Crystal structure of A3B3 complex of V-ATPase from Thermus thermophilus

Maher, M.J.; Akimoto, Satoru; Iwata, Momi; Nagata, Koji; Hori, Yoshiko; Yoshida, Masasuke; Yokoyama, Shigeyuki; Iwata, So; Yokoyama, Ken

doi:10.1038/emboj.2009.310

Cited by 61 publications

(61 citation statements)

References 39 publications

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“…In T. thermophilus V-ATPase, it has been shown that the BA interface is involved in ATP binding and catalysis, whereas the AB groove does not participate in either function (45). If the a-B interaction is purely structural, as in a stator function, one might argue that it is more likely to occur at the AB interface, so as to avoid interference with ATP binding and hydrolysis and the local conformational changes that ensue.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of Osteoclast Bone Resorption by Disrupting Vacuolar H+-ATPase a3-B2 Subunit Interaction

Kartner¹,

Yao²,

Li³

et al. 2010

Journal of Biological Chemistry

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Vacuolar H؉ -ATPases (V-ATPases) are highly expressed in ruffled borders of bone-resorbing osteoclasts, where they play a crucial role in skeletal remodeling. To discover protein-protein interactions with the a subunit in mammalian V-ATPases, a GAL4 activation domain fusion library was constructed from an in vitro osteoclast model, receptor activator of NF-B ligand-differentiated RAW 264.7 cells. This library was screened with a bait construct consisting of a GAL4 binding domain fused to the N-terminal domain of V-ATPase a3 subunit (NTa3), the a subunit isoform that is highly expressed in osteoclasts (a1 and a2 are also expressed, to a lesser degree, whereas a4 is kidney-specific). One of the prey proteins identified was the V-ATPase B2 subunit, which is also highly expressed in osteoclasts (B1 is not expressed). Further characterization, using pulldown and solid-phase binding assays, revealed an interaction between NTa3 and the C-terminal domains of both B1 and B2 subunits. Dual B binding domains of equal affinity were observed in NTa, suggesting a possible model for interaction between these subunits in the V-ATPase complex. Furthermore, the a3-B2 interaction appeared to be moderately favored over a1, a2, and a4 interactions with B2, suggesting a mechanism for the specific subunit assembly of plasma membrane V-ATPase in osteoclasts. Solid-phase binding assays were subsequently used to screen a chemical library for inhibitors of the a3-B2 interaction. A small molecule benzohydrazide derivative was found to inhibit osteoclast resorption with an IC 50 of ϳ1.2 M on both synthetic hydroxyapatite surfaces and dentin slices, without significantly affecting RAW 264.7 cell viability or receptor activator of NF-B ligand-mediated osteoclast differentiation. Further understanding of these interactions and inhibitors may contribute to the design of novel therapeutics for bone loss disorders, such as osteoporosis and rheumatoid arthritis. V-ATPases2 are proton pumps ubiquitous in eukaryotic cells, where they acidify numerous intracellular membrane compartments, including Golgi, endosomes, lysosomes, clathrin-coated vesicles, chromaffin granules, and insulin secretory granules (reviewed in Refs. 1-11). V-ATPases also pump protons across the plasma membrane into the extracellular space in a variety of specialized cells, including renal duct intercalated cells, clear cells of the epididymis, and osteoclasts. Here they are involved in functions, including pH homeostasis, sperm maturation, and bone resorption and remodeling. Mutations in V-ATPase subunits lead to diseases, such as renal tubular acidosis and osteopetrosis. Furthermore, their inappropriate activity can contribute to osteoporosis and tumor metastasis (12-15).V-ATPases are multisubunit molecular motors, structurally analogous to the F 1 F 0 -ATP synthases (F-ATPases), but working "in reverse" (16 -20). Thus, V-ATPases create proton gradients across membranes by utilizing the energy of ATP hydrolysis, rather than utilizing the potential energy of proton gradients to ...

