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

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 A 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.

Figure 1

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.

Figure 2

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.

Figure 3

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.

Figure 4

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.

Figure 5

Nucleotide-binding sites. (A) The bottom view of V₁-ATPase. The difference (F_nucleotide−F_free) 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 A_N and B_N. 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 A_N′ and B_N′. (D) The subunit interface between A_W and B_W.