Dimeric Core Structure of Modular Stator Subunit E of Archaeal H+-ATPase

Lokanath, N.K.; Matsuura, Yoshinori; Kuroishi, Chizu; Takahashi, Naoko; Kunishima, N.

doi:10.1016/j.jmb.2006.11.088

Cited by 37 publications

(85 citation statements)

References 45 publications

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“…Although the archaeal EG heterodimer is shorter in length and more stable than yeast EG, their secondary structure predictions and biological functions are similar (50,51), indicating that they may be used as a structural model for eukaryotic EG. NMR spectroscopy and domain mapping experiments (50) showed that although subunit G is entirely composed of ␣ helix, subunit E contains a globular C-terminal domain of mixed ␣␤ character as seen earlier in the crystal structure of the isolated domain (52). Overall, the EG heterodimer is elongated with the N termini involved in a coiled coil, whereas the C-terminal globular domain of the E subunit is in contact only with the very C-terminal ␣ helix of the G subunit (50).…”

Section: Discussionmentioning

confidence: 64%

Domain Characterization and Interaction of the Yeast Vacuolar ATPase Subunit C with the Peripheral Stator Stalk Subunits E and G

Oot

Wilkens

2010

Journal of Biological Chemistry

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To determine which subunit C domain binds EG with high affinity, we have generated C head and C foot and characterized their interaction with subunit EG heterodimer. Our findings indicate that the high affinity site for EGC interaction is C head . In addition, we provide evidence that the EGC head interaction greatly stabilizes EG heterodimer.2 is a ubiquitous multisubunit enzyme that couples the free energy of ATP hydrolysis to active proton transport. In all eukaryotic cells, V-ATPase function sets up an electrochemical potential and acidifies intracellular compartments (1-5). These functions make the V-ATPase a crucial enzyme involved in intracellular traffic, vesicular transport, endo/exocytosis, and pH homeostasis. In the specialized cells of higher eukaryotes, plasma membrane-associated V-ATPases serve to acidify the extracellular space. The malfunction of the V-ATPase in these cells has been linked to a range of diseases including osteoporosis, diabetes, renal tubular acidosis, and cancer (6 -9). As shown in EM images and reconstructions, the V-ATPase is bilobular in overall structure, typical of the rotary ATPases (10 -13). These two lobes are composed of distinct functional domains, a soluble catalytic sector, V 1 , and a membrane integral proton pore, V o . The V 1 sector contains subunits A 3 B 3 (C)DE 3 FG 3 H, and the V o is made of ac 3-4 cЈcЉde. Most V-ATPase subunits have functional and structural counterparts in the A-and F-type motors; however, only the nucleotide-binding and proteolipid subunits share significant sequence identity. Much like in the F-ATP synthase, the AB subunits come together in an alternating arrangement to form a catalytic hexamer with nucleotide-binding sites located at the AB interfaces (14, 15). ATP hydrolysis-driven rotation of a central rotor domain, composed of subunits DFd and a ring of the proteolipid subunits, c 3-4 cЈcЉ, is coupled to proton translocation along the interface of the proteolipid ring and the C-terminal domain of the a subunit. During catalysis, three peripheral stalks (each formed by a subunit EG heterodimer) in conjunction with subunits C and H and the N-terminal domain of subunit a (a NT ), resist the torque of rotation, thus keeping the A 3 B 3 hexamer static and allowing for rotation to be productive.Unlike F-ATPase, the activity of eukaryotic V-ATPase is regulated by a unique mechanism of reversible dissociation, a process first described for the enzymes from yeast and insect (16, 17) but more recently also found in cells of higher animals (18 -20). In yeast under conditions of nutrient deprivation, the V 1 sector dissociates from the V o sector, and both are functionally silenced (21,22). Although dissociated, a single V 1 subunit, C, is released into the cytosol and is reincorporated into the enzyme upon nutrient readdition (16). Because the C subunit has been proposed to be a molecular switch for controlled enzyme dissociation, the interactions between the C subunit and its intraenzyme binding partners are of particular interest.The crystal ...

