Subunit b-Dimer of the Escherichia coli ATP Synthase Can Form Left-Handed Coiled-Coils (original) (raw)
Related papers
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.
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.
Conformational changes in the Escherichia coli ATP synthase b-dimer upon binding to F1-ATPase
Journal of Bioenergetics and Biomembranes, 2008
Conformational changes within the subunit bdimer of the E. coli ATP synthase occur upon binding to the F 1 sector. ESR spectra of spin-labeled b at room temperature indicated a pivotal point in the b-structure at residue 62. Spectra of frozen b ± F 1 and calculated interspin distances suggested that where contact between b 2 and F 1 occurs (above about residue 80), the structure of the dimer changes minimally. Between b-residues 33 and 64 inter-subunit distances in the F 1 -bound b-dimer were found to be too large to accommodate tightly coiled coil packing and therefore suggest a dissociation and disengagement of the dimer upon F 1 -binding. Mechanistic implications of this "bubble" formation in the tether domain of ATP synthase b 2 are discussed.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2009
The structure and functional role of the dimeric external stalk of F o F 1 -ATP synthases have been very actively researched over the last years. To understand the function, detailed knowledge of the structure and protein packing interactions in the dimer is required. In this paper we describe the application of structural prediction and molecular modeling approaches to elucidate the structural packing interaction of the cyanobacterial ATP synthase external stalk. In addition we present biophysical evidence derived from ESR spectroscopy and site directed spin labeling of stalk proteins that supports the proposed structural model. The use of the heterodimeric bb′ dimer from a cyanobacterial ATP synthase (Synechocystis sp. PCC 6803) allowed, by specific introduction of spin labels along each individual subunit, the evaluation of the overall tertiary structure of the subunits by calculating inter-spin distances. At defined positions in both b and b′ subunits, reporter groups were inserted to determine and confirm inter-subunit packing. The experiments showed that an approximately 100 residue long section of the cytoplasmic part of the bb′-dimer exists mostly as an elongated α-helix. The distant C-terminal end of the dimer, which is thought to interact with the δ-subunit, seemed to be disordered in experiments using soluble bb′ proteins. A left-handed coiled coil packing of the dimer suggested from structure prediction studies and shown to be feasible in molecular modeling experiments was used together with the measured inter-spin distances of the inserted reporter groups determined in ESR experiments to support the hypothesis that a significant portion of the bb′ structure exists as a left-handed coiled coil.
Crystal structure of the ϵ subunit of the proton-translocating ATP synthase from Escherichia coli
Structure, 1997
Background: Proton-translocating ATP synthases convert the energy generated from photosynthesis or respiration into ATP. These enzymes, termed F 0 F 1-ATPases, are structurally highly conserved. In Escherichia coli, F 0 F 1-ATPase consists of a membrane portion, F 0 , made up of three different polypeptides (a, b and c) and an F 1 portion comprising five different polypeptides in the stoichiometry α 3 β 3 γδε. The minor subunits γ, δ and ε are required for the coupling of proton translocation with ATP synthesis; the ε subunit is in close contact with the α, β, γ and c subunits. The structure of the ε subunit provides clues to its essential role in this complex enzyme. Results: The structure of the E. coli F 0 F 1-ATPase ε subunit has been solved at 2.3 Å resolution by multiple isomorphous replacement. The structure, comprising residues 2-136 of the polypeptide chain and 14 water molecules, refined to an R value of 0.214 (R free = 0.288). The molecule has a novel fold with two domains. The N-terminal domain is a β sandwich with two fivestranded sheets. The C-terminal domain is formed from two α helices arranged in an antiparallel coiled-coil. A series of alanine residues from each helix form the central contacting residues in the helical domain and can be described as an 'alanine zipper'. There is an extensive hydrophobic contact region between the two domains providing a stable interface. The individual domains of the crystal structure closely resemble the structures determined in solution by NMR spectroscopy. Conclusions: Sequence alignments of a number of ε subunits from diverse sources suggest that the C-terminal domain, which is absent in some species, is not essential for function. In the crystal the N-terminal domains of two ε subunits make a close hydrophobic interaction across a crystallographic twofold axis. This region has previously been proposed as the contact surface between the ε and γ subunits in the complete F 1-ATPase complex. In the crystal structure, we observe what is apparently a stable interface between the two domains of the ε subunit, consistent with the fact that the crystal and solution structures are quite similar despite close crystal packing. This suggests that a gross conformational change in the ε subunit, to transmit the effect of proton translocation to the catalytic domain, is unlikely, but cannot be ruled out.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2008
