Oligomycin Sensitivity Conferring Protein of Mitochondrial ATP Synthase:  Deletions in the N-Terminal End Cause Defects in Interactions with F<sub>1</sub>, while Deletions in the C-Terminal End Cause Defects in Interactions with F<sub>o</sub>

The stator function in ATP synthase was studied by a combined mutagenesis and fluorescence approach. Specifically, binding of ␦-subunit to ␦-depleted F 1 was studied. A plausible binding surface on ␦-subunit was identified from conservation of amino acid sequence and the high resolution NMR structure. Specific mutations aimed at modulating binding were introduced onto this surface. Affinity of binding of wild-type and mutant ␦-subunits to ␦-depleted F 1 was determined quantitatively using the fluorescence signals of natural ␦-Trp-28, inserted ␦-Trp-11, or inserted ␦-Trp-79. The results demonstrate that helices 1 and 5 in the N-terminal domain of the ␦-subunit provide the F 1 -binding surface of ␦. Unexpectedly, mutations that impaired binding between F 1 and ␦ were found to not necessarily impair ATP synthase activity. Further investigation revealed that inclusion of the soluble cytoplasmic domain of the b subunit substantially enhanced affinity of binding of ␦-subunit to F 1 . The new data show that the stator is "overengineered" to resist rotor torque during catalysis.ATP synthase is the membrane enzyme responsible for ATP synthesis in oxidative and photophosphorylation of prokaryotes and eukaryotes, and also for ATP-driven proton pumping to generate the transmembrane proton gradient in bacterial membranes. In Escherichia coli, ATP synthase consists of a complex of eight subunits, ␣ 3 ␤ 3 ␥␦⑀ab 2 c n . It was defined in earlier times in terms of a membrane-peripheral F 1 sector (␣ 3 ␤ 3 ␥␦⑀) containing three catalytic sites, and a membrane-embedded F 0 sector (ab 2 c n ), which carries out transmembrane proton transport. Recent work has demonstrated that the enzyme functions as a rotary motor. The centrally located "rotor" consists of subunits ␥⑀c n . At the top, it rotates inside the ␣ 3 ␤ 3 hexagon, and thus modifies the activities of the catalytic sites; at the base, it rotates against subunit a, thereby facilitating proton movement. In this way, the energy of the proton gradient is transduced into the energy of ATP synthesis/hydrolysis. Understanding the mechanism by which this occurs is currently of major interest. To ensure that subunits a and ␣ 3 ␤ 3 remain firmly fixed in relation to each other they are connected by a peripheral structure, the "stator" stalk, consisting of subunits b 2 and ␦. For recent reviews of the structure and function of ATP synthase, see Refs. 1-3.The stator must be able to resist strain resulting from rotor torque, thus its construction is of considerable importance. The dimer of b-subunits forms an elongated helical connection between subunit a and the C-terminal domain of the ␦-subunit, it lies at one side of the ␣ 3 ␤ 3 hexagon, and its functional domains have been well characterized (4 -6). There may exist functional interactions between b 2 and the ␣ 3 ␤ 3 hexagon (7). Currently only partial high-resolution structure has been reported for the b subunit (8, 9). The ␦-subunit has been shown by electron microscopy to bind to the very top ("crown") of the ␣ 3 ␤ 3 hexagon (10),...

Section: Discussionmentioning

confidence: 99%

Identification of the F1-binding Surface on the δ-Subunit of ATP Synthase

Weber

Wilke-Mounts

Senior

2003

“…Together these subunits are believed to form a "second stalk" reaching from the membrane to near the top of the F 1 sector, which may function to hold the ␣ 3 ␤ 3 subunits stationary during rotation of ␥ and ⑀ driven by proton flow through F 0 (48,49). Earlier truncation and mutagenesis studies showed that the C terminus of b was essential for proper assembly of ATP synthase (10) and implied that the C terminus of ␦ played a role in interaction of F 1 with the F 0 sector (21)(22)(23)(24).…”

Section: Discussionmentioning

confidence: 99%

“…Membranes of mutant E. coli strains expressing truncated forms of ␦ showed little ATPase activity (21), suggesting that F 1 cannot bind to the membrane in the absence of ␦. In truncation and mutagenesis studies using the mitochondrial (22,23) and yeast (24) homologues of ␦, called OSCP 1 for oligomycin sensitivity conferring protein, the C-terminal region of OSCP was implicated in F 0 binding.…”

mentioning

confidence: 99%

The b and δ Subunits of the Escherichia coli ATP Synthase Interact via Residues in their C-terminal Regions

McLachlin¹,

Bestard

Dunn

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 .In the process of oxidative phosphorylation or photophosphorylation, the electron transport chain generates a transmembrane proton gradient. The ATP synthase, or F 1 F 0 -ATPase, allows protons to flow down this electrochemical gradient and uses the energy obtained to synthesize ATP (for reviews, see Refs. 1-4). Under appropriate conditions ATP synthase can hydrolyze ATP to pump protons. The enzyme is composed of two sectors; the F 0 sector is membrane-integral and is responsible for proton translocation, and the F 1 sector is attached to the membrane via F 0 and houses the catalytic sites for ATP synthesis. F 1 is easily detached from the membrane and can be purified as a soluble protein with ATPase activity.ATP synthases contain at least eight types of subunits. In the relatively simple enzyme from Escherichia coli, the F 1 sector has the stoichiometry ␣ 3 ␤ 3 ␥ 1 ␦ 1 ⑀ 1 , whereas F 0 is composed of three subunits of stoichiometry a 1 b 2 c 9 -12 . The a and c subunits, but not b, contain residues essential for the translocation of protons across the membrane (3). The 156-residue b subunit is believed to span the membrane once at its hydrophobic N terminus, whereas the remainder of the protein is very hydrophilic. b is thought to exist as a dimer in the complex (5-7), and proteolysis studies have shown that the hydrophilic region of b is required for the association of F 1 with the membrane (7-9). Removal of two residues from the C terminus of b disrupts normal assembly of the complex (10), as does mut...

“…The key sites for this interaction appear to be in the C terminus of ␦. C-terminal truncations of as few as 4 -6 residues from either ␦ or OSCP prevent the rebinding of ECF 1 or MF 1 , respectively, to F 0 (32,33).…”

Section: Discussionmentioning

confidence: 99%

The Subunit δ-Subunit b Domain of the Escherichia coli F1F0 ATPase

Rodgers

Wilkens

Aggeler

et al. 1997

To examine whether subunit b is a monomor or dimer in intact ECF 1 F 0 , CuCl 2 was used to induce cross-link formation in the mutants bS60C, bQ104C, bA128C, bG131C, and bS146C. With the exception of bS60C, CuCl 2 treatment resulted in formation of b subunit dimers in all mutants. Cross-linking yield was independent of nucleotide conditions and did not affect ATPase activity. These results show the b subunit to be dimeric for a large portion of the C terminus, with residues 124 -131 likely forming a pair of parallel ␣ helices.