Conformational changes in the Escherichia coli ATP synthase b-dimer upon binding to F1-ATPase (original) (raw)
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Structure of the Cytosolic Part of the Subunit b-Dimer of Escherichia coli F0F1-ATP Synthase
Biophysical Journal, 2008
The structure of the external stalk and its function in the catalytic mechanism of the F 0 F 1 -ATP synthase remains one of the important questions in bioenergetics. The external stalk has been proposed to be either a rigid stator that binds F 1 or an elastic structural element that transmits energy from the small rotational steps of subunits c to the F 1 sector during catalysis. We employed proteomics, sequence-based structure prediction, molecular modeling, and electron spin resonance spectroscopy using site-directed spin labeling to understand the structure and interfacial packing of the Escherichia coli b -subunit homodimer external stalk. Comparisons of bacterial, cyanobacterial, and plant b-subunits demonstrated little sequence similarity. Supersecondary structure predictions, however, show that all compared b-sequences have extensive heptad repeats, suggesting that the proteins all are capable of packing as left-handed coiled-coils. Molecular modeling subsequently indicated that b 2 from the E. coli ATP synthase could pack into stable left-handed coiled-coils. Thirty-eight substitutions to cysteine in soluble b-constructs allowed the introduction of spin labels and the determination of intersubunit distances by ESR. These distances correlated well with molecular modeling results and strongly suggest that the E. coli subunit b-dimer can stably exist as a left-handed coiled-coil.
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 .
Journal of Bacteriology, 2009
Subunit b, the peripheral stalk of bacterial F 1 F o ATP synthases, is composed of a membrane-spanning and a soluble part. The soluble part is divided into tether, dimerization, and ␦-binding domains. The first solution structure of b30-82, including the tether region and part of the dimerization domain, has been solved by nuclear magnetic resonance, revealing an ␣-helix between residues 39 and 72. In the solution structure, b30-82 has a length of 48.07 Å. The surface charge distribution of b30-82 shows one side with a hydrophobic surface pattern, formed by alanine residues. Alanine residues 61, 68, 70, and 72 were replaced by single cysteines in the soluble part of subunit b, b22-156. The cysteines at positions 61, 68, and 72 showed disulfide formation. In contrast, no cross-link could be formed for the A70C mutant. The patterns of disulfide bonding, together with the circular dichroism spectroscopy data, are indicative of an adjacent arrangement of residues 61, 68, and 72 in both ␣-helices in b22-156. ATP synthesis by oxidative phosphorylation or photophosphorylation is a multistep membrane-located process that provides the bulk of cellular energy in eukaryotes and many prokaryotes. The majority of ATP synthesis is accomplished by the enzyme ATP synthase (EC 3.6.1.34), also called F 1 F o ATP synthase, which, in its simplest form, as in bacteria, is composed of eight different subunits (␣ 3 ,  3 , ␥, ␦, ε, a, b 2 , and c 9-12). This multisubunit complex is divided into the F 1 headpiece, ␣ 3 : 3 , and a membrane-embedded ion-translocating part known as F o , to which F 1 is attached by a central and a peripheral stalk (1, 5, 25). ATP is synthesized or hydrolyzed on the ␣ 3 : 3 hexamer, and the energy provided for or released during that process is transmitted to the membrane-bound F o sector, consisting of subunits a and c and part of subunit b (30, 31). The energy coupling between the two active domains occurs via the stalk part(s) (6). The central stalk is made of subunits ␥ and ε, and the peripheral stalk is formed by subunits ␦ and b. The peripheral stalk, which lies at the edge of the multisubunit assembly of the F 1 F o ATP synthase, acts as a stator to counter the tendency of the ␣ 3 : 3 hexamer to follow the rotation of the central stalk and the attached c-ring, and to anchor the membrane-embedded a subunit (17, 36). In Escherichia coli, subunit b with its 156 residues extends with its soluble part (b sol ; b21-156) from the top of the F 1 sector down, into, and across the membrane, where it is associated with subunit a (2, 15, 32, 34). The 156-residue b subunit has been divided into four functional domains (28). They are, in order from the N to the C terminus; the membrane domain, the tether region, the dimerization domain, and the ␦-binding
