Cross-talk in the A1-ATPase from Methanosarcina mazei Gö1 Due to Nucleotide Binding

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In Archaea, bacteria, and eukarya, ATP provides metabolic energy for energy-dependent processes. It is synthesized by enzymes known as A-type or F-type ATP synthase, which are the smallest rotatory engines in nature (Yoshida, M., Muneyuki, E., and Hisabori, T. ATP synthases/ATPases are present in every life form and are the most important enzymes for the energy metabolism of the cell (1). They catalyze the formation of ATP at the expense of the transmembrane electrochemical ion gradient. They arose from a common ancestor that underwent structural and functional changes leading to three distinct classes of A 1 A 0 , 1 F 1 F 0 , and V 1 V 0 ATP synthases/ATPases. The V-type ATPases, found in organelles of eukaryotes, lost their ability to synthesize ATP. Their function is to create steep ion gradients at the expense of ATP hydrolysis (2). Archaea contain ATPases, the A 1 A 0 ATP synthases, that are structurally similar to V 1 V 0 ATPases but synthesize ATP like the F-type ATPases. The genomic sequences available today show that the overall subunit composition of the A 1 A 0 ATP synthases is very similar to the V 1 V 0 ATPases. For example, the A 1 A 0 ATP synthases contain duplicated and even triplicated K subunits (proteolipids) (3, 4). The A 1 A 0 ATP synthase has at least nine subunits (A 3 B 3 CDEFHIK X ), but the actual subunit stoichiometry, especially regarding the proteolipid subunits K in A ATPases, is different in various organisms (12, 6, 4, or as suggested by genomic data, only 1 (5)). The A 1 A 0 ATP synthase is composed of a water-soluble A 1 ATPase and an integral membrane subcomplex, A 0 . ATP is synthesized or hydrolyzed on the A 1 headpiece consisting of an A 3 B 3 domain, and the energy that is provided for or released during that process is transmitted to the membrane-bound A 0 domain (4). The energy coupling between the two active domains occurs via the so-called stalk part, an assembly proposed to be composed of the subunits C, D, and F (6, 7). Insight in the molecular structure of the A-type ATP synthases comes from small-angle x-ray scattering data of the A 1 ATPase from Methanosarcina mazei Gö1 whose A 1 domain is made up of the five different subunits, A 3 B 3 CDF (8). The data have shown that the hydrated A 1 ATPase is rather elongated with a headpiece of 10 ϫ 9.4 nm in dimension and a stalk of ϳ8.4 nm in length. A comparison of the central stalk of this A 1 complex with bacterial F 1 and eukaryotic V 1 ATPase indicates different lengths of the stalk domain (8, 9). Image processing of electron micrographs of negatively stained A 1 ATPase from M. mazei Gö1 (7) has revealed that the headpiece consists of a pseudohexagonal arrangement of six masses surrounding a seventh mass. These barrel-shaped masses of ϳ3.2 and 2.8 nm in diameter and 7.5 and 5.0 nm in length, which consist of the major subunits A and B, are arranged in an alternating manner (7). The hexagonal barrel of subunits A and B encloses a cavity of ϳ2.3 nm in the middle in which part of the central stalk is asymmetrically located...

Section: Subunit Composition and Electronmentioning

confidence: 99%

mentioning

confidence: 99%

Structure and Subunit Arrangement of the A-type ATP Synthase Complex from the Archaeon Methanococcus jannaschii Visualized by Electron Microscopy

Coskun

Chaban

Lingl

et al. 2004

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“…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.…”

mentioning

confidence: 99%

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

Vonck

Pisa

Morgner

et al. 2009

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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...