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Section: Discussionmentioning

confidence: 99%

Inhibition of Osteoclast Bone Resorption by Disrupting Vacuolar H+-ATPase a3-B2 Subunit Interaction

Kartner¹,

Yao²,

Li³

et al. 2010

Journal of Biological Chemistry

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“…ATP hydrolysis induces the rotation of the central stalk (DFd complex) and an attached membrane c ring, which causes ion pumping at the interface between the c ring and a subunit (9). To understand the precise function and rotational mechanism of V-ATPase, the details of these subunit structures and their subunit-subunit interactions should be elucidated.The crystal structures of several subunits (C and F) and subcomplexes (A 3 B 3 and EG) of T. thermophilus V-ATPase have been obtained at high resolution (5,(12)(13)(14). The crystal structure of the Tt-A 3 B 3 DF complex of the ATP synthase was recently obtained, although the structure was built as a polyalanine model at 4.5-Å resolution, and the foot portion (residues 56-131) of the axial D subunit was missing (15).…”

mentioning

confidence: 99%

“…The crystal structures of several subunits (C and F) and subcomplexes (A 3 B 3 and EG) of T. thermophilus V-ATPase have been obtained at high resolution (5,(12)(13)(14). The crystal structure of the Tt-A 3 B 3 DF complex of the ATP synthase was recently obtained, although the structure was built as a polyalanine model at 4.5-Å resolution, and the foot portion (residues 56-131) of the axial D subunit was missing (15).…”

mentioning

confidence: 99%

Crystal structure of the central axis DF complex of the prokaryotic V-ATPase

Saijo

Arai

Hossain

et al. 2011

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

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V-ATPases function as ATP-dependent ion pumps in various membrane systems of living organisms. ATP hydrolysis causes rotation of the central rotor complex, which is composed of the central axis D subunit and a membrane c ring that are connected by F and d subunits. Here we determined the crystal structure of the DF complex of the prokaryotic V-ATPase of Enterococcus hirae at 2.0-Å resolution. The structure of the D subunit comprised a long lefthanded coiled coil with a unique short β-hairpin region that is effective in stimulating the ATPase activity of V 1 -ATPase by twofold. The F subunit is bound to the middle portion of the D subunit. The C-terminal helix of the F subunit, which was believed to function as a regulatory region by extending into the catalytic A 3 B 3 complex, contributes to tight binding to the D subunit by forming a three-helix bundle. Both D and F subunits are necessary to bind the d subunit that links to the c ring. From these findings, we modeled the entire rotor complex (DFdc ring) of V-ATPase.I on-transporting rotary ATPases are divided into three types based on their function and taxonomic origin: F-, V-, and A-type ATPases. F-ATPases function as ATP synthases in mitochondria, chloroplasts, and oxidative bacteria (1). V-ATPases function as proton pumps in the membranes of acidic organelles and plasma membranes of eukaryotic cells (2). A-ATPases in archaea function as ATP synthases similar to the F-ATPases (the "A" designation in A-type refers to archaea), although the structure and subunit composition of A-ATPases are more similar to those of V-ATPases (3). These ATPases possess an overall similar structure that is composed of a hydrophilic catalytic portion (F 1 -, V 1 -, or A 1 -ATPase) and a membrane-embedded ion-transporting portion (F o -, V o -, or A o -ATPase), and they have a similar reaction mechanism that occurs through rotation (2).V-ATPases are found in bacteria, such as Thermus thermophilus and Enterococcus hirae. In T. thermophilus, V-ATPase physiologically functions as an ATP synthase (4). Therefore, it has sometimes been called an A-ATPase, although T. thermophilus is a eubacterium that does not belong to archaea (5). However, the E. hirae V-ATPase acts as a primary ion pump (6), which transports Na þ or Li þ instead of H þ (7). The enzyme is composed of nine subunits (Eh-A, -B, -d, -D, -E, -F, -G, -a, -c; previously designated as NtpA, -B, -C, -D, -E, -G, -F, -I, -K) having amino acid sequences that are homologous to those of the corresponding subunits (A, B, d, D, E, F, G, a, c) of eukaryotic V-ATPases (7) (see Fig. 1). The core of the V 1 domain of this ATPase is composed of a hexameric arrangement of alternating A and B subunits responsible for ATP binding and hydrolysis (8). The V o domain, in which rotational energy is converted to drive Na þ translocation, is composed of oligomers of the 16-kDa c subunits and an a subunit (9, 10). The V 1 and V o domains are connected by a central stalk, which is composed of D, F, and d subunits, and two peripheral stalks, which a...

