⑀S108C cross-linking shows a sensitivity to different nucleotides but does so over a much briefer period of time (Fig.…”

Subunit Interactions in the Clathrin-coated Vesicle Vacuolar (H+)-ATPase Complex

“…It will be important to test whether these or other subunit contacts are in fact altered during turnover of the enzyme. Such catalysis-dependent changes in subunit contacts have in fact been demonstrated for the F-ATPase complex using covalent cross-linking (22,51). The picture presented in Fig.…”

The vacuolar H+-ATPase: a universal proton pump of eukaryotes

“…The γ subunit also interfaces with the ε subunit [217], and both of these subunits in turn contact both the β subunit in F " [218] and subunit c in the F o sector [143,219,220]. Both subunits certainly undergo conformational changes during the catalytic cycle [210,221,222].…”