“…5A) which can be cross-linked to the nucleotide-binding subunits A and B. Recently, CuCl 2 -mediated disulfide formation indicated that the N and C termini of subunit D are in close proximity with the catalytic A subunit after addition of MgADP or the hydrolyzable MgATP (28). Taken together, the nucleotide-dependent alterations of the stalk subunits C, D, and F described and the close proximity of these subunits to the major subunits A and/or B would provide coupling between the catalytic site events via subunit D into the central CDF stalk domain, and thereby the physical and structural linkage between the A 3 B 3 headpiece and the ion-conducting A 0 part.…”

Section: Discussionmentioning

confidence: 99%

“…In summary, the first three-dimensional reconstruction of the A 1 -ATPase from M. mazei Gö1 presented provides the structural basics toward a fuller understanding of the mechanistic events occurring in this enzyme (28). Both features, a large cavity surrounded by the A 3 B 3 barrel and the shaft inside this cavity and presumably formed by subunit D, are similar to the structural elements in the related F-and V-ATPases, in which they facilitate the rotational movements inside these complexes.…”

Section: Discussionmentioning

confidence: 99%

Three-dimensional Organization of the Archaeal A1-ATPase from Methanosarcina mazei Gö1

Coskun

Radermacher

Müller

et al. 2004

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Methanogenic, halophilic, and thermophilic Archaea synthesize ATP by means of ion gradient-driven phosphorylation. Although it was speculated for some time, due to the lack of indepth information, that the ATP synthases of Archaea may be either F 1 F 0 -or V 1 V 0 -like enzymes, it is now clear that they evolved as a separate class of ATPases/ATP synthases, the A 1 A 0 -ATPase/synthases (1-4). This class of enzymes is different from F 1 F 0 -or V 1 V 0 -ATPases by function, subunit composition, regulation, and structure (1). The A 1 A 0 -ATPase has at least nine subunits (A 3 :B 3 :C:D:E:F:H:I:K x ), but the actual subunit stoichiometry, especially regarding the proteolipid subunits K in AATPases, is different in various organisms (12, 6, 4 or, as suggested by genomic data, only 1 (5)). As suggested by its bipartite name, the A 1 A 0 -ATPase is composed of a water-soluble A 1 -ATPase and an integral membrane subcomplex, A 0 . ATP is synthesized or hydrolyzed on the A 1 headpiece, consisting of an A 3 B 3 domain, and the energy provided for or released during that process is transmitted to the membrane-bound A 0 domain (1). The energy coupling between the two active domains occurs via the so-called stalk part, an assembly proposed to be composed by the subunits C, D, and F (2). The archaeal A 1 A 0 -ATPase/synthase is regarded as a chimeric protein in which the membrane domain is closely related to F 1 F 0 -ATP synthases but the catalytic subunits closely to V 1 V 0 -ATPases (3, 4).The A 1 -ATPase from Methanosarcina mazei Gö1 is made up of at least the five different subunits A-D and F with apparent molecular masses of 65, 54, 41, 28, and 9 kDa (6). The enzyme, as shown by small angle x-ray scattering data, consists of an ϳ10-nm long headpiece and an 8.5-nm high and 6.0-nm diameter stalk (7). A comparison of the central stalk of this A 1 complex with bacterial F 1 -and V 1 -ATPase indicates different lengths of the stalk domain (7-9). The prevailing view is, however, that ATP synthesis/hydrolysis in the A 1 headpiece is coupled to ion flow in A 0 through rotational movements of the central stalk subunit(s) as demonstrated for the F 1 (reviewed in Refs. 10 and 11) and the Thermus thermophilus A 1 /V 1 -ATPase (12). (Note, the so-called V 1 V 0 -ATPase from T. thermophilus, which also synthesizes ATP, is of archaeal origin (13, 14); therefore, the ATPase headpiece will be considered A 1 /V 1 -ATPase throughout this work.)Here we describe a modified isolation procedure of the A 1 -ATPase from M. mazei Gö1, which facilitates the first threedimensional reconstruction of this enzyme by using tilt pairs of negatively stained molecules. The structure adds to the emerging picture of A 1 in which three copies of A and B subunits are arranged as a hexagonal barrel, enclosing a large cavity in which a shaft is asymmetrically located. This three-dimensional model allows comparison with structural models of related F 1 -and V 1 -ATPase determined by crystallography (15) and electron microscopy (16,17). Furthermore, insights i...