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. 2009 Sep 25;10(11):1228–1234. doi: 10.1038/embor.2009.202

Inter-subunit interaction and quaternary rearrangement defined by the central stalk of prokaryotic V1-ATPase

Nobutaka Numoto 1,†,*, Yu Hasegawa 1,*, Kazuki Takeda 1, Kunio Miki 1,2,a
PMCID: PMC2775172  PMID: 19779483

Abstract

V-type ATPases (V-ATPases) are categorized as rotary ATP synthase/ATPase complexes. The V-ATPases are distinct from F-ATPases in terms of their rotation scheme, architecture and subunit composition. However, there is no detailed structural information on V-ATPases despite the abundant biochemical and biophysical research. Here, we report a crystallographic study of V1-ATPase, from Thermus thermophilus, which is a soluble component consisting of A, B, D and F subunits. The structure at 4.5 Å resolution reveals inter-subunit interactions and nucleotide binding. In particular, the structure of the central stalk composed of D and F subunits was shown to be characteristic of V1-ATPases. Small conformational changes of respective subunits and significant rearrangement of the quaternary structure observed in the three AB pairs were related to the interaction with the straight central stalk. The rotation mechanism is discussed based on a structural comparison between V1-ATPases and F1-ATPases.

Keywords: V-ATPase, asymmetric, crystal structure, rotation, vacuolar type

Introduction

V-type ATPases (V-ATPases) couple the transfer of protons or sodium ions across the membrane with ATP hydrolysis or synthesis through a rotary catalytic mechanism (Yoshida et al, 2001; Forgac, 2007). V-ATPases occur in the membranes of acidic organelles, such as lysosomes and endosomes in eukaryotic cells, maintaining acidic pH by pumping protons coupled with ATP hydrolysis. V-ATPases are also found in the plasma membranes of archaea and some eubacteria (Yokoyama et al, 1990). These prokaryotic V-ATPases and closely related A-ATPases (Grüber & Marshansky, 2008) are primarily responsible for ATP synthesis, which is the reverse of the ATP-driven proton pumping reaction.

V-ATPases are thought to originate from an ancestral enzyme in common with F-ATPases (Mulkidjanian et al, 2007), because they have similar structural and functional features (Noji et al, 1997; Imamura et al, 2003). Both enzymes consist of water-soluble components (V1-ATPases and F1-ATPases, respectively), catalyse ATP hydrolysis and synthesis, and contain membrane-embedded components (Vo and Fo, respectively) involved in proton and ion pumping. These two components are linked through a central stalk (a rotor) and peripheral stalks (stators).

The V-ATPase of the thermophilic eubacterium, Thermus thermophilus, synthesizes ATP (Yokoyama et al, 1998), whereas isolated soluble V1-ATPase has been shown to exert ATP hydrolysis activity, as in the case of F1-ATPase. The subunit compositions for V1-ATPases and F1-ATPases are A3B3DF and α3β3γδɛ, respectively (Yokoyama et al, 2000, 2003; Yoshida et al, 2001; Imamura et al, 2003). Despite the similar structural compositions of V1-ATPases and F1-ATPases, detailed analyses of the rotation kinetics have revealed divergence in the torque and rotation steps. The estimated torque of F1-ATPase is about 46 pN nm (Yasuda et al, 1998; Imamura et al, 2005), whereas that of V1-ATPase is approximately 35 pN nm (Hirata et al, 2003; Imamura et al, 2005). The rotation proceeds in 120° steps in F1-ATPase, and the 120° rotation can be separated into 80° and 40° sub-steps during hydrolysis of one molecule of ATP (Shimabukuro et al, 2003). By contrast, V1-ATPase shows 120° rotation without sub-steps (Imamura et al, 2005). These observations suggest strongly that V1-ATPases and F1-ATPases utilize different rotation mechanisms due to the different subunit interactions, structural transitions and torque generation mechanism.