Biochemistry, 2002
The b subunit of E. coli F 0 F 1 -ATPase links the peripheral F 1 subunits to the membrane-integral F 0 portion and functions as a "stator", preventing rotation of F 1 . The b subunit is present as a dimer in ATP synthase, and residues 62-122 are required to mediate dimerization. To understand how the b subunit dimer is formed, we have studied the structure of the isolated dimerization domain, b 62-122 . Analytical ultracentrifugation and solution small-angle X-ray scattering (SAXS) indicate that the b 62-122 dimer is extremely elongated, with a frictional ratio of 1.60, a maximal dimension of 95 Å, and a radius of gyration of 27 Å, values that are consistent with an R-helical coiled-coil structure. The crystal structure of b 62-122 has been solved and refined to 1.55 Å. The protein crystallized as an isolated, monomeric R helix with a length of 90 Å. Combining the crystal structure of monomeric b 62-122 with SAXS data from the dimer in solution, we have constructed a model for the b 62-122 dimer in which the two helices form a coiled coil with a right-handed superhelical twist. Analysis of b sequences from E. coli and other prokaryotes indicates conservation of an undecad repeat, which is characteristic of a right-handed coiled coil and consistent with our structural model. Mutation of residue Arg-83, which interrupts the undecad pattern, to alanine markedly stabilized the dimer, as expected for the proposed two-stranded, right-handed coiled-coil structure.
Site-directed Cross-linking of b to the α, β, anda Subunits of the Escherichia coli ATP Synthase
Journal of Biological Chemistry, 2000
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 F 1 F 0 was attempted. Cross-linking in both the full-length and truncated forms of b was obtained at positions 92 (to ␣ and ), and 109 and 110 (to ␣ only). Mass spectrometric analysis of peptide fragments derived from the b 24-156 A92C crosslink revealed that cross-linking took place within the region of ␣ between Ile-464 and Met-483. This result indicates that the b dimer interacts with the ␣ subunit near a non-catalytic ␣/ interface. A cysteine residue introduced in place of the highly conserved arginine at position 36 of the b subunit could be cross-linked to the a subunit of F 0 in membrane-bound ATP synthase, implying that at least 10 residues of the polar domain of b are adjacent to residues of a. Sites of cross-linking between b 24-156 A92C and  as well as b 24-156 I109C and ␣ are proposed based on the mass spectrometric data, and these sites are discussed in terms of the structure of b and its interactions with the rest of the complex. 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
The Dimerization Domain of the b Subunit of the Escherichia coli F1F0-ATPase
Journal of Biological Chemistry, 1999
In this study a series of N- and/or C-terminal truncations of the cytoplasmic domain of the b subunit of the Escherichia coli F(1)F(0) ATP synthase were tested for their ability to form dimers using sedimentation equilibrium ultracentrifugation. The deletion of residues between positions 53 and 122 resulted in a strongly decreased tendency to form dimers, whereas all the polypeptides that included that sequence exhibited high levels of dimer formation. b dimers existed in a reversible monomer-dimer equilibrium and when mixed with other b truncations formed heterodimers efficiently, provided both constructs included the 53-122 sequence. Sedimentation velocity and (15)N NMR relaxation measurements indicated that the dimerization region is highly extended in solution, consistent with an elongated second stalk structure. A cysteine introduced at position 105 was found to readily form intersubunit disulfides, whereas other single cysteines at positions 103-110 failed to form disulfides either with the identical mutant or when mixed with the other 103-110 cysteine mutants. These studies establish that the b subunit dimer depends on interactions that occur between residues in the 53-122 sequence and that the two subunits are oriented in a highly specific manner at the dimer interface.