A dimer of 156-residue b subunits forms the peripheral stator stalk of eubacterial ATP synthase. Dimerization is mediated by a sequence with an unusual 11-residue (hendecad) repeat pattern, implying a right-handed coiled coil structure. We investigated the potential for producing functional chimeras in the b subunit of Escherichia coli ATP synthase by replacing parts of its sequence with corresponding regions of the b subunits from other eubacteria, sequences from other polypeptides having similar hendecad patterns, and sequences forming left-handed coiled coils. Replacement of positions 55-110 with corresponding sequences from Bacillus subtilis and Thermotoga maritima b subunits resulted in fully functional chimeras, judged by support of growth on nonfermentable carbon sources. Extension of the T. maritima sequence N-terminally to position 37 or C-terminally to position 124 resulted in slower but significant growth, indicating retention of some capacity for oxidative phosphorylation. Portions of the dimerization domain between 55 and 95 could be functionally replaced by segments from two other proteins having a hendecad pattern, the distantly related E subunit of the Chlamydia pneumoniae V-type ATPase and the unrelated Ag84 protein of Mycobacterium tuberculosis. Extension of such sequences to position 110 resulted in loss of function. None of the chimeras that incorporated the leucine zipper of yeast GCN4, or other left-handed coiled coils, supported oxidative phosphorylation, but substantial ATP-dependent proton pumping was observed in membrane vesicles prepared from cells expressing such chimeras. Characterization of chimeric soluble b polypeptides in vitro showed their retention of a predominantly helical structure. The T. maritima b subunit chimera melted cooperatively with a midpoint more than 20°C higher than the normal E. coli sequence. The GCN4 construct melted at a similarly high temperature, but with much reduced cooperativity, suggesting a degree of structural disruption. These studies provide insight into the structural and sequential requirements for stator stalk function.
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
Journal of Biological Chemistry, 2004
The H ؉-translocating F 0 F 1-ATP synthase of Escherichia coli functions as a rotary motor, coupling the transmembrane movement of protons through F 0 to the synthesis of ATP by F 1. Although the ⑀ subunit appears to be tightly associated with the ␥ subunit in the central stalk region of the rotor assembly, several studies suggest that the C-terminal domain of ⑀ can undergo significant conformational change as part of a regulatory process. Here we use disulfide cross-linking of substituted cysteines on functionally coupled ATP synthase to characterize interactions of ⑀ with an F 0 component of the rotor (subunit c) and with an F 1 component of the stator (subunit ). Oxidation of the engineered F 0 F 1 causes formation of two disulfide bonds, D380C-⑀S108C and ⑀E31C-cQ42C, to give a -⑀-c cross-linked product in high yield. The results demonstrate the ability of ⑀ to span the central stalk region from the surface of the membrane (⑀-c) to the bottom of F 1 (-⑀) and suggest that the conformation detected here is distinct from both the "closed" state seen with isolated ⑀ (Uhlin, U., Cox, G. B., and Guss, J. M. (1997) Structure 5, 1219-1230) and the "open" state seen in a complex with a truncated form of the ␥ subunit (Rodgers, A. J., and Wilce, M. C. (2000) Nat. Struct. Biol. 7, 1051-1054). The kinetics of -⑀ and ⑀-c cross-linking were studied separately using F 0 F 1 containing one or the other matched cysteine pair. The rate of cross-linking at the ⑀/c (rotor/rotor) interface is not influenced by the type of nucleotide added. In contrast, the rate of -⑀ cross-linking is fastest under ATP hydrolysis conditions, intermediate with MgADP, and slowest with MgAMP-PNP. This is consistent with a regulatory role for a reversible /⑀ (stator/rotor) interaction that blocks rotation and inhibits catalysis. Furthermore, the rate of -⑀ cross-linking is much faster than that indicated by previous studies, allowing for the possibility of a rapid response to regulatory signals.
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 .
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