Crystal structures of A and B subunits of A-ATPase (Maegawa et al, 2006; Schäfer et al 2006; Kumar et al, 2008), and the F subunit of V-ATPase (Makyio et al, 2005) have been determined in their isolated states. However, structural information for the D subunit (homologous to the γ-subunit of F1-ATPase) is still unclear. Electron microscopic studies at about 20 Å resolution have provided only limited information about the subunit interactions (Bernal & Stock, 2004; Zhang et al, 2008). As the asymmetrical interaction between the central stalk and cylindrical A3B3 hexamer is thought to be of primary importance in the rotation mechanism, as in the case of F1-ATPase (Yoshida et al, 2001; Kabaleeswaran et al, 2009), the detailed structure of the whole V1-ATPase complex is indispensable for understanding the rotation mechanism.

Here, we report the crystal structure of V1-ATPase from T. thermophilus. The rotation mechanism is discussed based on the interactions between subunits, and several meaningful differences have been observed between V1-ATPases and F1-ATPases in this regard.

Results And Discussion

Structure determination

Intact V1-ATPase (Fig 1A) was crystallized in nucleotide-free and nucleotide-bound forms. For the nucleotide-bound form, crystals were obtained by co-crystallization with Mg2+ ADP and aluminium fluoride (AlFx). The structures were determined by using a combination of molecular replacement and multiple isomorphous replacement with anomalous scattering methods (Fig 1B; supplementary Table S1 online). The nucleotide-free structure was built as poly-alanine models and finally refined at 4.8 Å resolutions. For the D subunit, simulated annealing with a tight secondary structure restraint was carried out to fit the models of long helices into the electron density. Refinements were carried out by using rigid body refinement with several rigid domain definitions in each subunit (supplementary information online), and B-factor refinement using the same domain definitions. Two molecules of V1-ATPases are contained in the asymmetric unit, and the final correlation coefficient of 0.920, for non-crystallographic symmetry averaging, indicates that these two complexes take almost the same conformation. The model of the nucleotide-bound form was built from the nucleotide-free structure and refined by using the same procedures as used for the nucleotide-free structure at 4.5 Å resolution (Fig 1C,D). The structures of these forms were almost the same, with an r.m.s.d. value of 0.44 Å for all Cα atoms (more than 3,266 atoms) in the superposition. Statistics are shown in Table 1.

Figure 1.

Figure 1

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Crystal structure of V1-ATPase from Thermus thermophilus. (A) Schematic representation of the subunit composition of V-ATPase. The plasma membrane is indicated as a grey band. V1-ATPase subunits are coloured. (B) Peaks on isomorphous and anomalous Fourier maps for a platinum derivative are shown as green and red meshes, whereas those for a mercury derivative are shown in purple and cyan. (C) An electron density map after phase combination is shown at the 1.2σ level. Electron density at one of the insertion domains of the A subunits (upper left of the molecule) is clearly observed. By contrast, corresponding electron densities at the other two domains are very weak (∼0.5σ level; upper right and back side of the molecule), probably due to the lack of crystal contact. Therefore, the models for these two domains were built according to the model for the well-defined domain, and drawn as a half-transparent description. (D) Close-up view of the contact regions between the carboxy-terminal domain of the A and B subunits and the D subunit.

Table 1.

Data collection, phasing and refinement statistics

  Nucleotide free Nucleotide bound
Data collection    
 Space group P321
 Unit-cell parameters (Å)    
  a 380.7 381.6
  c 148.0 147.7
 Resolutions (Å) 50–4.80 (4.97–4.80) 50–4.50 (4.66–4.50)
 Number of observations 297,505 359,640
 Number of unique  reflections 57,305 70,598
 Completeness (%) 95.2 (89.8) 96.9 (92.7)
 Average I/σ(I) 13.4 (2.1) 10.3 (2.3)
 Redundancy 5.2 (2.6) 5.1 (2.8)
 Rsym* (%) 6.8 (37.5) 8.7 (41.7)
Phasing    
 Combined figure of merit† 0.72  
 NCS correlation coefficient 0.920  
Refinement    
 Protein residues 6,532 6,532
 ADP molecules 0 4
 R‡/Rfree§ (%) 44.1/45.2 43.0/43.6
Values in parentheses are for the highest resolution shell; * Rsym = ∑∑i ∣ I (h) − I (h)i ∣∑∑i I (h), where I(h) is the mean intensity; †Figure of merit is 〈cos (Δαh)〉, where Δαh is the error in the phase angle; ‡ R = ∑∣ Fo − Fc∣/∑∣Fo∣, where Fo is the observed structure factor amplitude and Fc is the calculated structure factor amplitude; §Rfree is as for R but calculated using a random set containing 5% of the data that were excluded during refinement.
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Overall structure