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Section: Discussionmentioning

confidence: 64%

Domain Characterization and Interaction of the Yeast Vacuolar ATPase Subunit C with the Peripheral Stator Stalk Subunits E and G

Oot

Wilkens

2010

Journal of Biological Chemistry

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“…S1c). The x-ray structure of the E C domain from P. horikoshii has recently been determined (13); it has an elongated shape ϳ75 Å long with ␣-helical and ␤-strand elements and forms a tight dimer in the crystal structure, with all the predicted secondary structure elements (supplemental Fig. S1c).…”

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

confidence: 99%

“…S1c). The ␣-helical N-terminal domain (E N ) is homologous to H and to the F 1 F 0 b-subunit and interacts with the H subunit (13). 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.…”

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

confidence: 99%

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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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“…A crystal structure of the central stalk subunit C was determined from the T. thermophilus enzyme (24). From the peripheral stalk, only the structure of the globular C-terminal domain of subunit E from P. horikoshii OT3 has been determined (25). A crystal structure has been determined for the proteolipid K-subunit ring for the V-ATPases from Enterococcus hirae (26), and atomic models have also been determined for eukaryotic V-ATPase subunits C (27) and H (28) from S. cerevisiae, which have no counterparts in the bacterial enzyme studied here.…”

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confidence: 99%

Structure of intact Thermus thermophilus V-ATPase by cryo-EM reveals organization of the membrane-bound V _O motor

Lau

Rubinstein

2010

Proc. Natl. Acad. Sci. U.S.A.

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The eubacterium Thermus thermophilus uses a macromolecular assembly closely related to eukaryotic V-ATPase to produce its supply of ATP. This simplified V-ATPase offers several advantages over eukaryotic V-ATPases for structural analysis and investigation of the mechanism of the enzyme. Here we report the structure of the complex at ∼16 Å resolution as determined by single particle electron cryomicroscopy (cryo-EM). The resolution of the map and our use of cryo-EM, rather than negative stain EM, reveals detailed information about the internal organization of the assembly. We could separate the map into segments corresponding to subunits A and B, the threefold pseudosymmetric C-subunit, a central rotor consisting of subunits D and F, the L-ring, the stator subcomplex consisting of subunits I, E, and G, and a micelle of bound detergent. The architecture of the V O region shows a remarkably small area of contact between the I-subunit and the ring of L-subunits and is consistent with a two half-channel model for proton translocation. The arrangement of structural elements in V O gives insight into the mechanism of torque generation from proton translocation. membrane protein | single particle analysis V acuolar-type ATPases (V-ATPases) in eukaryotes function as ATP-driven proton pumps that acidify intracellular compartments including lysosomes, endosomes, and secretory vesicles. This acidification, in turn, affects diverse processes including protein sorting and degradation, overall ion homeostasis, and protection of cells from oxidative stress (1). Extracellular acidification by V-ATPases is linked to tumor invasion and metastasis and osteoporosis (2). F-type ATP synthases and V-type ATPases are evolutionarily related but differ in the details of subunit composition and arrangement. Both F-and V-type ATPases use a rotary catalytic mechanism where proton translocation through the membranebound F O or V O region, respectively, generates a torque on a rotor subcomplex that drives ATP synthesis in the F 1 or V 1 region. The enzymes can also run in the opposite direction with ATP hydrolysis in the F 1 or V 1 region resulting in proton pumping through F O or V O . This mechanism has been the subject of a large body of research for F-type ATP synthases (e.g., 3-5), but there has also been direct demonstration of rotary catalysis for V-ATPases (6). Some archaea and eubacteria use a complex more closely related to VATPase than F-type ATP synthase, sometimes called an A-ATPase, to generate their supply of ATP (7).The V-ATPase from the eubacterium Thermus thermophilus is composed of nine different subunits with a stoichiometry of A 3 B 3 CDE 2 FG 2 IL 12 (Fig. S1). Subunit nomenclature for this family of enzymes differs between F-and V-type complexes and from organism to organism. Where the eukaryotic ATP synthase F 1 catalytic region consists of α 3 β 3 γδε, the V-ATPase V 1 catalytic region consists of B 3 A 3 DF with no equivalent of the ε-subunit. The catalytic A-subunit of V-ATPase is homologous to the F-type ATP synthas...

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Dimeric Core Structure of Modular Stator Subunit E of Archaeal H+-ATPase

Cited by 37 publications

References 45 publications

Domain Characterization and Interaction of the Yeast Vacuolar ATPase Subunit C with the Peripheral Stator Stalk Subunits E and G

Domain Characterization and Interaction of the Yeast Vacuolar ATPase Subunit C with the Peripheral Stator Stalk Subunits E and G

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

Structure of intact Thermus thermophilus V-ATPase by cryo-EM reveals organization of the membrane-bound V _O motor

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Dimeric Core Structure of Modular Stator Subunit E of Archaeal H+-ATPase

Cited by 37 publications

References 45 publications

Domain Characterization and Interaction of the Yeast Vacuolar ATPase Subunit C with the Peripheral Stator Stalk Subunits E and G

Domain Characterization and Interaction of the Yeast Vacuolar ATPase Subunit C with the Peripheral Stator Stalk Subunits E and G

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

Structure of intact Thermus thermophilus V-ATPase by cryo-EM reveals organization of the membrane-bound V O motor

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Structure of intact Thermus thermophilus V-ATPase by cryo-EM reveals organization of the membrane-bound V _O motor