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

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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...

supporting

confidence: 69%

Section: Methodsmentioning

confidence: 99%

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

Kish-Trier

Wilkens

2009

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“…CD spectroscopy experiments showed that, upon NtpE-F complex formation, there was an increase in ␣-helical secondary structure, most likely due to ␣-helical coiled-coil formation between the two subunits as indicated by stronger minima at 208 and 222 nm and by the increased ratio of 222 / 208 . The dissociation of the respective dimer of each subunit is likely to take place during formation of the interaction between NtpE and NtpF, as shown for studies on other V (or A)-ATPases (8,36).…”

Section: Figure 6 Saxs Profiles Of the Ntpe-f-imentioning

confidence: 82%

“…This characteristic increase of the ratio is often observed when two helical elements interact to form higher order structures such as coiled coils (35). Interaction of the corresponding subunits from yeast V-ATPase and Thermoplasma acidophilum A-ATPase (resembling V-ATPase structurally) to form a higher order coiled-coil has been previously described (8,36). NtpI…”

Section: Subunit-subunit Interactions Assessed By Pulldown Assay and mentioning

confidence: 99%

Interaction and Stoichiometry of the Peripheral Stalk Subunits NtpE and NtpF and the N-terminal Hydrophilic Domain of NtpI of Enterococcus hirae V-ATPase

Yamamoto

Unzai

Saijo

et al. 2008

The vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain and an integral membrane domain connected by a central stalk and a few peripheral stalks. The number and arrangement of the peripheral stalk subunits remain controversial. The peripheral stalk of Na ؉ -translocating V-ATPase from Enterococcus hirae is likely to be composed of NtpE and NtpF (corresponding to subunit G of eukaryotic V-ATPase) subunits together with the N-terminal hydrophilic domain of NtpI (corresponding to subunit a of eukaryotic V-ATPase). Here we purified NtpE, NtpF, and the N-terminal hydrophilic domain of NtpI (NtpI Nterm ) as separate recombinant His-tagged proteins and examined interactions between these three subunits by pulldown assay using one tagged subunit, CD spectroscopy, surface plasmon resonance, and analytical ultracentrifugation. NtpI Nterm directly bound NtpF, but not NtpE. NtpE bound NtpF tightly. NtpI Nterm bound the NtpE-F complex stronger than NtpF only, suggesting that NtpE increases the binding affinity between NtpI Nterm and NtpF. Purified NtpE-F-I Nterm complex appeared to be monodisperse, and the molecular masses estimated from analytical ultracentrifugation and small-angle x-ray scattering (SAXS) indicated that the ternary complex is formed with a 1:1:1 stoichiometry. A low resolution structure model of the complex produced from the SAXS data showed an elongated "L" shape. The vacuolar ATPases (V-ATPases)2 function as ATP-dependent proton pumps in the membranes of acidic organelles and in plasma membranes of eukaryotic cells. This acidification is involved in concentration of neurotransmitters, processing of secretory proteins, endocytosis, and other important cellular processes (1). The V-ATPase contains a globular catalytic domain, V 1 , which hydrolyzes ATP, attached by central and peripheral stalks to an integral membrane domain, V o , which pumps ions across the membrane. ATP hydrolysis generates rotation of the central stalk and an attached membrane ring of hydrophobic subunits. Ions are pumped through a pathway at the interface between the rotating ring and a static membrane component, which is linked to the outside of the V 1 domain by the peripheral stalk (1).In yeast, the V 1 domain contains subunits A-H, while the membrane-bound V o is made of subunit a, c, cЈ, cЉ, d, and e. The core of the V 1 domain is composed of a hexameric arrangement of alternating A and B subunits responsible for ATP binding and hydrolysis. The V o domain consists of a ring of proteolipid subunits (c, cЈ, and cЉ) adjacent to subunits a and e. The V 1 and V o domains are connected by a central stalk, composed of subunits D and F of V 1 and subunit d of V o , and a few peripheral stalks, composed of subunits C, E, G, and H together with N-terminal domain of subunit a (1). Because V-ATPases function in various physiological processes, regulation of their activity is very important. The peripheral stalk subunits have been shown to play an important role in the regulation of the enzyme via a reversible dissociation an...

“…The equivalent E subunit in yeast V-type ATPase can be cross-linked to the full-length of the outside of the B subunit (53,62), placing E in the peripheral stalk. A recent study shows that the E and H subunit from Thermoplasma acidophilum, when expressed together or reconstituted, form a stable heterodimer with high affinity (63), suggesting that the H or E homodimers formed in solution or in crystals of the isolated subunit are artifacts. This was confirmed in an EM study of the T. thermophilus ATPase, where antibodies against an EG complex (equivalent to EH in archaea) attached to both sides of the head, while not or weakly binding to isolated E or G (64).…”

Section: Purification and Characterization Of The A 1 A 0 Atp Synthasmentioning

confidence: 99%

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

Vonck

Pisa

Morgner

et al. 2009

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...