V1-ATPase has dimensions of approximately 120 Å (height) and 120 Å (diameter) at its maximum (Fig 2A,B). The central stalk composed of the D and F subunits protrudes about 40 Å from the A3B3 sub-assembly, similarly to that of F1-ATPase consisting of γ-, δ- and ɛ-subunits. The most marked features of V1-ATPase are attributed to the structure of the catalytic A subunit. An insertion domain between the amino-terminal β-barrel domain and nucleotide-binding domain is located outward from the spherical body of the A3B3 hexamer (Fig 2C). In addition, the carboxy-terminal helices of the A subunit also protrude. Owing to these protruding domains, the overall shape of the A3B3 hexamer is trapezoidal, with an increase of 20 Å in the maximum diameter compared with that of F1-ATPase.

Figure 2.

Figure 2

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Ribbon diagrams of V1-ATPase. (A) A side view of V1-ATPase. The A, B, D and F subunits are shown in green, yellow, purple and cyan, respectively. Approximate dimensions are indicated. (B) V1-ATPase shown in (A) is rotated by about 60° around the perpendicular axis. (C) A top view looking towards the membrane of V1-ATPase. All panels show the nucleotide-bound form.

The A3B3 hexamer is assembled asymmetrically around a central stalk. Superposition (Fig 3A) and comparison of buried surface areas (supplementary Table S2 online) of three AB pairs with three αβ pairs clearly indicate that the AWBW pair forms a wide-open conformation. This also features in an αEβE pair, in which the active site is located at the interface region. By contrast, the ANBN and AN′BN′ pairs form a narrowly closed conformation, as do the αTPβTP and αDPβDP pairs. The AWBW pair can be superposed with a significantly lower r.m.s.d. value on the αEβE pair than on the αTPβTP or αDPβDP pair of bovine F1-ATPase (supplementary Table S3 online). Consequently, ANBN and AN′BN′ pairs correspond to αTPβTP and αDPβDP pairs with the lowest r.m.s.d. values, respectively. We use the notations of the A and B subunits based on the quaternary state of each AB pair in this paper.

Figure 3.

Figure 3

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Ternary and quaternary structure of A and B subunits. (A) A sequential view of rearrangement at the interface between A (in green) and B (in yellow) subunits. The α- and β-subunits of F1-ATPase are shown in grey for comparison. Some incompatibility is observed between V1-ATPase and F1-ATPase in their carboxy-terminal domains, as additional helices are attached to the catalytic A subunit in V1-ATPase and the non-catalytic α-subunit in F1-ATPase. (B) A superposition of three catalytic A subunits. (C) A superposition of three non-catalytic B subunits. (D) A superposition of three AB pairs. The fit is carried out over B subunits.

The superposition of the three catalytic A subunits does not reveal the open–closed transition observed in the β-subunit of F1-ATPase (Fig 3B). However, a superposition of three B subunits indicates that only the BW subunit adopts the open conformation (Fig 3C). In contrast to the β-subunit of F1-ATPase, however, the BW subunit shows only small deviations from other B subunits.

Some significant differences are seen between the structures of A and B subunits in V1-ATPase complex and the previously reported isolated structure. The N-terminal β-barrel domains of both subunits and the interface regions between the D subunit show primarily large deviations between complex and isolated structures (supplementary Fig S1 online). These facts indicate that sufficiently functional conformations of A and B subunits could be achieved only after the formation of the V1 complex, or, at least, the A3B3 sub-complex.