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“…Surprisingly, even in the absence of nucleotide, the three catalytic sites formed by the same AB pair types show different conformations from one another. Previous reports of the apo structures of the thermophilic α 3 β 3 F 1 motor [18] and the A 3 B 3 unit of the V 1 motor [21] both showed 3-fold rotational symmetry. Therefore, our structure is the first report of a motor protein structure with asymmetrical arrangement at the catalytic head.…”

Section: Asymmetrical Crystal Structures Of a 3 B 3 Complexmentioning

confidence: 99%

Operating Principles of Rotary Molecular Motors: Differences between F<sub>1</sub> and V<sub>1</sub> Motors

Murata

2014

Biophysics

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Among the many types of bioenergy-transducing machineries, F-and V-ATPases are unique bio-and nanomolecular rotary motors. The rotational catalysis of F 1 -ATPase has been investigated in detail, and molecular mechanisms have been proposed based on the crystal structures of the complex and on extensive singlemolecule rotational observations. Recently, we obtained crystal structures of bacterial V 1 -ATPase (A 3 B 3 and A 3 B 3 DF complexes) in the presence and absence of nucleotides. Based on these new structures, we present a novel model for the rotational catalysis mechanism of V 1 -ATPase, which is different from that of F 1 -ATPases.Key words: V-ATPase, F-ATPase, ATP, Crystal structure, molecular mechanism Several mechanochemical energy transducers or molecular motors are present in living cells. These include linear motors such as myosins that function in muscle contraction and kinesins/dyneins that utilize the energy from ATP hydrolysis for vesicle transport. Molecular rotary motors, which include F-ATPases and V-ATPases, are another class of motors. This class utilizes the energy from ATP hydrolysis and the ionic current for axis rotation; flagellar motors also utilize ionic currents for rotation. F-and V-ATPases resemble one another and consist of a hydrophilic rotary motor region (F 1 and V 1 ) and a hydrophobic membrane embedded region (F o and V o ). Rotation of the motor axis is coupled to ATP hydrolysis, whereas rotation of the embedded region drives proton transport across membranes. In this review, we focus on the rotary motor regions (F 1 and V 1 ). Although numerous structural and biophysical studies have been conducted, the molecular rotational mechanism of the F 1 motor is not fully understood. We also introduce our recent achievements in the three-dimensional structural analysis of the V 1 motor and discuss the unresolved questions related to the molecular mechanism of F 1 motors. This review is an extension of a previous Japanese review by Murata (2014) [1], which discusses recent progress in determining the rotation mechanism of mammalian F 1 motors [2,3] in addition.

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Crystal structure of A3B3 complex of V-ATPase from Thermus thermophilus

Cited by 61 publications

References 39 publications

Inhibition of Osteoclast Bone Resorption by Disrupting Vacuolar H+-ATPase a3-B2 Subunit Interaction

Inhibition of Osteoclast Bone Resorption by Disrupting Vacuolar H+-ATPase a3-B2 Subunit Interaction

Crystal structure of the central axis DF complex of the prokaryotic V-ATPase

Operating Principles of Rotary Molecular Motors: Differences between F<sub>1</sub> and V<sub>1</sub> Motors

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