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

Rodgers, Andrew J.W.; Wilkens, Stephan; Aggeler, Robert; Morris, Mitchell; Howitt, Susan; Capaldi, Roderick A.

doi:10.1074/jbc.272.49.31058

Cited by 73 publications

(38 citation statements)

References 36 publications

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“…4A). Because disulfide bond formation studies indicate that the two b subunits are largely parallel throughout their lengths (7,20,28), the two A92C residues should be proximal in the b dimer and therefore the sites of cross-linking on ␣ and ␤ should also be close together. Therefore, residues ␤-(113-156) are probably too far away to contain the site of cross-linking.…”

Section: Figmentioning

confidence: 99%

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Site-directed Cross-linking of b to the α, β, anda Subunits of the Escherichia coli ATP Synthase

McLachlin¹,

Coveny

Clark

et al. 2000

Journal of Biological Chemistry

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The b subunit dimer of the Escherichia coli ATP synthase, along with the ␦ subunit, is thought to act as a stator to hold the ␣ 3 ␤ 3 hexamer stationary relative to the a subunit as the ␥⑀c 9 -12 complex rotates. Despite their essential nature, the contacts between b and the ␣, ␤, and a subunits remain largely undefined. We have introduced cysteine residues individually at various positions within the wild type membrane-bound b subunit, or within b 24 -156 , a truncated, soluble version consisting only of the hydrophilic C-terminal domain. The introduced cysteine residues were modified with a photoactivatable cross-linking agent, and cross-linking to subunits of the F 1 sector or to complete ATP synthase, or F 1 F 0 -ATPase, utilizes a transmembrane proton gradient to synthesize ATP and is responsible for the final step in oxidative phosphorylation and photophosphorylation. The enzyme (reviewed in Refs. 1-3) is composed of two sectors. The membrane-integral F 0 sector is a proton pore, and in Escherichia coli has a subunit composition of ab 2 c 9 -12 . The membrane-peripheral F 1 sector has a subunit stoichiometry of ␣ 3 ␤ 3 ␥␦⑀. A key feature of the F 1 sector, as seen in the bovine heart mitochondrial crystal structure (4), is that the ␣ and ␤ subunits alternate in a ring around a lengthy pair of ␣-helices of ␥. Each ␤ subunit bears one catalytic nucleotide-binding site, while non-catalytic nucleotide-binding sites are found on the ␣ subunits. These nucleotide-binding sites are located close to the interfaces between ␣ and ␤ subunits, with one site near each of the six interfaces.Subunits from each sector contribute to the formation of two stalks that join F 1 and F 0 . The ␥ and ⑀ subunits form the central stalk, rotation of which is believed to be caused by translocation of protons across the membrane by the a and c subunits. This rotation is thought to cause conformational changes in the catalytic sites, driving synthesis of ATP (1). It is believed that the ␣ and ␤ subunits are prevented from rotating by a peripheral stalk consisting of ␦ and the two b subunits (5, 6) that joins the ␣ 3 ␤ 3 complex to the a subunit.In recent years the interaction of the b dimer and ␦ has been well established by a variety of evidence (7-10). The ␦ subunit appears to be located near the crown of the F 1 complex, the part of F 1 furthest from the membrane (11-15). Because b has a single membrane-spanning region at its N terminus, the remainder of the subunit must span a distance of over 100 Å to come in contact with ␦. Consistent with this proposed arrangement, the region of interaction between b and ␦ has been localized to the C terminus of b (10, 16). The hydrophilic domain of b by itself is mostly ␣-helical as measured by circular dichroism (17), and an isolated complex composed of ␦ with the hydrophilic domain of b was demonstrated by sedimentation velocity ultracentrifugation to be highly extended (9). The dimerization domain of b, encompassing residues 53-122, was also shown to be highly extended (18). Therefore the b 2...

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Section: Figmentioning

confidence: 99%

“…In recent years the interaction of the b dimer and ␦ has been well established by a variety of evidence (7)(8)(9)(10). The ␦ subunit appears to be located near the crown of the F 1 complex, the part of F 1 furthest from the membrane (11)(12)(13)(14)(15).…”

mentioning

confidence: 99%

Site-directed Cross-linking of b to the α, β, anda Subunits of the Escherichia coli ATP Synthase

McLachlin¹,

Coveny

Clark

et al. 2000

Journal of Biological Chemistry

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“…However, recent evidence has suggested that the ␦ subunit may be positioned slightly to the side of F 1 in association with a single ␣ subunit (13)(14)(15)(16). The C-terminal region of the ␦ subunit is in direct contact with the extreme C-terminal end of the b dimer (1,(17)(18)(19)(20). The b subunit dimer constitutes the majority of the peripheral stalk stretching from within the membrane to near the top of F 1 (21).…”

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confidence: 99%

Integration of b Subunits of Unequal Lengths into F1F0-ATP Synthase

Grabar

Cain

2003

Journal of Biological Chemistry

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In Escherichia coli the peripheral stalk of F 1 F 0 -ATP synthase consists of a parallel dimer of identical b subunits. However, the length of the two b subunits need not be fixed. This led us to ask whether it is possible for two b subunits of unequal length to dimerize in a functional enzyme complex. A two-plasmid expression system has been developed that directs production of b subunits of unequal lengths in the same cell. Two b subunits differing in length have been expressed with either a histidine or V5 epitope tag to facilitate nickelaffinity resin purification (Ni-resin) and Western blot analysis. The epitope tags did not materially affect enzyme function. The system allowed us to determine whether the different b subunits segregate to form homodimers or, conversely, whether a heterodimer consisting of both the shortened and lengthened b subunits can occur in an intact enzyme complex. Experiments expressing different b subunits lengthened and shortened by up to 7 amino acids were detected in the same enzyme complex. The V5-tagged b subunit shortened by 7 amino acids (b ⌬7-V5 ) was detected in Ni-resin-purified membrane preparations only when coexpressed with a histidine-tagged b subunit in the same cell. The results demonstrate that the enzyme complex can tolerate a size difference between the two b subunits of up to 14 amino acids. Moreover, the experiments demonstrated the feasibility of constructing enzyme complexes with non-identical b subunits that will be valuable for research requiring specific chemical modification of a single b subunit.

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“…The obvious interpretation is that multiple protein-protein interactions between the two b subunits likely provide these structural determinants. There is ample evidence for direct contacts within the dimerization domain (28,29). To date, there is no evidence of intimate interactions between the two b subunits within the tether domain between positions K23 and D53.…”

Section: Discussionmentioning

confidence: 99%