Central stalk

Long coiled-coil helices of the D subunit are clearly seen in the electron density maps (Fig 4A; supplementary Fig S2 online). The lengths of the two helices are about 80 Å for the N-terminal helix (direction was determined in comparison with the structure and amino-acid sequence of the γ-subunit of F1-ATPase) and 110 Å for the C-terminal helix. Superposition with the γ-subunit of bovine F1-ATPase reveals the uncurved feature of these helices of V1-ATPase (Fig 4A).

Figure 4.

Figure 4

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Structure of the central stalk. (A) The coiled-coil helices of the D subunit (purple) are superposed with those of the γ-subunit of F1-ATPase (green). Superposition was carried out by using the nucleotide-binding domains of the catalytic subunits between V1-ATPase and F1-ATPase (Gibbons et al, 2000). A blue mesh indicates the model-omitted electron density maps for the coiled-coil helices of the D subunit contoured at the 1.0σ level. Interface regions to the carboxy-terminal domain of the A and B subunits are indicated with black arrows. (B) An electron density around the F subunit in an omit map is shown as a blue mesh at the 1.0σ level. The F and D subunits are shown as cyan and purple tubes, respectively. Peaks on the isomorphous and anomalous difference Fourier map (contoured 4.0σ and 3.0σ level, respectively) of the platinum derivative are indicated as green and red surfaces. Cα atoms of the methionine residues are shown as orange spheres as a reference for the platinum-binding sites. (C) The D (purple) and F (cyan) subunits are superposed with the γ-subunit of F1-ATPase (grey). The superposition was carried out by fitting the α/β-domain of the γ-subunit to the F subunit. (D) The foot portion of the central stalk. Residual electron densities (1.2σ level) around the stalk are represented as yellow surfaces. Side chains of Glu 8 and Ala 75 are represented as red sticks.

A significant electron density around the foot of the protruding region of the central stalk can be seen, and residues 1–75 of the structure of the F subunit (Makyio et al, 2005) are successfully placed in the electron density map (Fig 4B). The interpretation is validated using isomorphous/anomalous peaks (5.3 and 4.3σ at Met 1, and 7.3 and 3.1σ at Met 90 for the isomorphous and anomalous difference Fourier maps, respectively) from platinum derivative near the methionine residues. This region corresponds to the α/β-domain of the γ-subunit of F1-ATPase. It is noteworthy that both the F subunit and α/β-domain of the γ-subunit show a Rossmann fold. However, the orientations differ from each other by about 90° (Fig 4C). The remaining density and another platinum peak near the N-terminal helix of the D subunit can be interpreted as the C-terminal helix (86–98) and Met 90 of the F subunit, respectively (Fig 4B). This conformation is not compatible with either the previously expected ‘extended' or ‘retracted' forms (supplementary Fig S3 online). However, our model for the F subunit is consistent for the cross-link experiments (Makyio et al, 2005) and reconstruction experiments (Imamura et al, 2004). Glu 8 of the F subunit is favourably located to be in contact with the C subunit, which is expected to be located between the central stalk of V1-ATPase and the rotor ring of Vo (Iwata et al, 2004; Numoto et al, 2004), and Ala 75 is actually placed at the interface between the D subunit and the F subunit (Fig 2D). Moreover, the C-terminal helix has direct interactions with the C-terminal regions of A and B subunits (Fig 4D), by which the F subunit can modulate ATPase activity, as reported previously (Imamura et al, 2004).

There are some remaining densities around the coiled-coil helices of the D subunit (Fig 4D) that have not been interpreted. These densities might correspond to the non-helical middle region of the D subunit. Taking these densities into account, the overall diameter of the foot of the central stalk is estimated to be about 30 Å, which is significantly smaller than that of F1-ATPase at about 50 Å. This smaller foot of the central stalk might be a feature of the interaction with the C subunit located between V1-ATPase and Vo as an adaptor, as there is no counterpart for the C subunit in F-ATPases.

