Structural and functional analysis of the coupling subunit F in solution and topological arrangement of the stalk domains of the methanogenic A1AO ATP synthase

Schäfer, Ingmar B.; Rößle, Manfred; Biuković, Goran; Müller, Volker; Grüber, Gerhard

doi:10.1007/s10863-006-9015-4

Cited by 36 publications

(76 citation statements)

References 47 publications

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“…Such an arrangement would be consistent with chemical cross-linking data that place the related EG heterodimer of the vacuolar ATPase along the surface of the B subunit (28). These results together with cross-linking experiments conducted with the A-ATPase from Methanosarcina mazei Gö1, which place the C-terminal region of the H subunit near the N-terminal region of one of the A subunits (29), suggest that the binding region for the EH (or EG) heterodimer is near a subunit AB interface as shown in Fig. 6D.…”

Section: Figure 5 Secondary Structure Analysis Of E Ct1 H Ct As Carrsupporting

confidence: 83%

Domain Architecture of the Stator Complex of the A1A0-ATP Synthase from Thermoplasma acidophilum

Kish-Trier

Wilkens

2009

Journal of Biological Chemistry

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A key structural element in the ion translocating F-, A-, and V-ATPases is the peripheral stalk, an assembly of two polypeptides that provides a structural link between the ATPase and ion channel domains. Previously, we have characterized the peripheral stalk forming subunits E and H of the A-ATPase from Thermoplasma acidophilum and demonstrated that the two polypeptides interact to form a stable heterodimer with 1:1 stoichiometry (Kish-Trier, E., Briere, L. K., Dunn, S. D., and Wilkens, S. (2008) J. Mol. Biol. 375, 673-685). To define the domain architecture of the A-ATPase peripheral stalk, we have now generated truncated versions of the E and H subunits and analyzed their ability to bind each other. The data show that the N termini of the subunits form an ␣-helical coiled-coil, ϳ80 residues in length, whereas the C-terminal residues interact to form a globular domain containing ␣-and ␤-structure. We find that the isolated C-terminal domain of the E subunit exists as a dimer in solution, consistent with a recent crystal structure of the related Pyrococcus horikoshii A-ATPase E subunit The archaeal ATP synthase (A 1 A 0 -ATPase), 2 along with the related F 1 F 0 -and V 1 V 0 -ATPases (proton pumping vacuolar ATPases), is a rotary molecular motor (1-4). The rotary ATPases are bilobular in overall architecture, with one lobe comprising the water-soluble A 1 , F 1 , or V 1 and the other comprising the membrane-bound A 0 , F 0 , or V 0 domain, respectively. The subunit composition of the A-ATPase is A 3 B 3 DE 2 FH 2 for the A 1 and CIK x for the A 0 . In the A 1 domain, the three A and B subunits come together in an alternating fashion to form a hexamer with a hydrophobic inner cavity into which part of the D subunit is inserted. Subunits D and F comprise the central stalk connection to A 0 , whereas two heterodimeric EH complexes are thought to form the peripheral stalk attachment to A 0 seen in electron microscopy reconstructions (5, 6). In the A 0 domain (subunits CIK x ), the K subunits (proteolipids) form a ring that is linked to the central stalk by the C subunit, whereas the cytoplasmic N-terminal domain of the I subunit probably mediates the binding of the EH peripheral stalks to A 0 , as suggested for the bacterial A/V-type enzyme (7). Although closer in structure to the proton-pumping V-ATPase, the A-ATPase functions in vivo as an ATP synthase, coupling ion motive force to ATP synthesis, most likely via a similar rotary mechanism as demonstrated for the bacterial A/V-and the vacuolar type enzymes (8, 9). During catalysis, substrate binding occurs sequentially on the three catalytic sites, which are formed predominantly by the A subunits. This is accompanied by conformation changes in the A 3 B 3 hexamer that are linked to the rotation of the embedded D subunit together with the rotor subunits F, C, and the proteolipid ring. Each copy of K contains a lipid-exposed carboxyl residue (Asp or Glu), which is transiently interfaced with the membrane-bound domain of I during rotation, thereby catalyzing ion tran...

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Section: Figure 5 Secondary Structure Analysis Of E Ct1 H Ct As Carrsupporting

confidence: 83%

Domain Architecture of the Stator Complex of the A1A0-ATP Synthase from Thermoplasma acidophilum

Kish-Trier

Wilkens

2009

Journal of Biological Chemistry

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“…For the whole enzyme, projections (18,67,68) and three-dimensional structures (19 -22) by EM are available for several species. Cross-linking experiments have clarified the positions of subunits relative to each other (53,54,61,62). The stoichiometry of the subunits is not yet firmly established.…”

Section: Discussionmentioning

confidence: 99%

Three-dimensional Structure of A1A0 ATP Synthase from the Hyperthermophilic Archaeon Pyrococcus furiosus by Electron Microscopy