Folding and stability of the b subunit of the F 1 F 0 ATP synthase
Protein Science, 2002
The F 1 F 0 ATP synthase is a reversible molecular motor that employs a rotary catalytic cycle to couple a chemiosmotic membrane potential to the formation/hydrolysis of ATP. The multisubunit enzyme contains two copies of the b subunit that form a homodimer as part of a narrow, peripheral stalk structure that connects the membrane (F 0 ) and soluble (F 1 ) sectors. The three-dimensional structure of the b subunit is unknown making the nature of any interactions or conformational changes within the F 1 F 0 complex difficult to interpret. We have used circular dichroism and analytical ultracentrifugation analyses of a series of N-and C-terminal truncated b proteins to investigate its stability and structure. Thermal denaturation of the b constructs exhibited distinct two-state, cooperative unfolding with T m values between 30 and 40°C. CD spectra for the region comprising residues 53-122 (b 53-122 ) showed 222 / 208 ס 0.99, which reduced to 0.92 in the presence of the hydrophobic solvent trifluoroethanol. Thermodynamic parameters for b 53-122 (⌬G, ⌬H and ⌬C p ) were similar to those reported for several nonideal, coiled-coil proteins. Together these results are most consistent with a noncanonical and unstable parallel coiled-coil at the interface of the b dimer.
The second stalk of Escherichia coli ATP synthase
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2000
Two stalks link the F 1 and F 0 sectors of ATP synthase. The central stalk contains the Q and O subunits and is thought to function in rotational catalysis as a rotor driving conformational changes in the catalytic K 3 L 3 complex. The two b subunits and the N subunit associate to form b 2 N, a second, peripheral stalk extending from the membrane up the side of K 3 L 3 and binding to the N-terminal regions of the K subunits, which are approx. 125 A î from the membrane. This second stalk is essential for binding F 1 to F 0 and is believed to function as a stator during rotational catalysis. In vitro, b 2 N is a highly extended complex held together by weak interactions. Recent work has identified the domains of b which are essential for dimerization and for interaction with N. Disulphide cross-linking studies imply that the second stalk is a permanent structure which remains associated with one K subunit or KL pair. However, the weak interactions between the polypeptides in b 2 N pose a challenge for the proposed stator function. ß
Dimerization Interactions of the b Subunit of the Escherichia coli F1F0-ATPase
Journal of Biological Chemistry, 1997
Site-directed mutagenesis and N-terminal truncations were used to examine dimerization interactions in the b subunit of Escherichia coli F1F0-ATPase. Individual cysteine residues were incorporated into bsyn, a soluble form of the protein lacking the membrane-spanning N-terminal domain, in two main areas: the heptad repeat region and the hydrophobic region which begins at residue Val-124. The tendencies of these cysteine residues to form disulfide bonds with the corresponding cysteine in the bsyn dimer were tested using disulfide exchange by glutathione and air oxidation catalyzed by Cu2+. Within the heptad repeat region, only cysteines at residues 59 and 60, which occupy the b and c positions of the heptad repeat, showed significant tendencies to form disulfides, a result inconsistent with a coiled-coil model for bsyn. Mixed disulfide formation most readily occurred with the S60C + L65C and A61C + L65C pairs. Cysteines at positions 124, 128, 132, and 139 showed strong tendencies to form disulfides with their mates in the dimer, suggesting a parallel alpha-helical interaction between the subunits in this region. Deletion of residues N-terminal to either Glu-34 or Asp-53 had no apparent effect on dimerization as determined by sedimentation equilibrium, while deletion of all residues N-terminal to Lys-67 produced a monomeric form. These results imply that residues 53-66 but not 24-52 are essential for bsyn dimerization. Taken together the results are consistent with a model in which the two b subunits interact in more than one region, including a parallel alignment of helices containing residues 124-139.