Nucleotide-binding sites

Significant peaks are seen in the difference (Fnucleotide−Ffree) electron density map at the nucleotide-binding sites of the A subunits (Fig 5A). In AN and AN′ subunits, spheroidal peaks (6.8σ and 6.7σ, the two largest peaks in the molecule) are observed at the P loops (Fig 5B,C). The superposition of the βTP and βDP subunits of ADP–AlF4−-bound F1-ATPase (Menz et al, 2001) reveals that the phosphate groups fit well with the density map, strongly suggesting that this density corresponds primarily to the bound phosphate groups of ADP. Indeed, the phase-combined, density-modified map at the nucleotide binding sites indicates that no nucleotides are bound in the nucleotide-free form (supplementary Fig S4A,C online). By contrast, a similar map for the nucleotide-bound form reveals apparently protruding electron density thought to be bound nucleotide (supplementary Fig S4B,D online). Although AlFx was included in the crystallization condition, at the present resolution it is difficult to evaluate whether bound nucleotides are ADP alone or ADP–AlFx complexes. The negative peak near the N′ site corresponds to the residues of a helix following the P loop, so that this peak might be due to some conformational change of the helix upon nucleotide binding. In fact, the structure of the P loop region in the A subunit might be flexible because large conformational changes (supplementary Fig S1 online) are observed between V1-ATPase and the isolated form (Maegawa et al, 2006).

Figure 5.

Figure 5

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Nucleotide-binding sites. (A) The bottom view of V1-ATPase. The difference (Fnucleotide−Ffree) map is shown as blue surfaces contoured at the +5.0σ level. The catalytic sites of the A subunits are indicated by red circles and possible nucleotide-binding sites of B subunits are indicated by blue circles. (B) The subunit interface between AN and BN. The difference map is shown as blue and yellow meshes contoured at the +5.0σ and −4.0σ levels, respectively. The side chain of Arg 360 and the model of ADP are drawn by the superposition of the structure of the F1-ATPase (Menz et al, 2001). (C) The subunit interface between AN′ and BN′. (D) The subunit interface between AW and BW.

However, only small positive and negative densities are seen in the difference map at the catalytic site of the AW subunit (Fig 5D). Therefore, we interpret these results as indications that nucleotides are bound in the AN and AN′ subunits, whereas no nucleotide is bound in the AW subunit. These properties of the electron density at the nucleotide-binding sites are consistent between two molecules in the asymmetric unit, except for their peak values.

Despite some evidence that the B subunit of V-ATPases binds to nucleotides (Schäfer & Meyering-Vos, 1992; Schäfer et al 2006; Kumar et al, 2008), there are no densities found in any of the non-catalytic B subunits (Fig 3A), indicating that no nucleotides are bound in the B subunits under our crystallization conditions.

Rotation mechanism of V-type ATPase

The active site of V1-ATPase consists not only of residues of the A subunit but also of Arg360 of the B subunit, which is conserved between V1-ATPases and F1-ATPases (Fig 5B–D). The ternary and quaternary structure of each AB pair in V1-ATPase is almost identical in the nucleotide-free and nucleotide-bound forms. Recently, it has been reported that the nucleotide-free structure of yeast F1-ATPase forms an asymmetric structure quite close to that of nucleotide-bound enzyme (Kabaleeswaran et al, 2009). However, the structure of the nucleotide-free α3β3 sub-complex of bacterial F1-ATPase, lacking the central stalk, reveals an almost completely threefold symmetric feature (Shirakihara et al, 1997). These facts imply that asymmetric interactions between the A3B3 hexamer and the central stalk are primarily responsible for the formation of the asymmetric quaternary structure of the AB pairs. As the quaternary arrangement of the three asymmetric AB pairs and αβ pairs are quite similar among V1-ATPase, bovine and yeast F1-ATPases from completely different crystal packing, these structures are unlikely to be the crystalline artefacts.