Vonck

Pisa

Morgner

et al. 2009

Journal of Biological Chemistry

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The archaeal ATP synthase is a multisubunit complex that consists of a catalytic A 1 part and a transmembrane, ion translocation domain A 0 . The A 1 A 0 complex from the hyperthermophile Pyrococcus furiosus was isolated. Mass analysis of the complex by laser-induced liquid bead ion desorption (LILBID) indicated a size of 730 ؎ 10 kDa. A three-dimensional map was generated by electron microscopy from negatively stained images. The map at a resolution of 2.3 nm shows the A 1 and A 0 domain, connected by a central stalk and two peripheral stalks, one of which is connected to A 0 , and both connected to A 1 via prominent knobs. X-ray structures of subunits from related proteins were fitted to the map. On the basis of the fitting and the LILBID analysis, a structural model is presented with the stoichiometry A 3 B 3 CDE 2 FH 2 ac 10 .Archaea produce ATP by an ATP synthase that is distinct from the well known F 1 F 0 -type ATP synthase occurring in bacteria, mitochondria, and chloroplasts. The A-type ATP synthases are more closely related to vacuolar (V-type) ATPase, which, however, is functionally different and acts as an ATPdriven ion pump (1). Some bacteria also harbor A-type ATP synthases, probably acquired by horizontal gene transfer (2, 3).Like F 1 F 0 ATP synthases and V 1 V 0 ATPases, A 1 A 0 ATP synthases consist of a soluble enzymatic head in the cytoplasm and a transmembrane ion-translocating domain, forming a pair of coupled rotary motors. There is clear homology in the main catalytic subunits of all types, showing a common evolutionary origin, but many of the peripheral subunits are unique for the distinct groups. The A-and V-type enzymes are considerably larger than the F-type (1).The catalytic heads are made up of three A and three B subunits, where A is the catalytic subunit equivalent to F-type ␤. The A subunit contains a 90-amino acid insert near the N terminus, known as the non-homologous region, which makes this subunit with Ϸ66 kDa considerably larger than its homologues (4 -7). The central stalk consists of the C, D, and F subunits, and the D subunit is thought to be the equivalent of the ␥ subunit, responsible for conformational changes in the nucleotide-binding pockets upon rotation (8, 9). The transmembrane domain contains a rotor of a number of identical c-subunits and part of the a-subunit. The a-subunit is large, comprising a transmembrane domain with seven to eight predicted transmembrane helices and a soluble domain, which probably forms part of the stator. In addition, the A-type ATP synthases contain subunits H and E (10).Structural information on the A-type and V-type ATPases has been forthcoming in recent years. For several subunits, x-ray structures have been determined, including isolated archaeal A (11) and B (12) subunits, the E subunit (13), and a bacterial A-type C subunit (14, 15). The archaeal F subunit has been solved by NMR spectroscopy (16), and the shape of an H subunit dimer is known from small angle x-ray scattering (17). Information about the whole complex has been pro...

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“…30,31 For example, in the bacterial flagellum, a protein with weak homology to subunit E, named FliH, has been found, and it has been shown that this protein can bind via its C-terminal domain to FliI, a flagellar component with homology to the F-ATPase catalytic β subunit. 31 On the other hand, a close proximity of subunits A and H near the top of the A 1 domain has been observed based on chemical cross-linking experiments, 32 suggesting that subunit H might also play a role in the interaction between peripheral stator and catalytic domain. Experiments to identify the interaction of peripheral stator and ATPase domain in the A-ATPase from T. acidophilum are ongoing in our laboratories.…”

Section: Discussionmentioning

confidence: 99%

The Stator Complex of the A1A0-ATP Synthase—Structural Characterization of the E and H Subunits

Kish-Trier

Briere

Dunn

et al. 2008

Journal of Molecular Biology

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Archaeal ATP synthase (A-ATPase) is the functional homolog to the ATP synthase found in bacteria, mitochondria and chloroplasts, but the enzyme is structurally more related to the proton-pumping vacuolar ATPase found in the endomembrane system of eukaryotes. We have cloned, overexpressed and characterized the stator-forming subunits E and H of the A-ATPase from the thermoacidophilic Archaeon, Thermoplasma acidophilum. Size exclusion chromatography, CD, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and NMR spectroscopic experiments indicate that both polypeptides have a tendency to form dimers and higher oligomers in solution. However, when expressed together or reconstituted, the two individual polypeptides interact with high affinity to form a stable heterodimer. Analyses by gel filtration chromatography and analytical ultracentrifugation show the heterodimer to have an elongated shape, and the preparation to be monodisperse. Thermal denaturation analyses by CD and differential scanning calorimetry revealed the more cooperative unfolding transitions of the heterodimer in comparison to those of the individual polypeptides. The data are consistent with the EH heterodimer forming the peripheral stalk(s) in the A-ATPase in a fashion analogous to that of the related vacuolar ATPase.

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Structural and functional analysis of the coupling subunit F in solution and topological arrangement of the stalk domains of the methanogenic A1AO ATP synthase

Cited by 36 publications

References 47 publications

Domain Architecture of the Stator Complex of the A1A0-ATP Synthase from Thermoplasma acidophilum

Domain Architecture of the Stator Complex of the A1A0-ATP Synthase from Thermoplasma acidophilum

Three-dimensional Structure of A1A0 ATP Synthase from the Hyperthermophilic Archaeon Pyrococcus furiosus by Electron Microscopy

The Stator Complex of the A1A0-ATP Synthase—Structural Characterization of the E and H Subunits

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