There are no significant open–closed transitions in the catalytic A subunits of V1-ATPase, although the large ternary structural change of the catalytic β-subunit, especially the open–closed transition of the C-terminal helical region, is responsible for the torque generation in F1-ATPase. Little rotation of the central stalk was observed between V1-ATPase and F1-ATPase when both structures are superposed. Therefore, it is plausible that the linear feature of the coiled-coil helices of the D subunit causes small ternary changes at A and B subunits. Thus, the torque is primarily generated by quaternary rearrangement at the interface between A and B subunits rather than the open–closed transition of the catalytic A subunit (supplementary Fig S5 online).

Speculation

The asymmetric quaternary structure of the three AB pairs and the α3β3 pairs are similar (Fig 3A). The main structural difference between V1-ATPase and F1-ATPase are the ternary changes in the catalytic subunits, which are thought to have the crucial function of rotating the γ-subunit. A lack of these ternary changes at the catalytic A subunits might be an explanation for the relatively small torque of V1-ATPase. These differences would provide the common essence of the rotary mechanism.

Methods

Purification and crystallization. T. thermophilus HB8 was grown as reported previously by Yokoyama et al (1990). Cells were disrupted by sonication. Membranes were precipitated by centrifugation and suspended in 50 mM Tris–HCl (pH 8.0) and 1 mM EDTA. A total of 50 ml chloroform was added and the solution was vigorously stirred for 30 min. The supernatant containing solubilized V1-ATPase was applied to columns of diethylaminoethyl-toyopearl 650S (TOSOH, Tokyo, Japan), Resource ISO (GE Healthcare, Uppsala, Sweden) and HiLoad 16/60 Superdex 200 (GE Healthcare). Protein was concentrated to 30 mg/ml in 10 mM Tris–HCl (pH 8.0) for crystallization experiments.

Crystals of V1-ATPase were obtained in a 1:1 mixture of protein and reservoir solution containing 100 mM Na-2-(N-morpholino)ethanesulphonic acid (pH 6.0), 1.6 M (NH4)2SO4, and 10% (v/v) dioxane, using the sitting-drop vapour-diffusion method at 20°C. For the nucleotide-bound enzyme, a final concentration of 1 mM Al(NO3)3 and KF was added to the protein solution before crystallization. After 45 min, ADP and MgCl2 were added to a final concentration of 10 mM. Heavy atom derivatives were prepared by soaking nucleotide-free crystals in solutions containing 5–10 mM of the heavy-atom compound of interest. Detailed information about protein purification and crystallization is described in the supplementary information online.

Structure solution and refinement. Before data collection, crystals were soaked in a cryoprotectant solution of 100 mM Na-2-(N-morpholino)ethanesulphonic acid. (pH 6.0), 1.9 M (NH4)2SO4 and 20% (v/v) glycerol and flash-frozen under a nitrogen gas stream at −183°C. X-ray diffraction experiments were carried out at beamlines BL17A in Photon Factory, and BL38B1, BL41XU and BL44XU in SPring-8. The structures were determined by a combination of molecular replacement and multiple isomorphous replacement with anomalous scattering methods. The models were built and refined as poly-alanine models (see main text). Further details about data processing, structure determination and statistics are described in the supplementary information online.

Coordinates. The structure of nucleotide-free and nucleotide-bound V1-ATPases have been deposited in the Protein Data Bank (accession codes 3A5D and 3A5C).

Supplementary information is available at EMBO reports online (http://www.emboreports.org).

Supplementary Material

Supplementary Information

embor2009202-s1.doc (8.3MB, doc)

Acknowledgments

We thank the beamline staff at BL17A in Photon Factory, and BL38B1, BL41XU and BL44XU in SPring-8 for their help during the data collection. We also thank M. Yoshida, K. Yokoyama, T. Nogi and T.A. Fukami for their contribution in the early stages of the project. This study was supported by a grant (to KM) from the National Project on Protein Structural and Functional Analyses.

Footnotes

The authors declare that they have no conflict of interest.

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