Domain movements of plasma membrane H(+)-ATPase: 3D structures of two states by electron cryo-microscopy

Abstract

H(+)-ATPase is a P-type ATPase that transports protons across membranes using the energy from ATP hydrolysis. This hydrolysis is coupled to a conformational change between states of the protein, in which the proton-binding site is alternately accessible to the two sides of the membrane with an altered affinity. When isolated from Neurospora crassa, H(+)-ATPase is a 600 kDa hexamer of identical 100 kDa polypeptides. We have obtained the three-dimensional structures of both ligand-free and Mg(2+)/ADP-bound states of this complex using single-particle electron cryo- microscopy. Structural comparisons, together with the difference map, indicate that there is a rearrangement of the cytoplasmic domain on Mg(2+)/ADP binding, which consists of a movement of mass towards the 6-fold axis causing the structure to become more compact, accompanied by a modest conformational change in the transmembrane domain.

Fig. 1. Simplified diagram for the enzymatic cycle of the proton pump coupled to ATP-hydrolysis. H⁺-ATPases are thought to have two ion (I_H+) access conformations (E1 and E2) regardless of the ligand binding, of which E1 takes up the ion in the cytoplasm while E2 releases it to the other side of the membrane, as marked in yellow. Substrate (red) induces the acyl-phosphate intermediate state E1∼P, which releases ADP before becoming E2-P, which is subsequently hydrolyzed.

Fig. 2. Electron micrographs of the H⁺-ATPase from N.crassa in the ligand-free (A and B) and the Mg²⁺/ADP-bound (C and D) states. The overall appearance of the particles prepared either in ice (A and C) or with negative staining (B and D) is very similar and not distinguishable without image processing. Scale bar = 500 Å. (A) Electron micrograph showing the particles embedded in a thin layer of vitreous ice. The samples are unstained so that protein density appears in black. (B) Negative staining of the H⁺-ATPase shown in (A). Protein densities that exclude the dense stain are visible in white. (C) Electron micrograph showing the H⁺-ATPase (in the presence of Mg²⁺/ADP) embedded in a thin layer of vitreous ice. (D) Negative staining of the H⁺-ATPase (in the presence of Mg²⁺/ADP) showing homogeneous particles.

Fig. 3. The H⁺-ATPase in the ligand-free state and its 6-fold internal symmetry. (A) Five characteristic projection views out of 50 class-sum images obtained by multivariate statistical analysis (I–V). Eulerian angles, ψ, θ and φ (VI). In terms of the 3D map which follows, (I) corresponds to the top view, (II–IV) to side views and (V) to an intermediate view. (B) The 3D angular reconstitution map displayed as a shaded surface. Slightly tilted side view with the membrane surface below. The volume of the molecule is 510 000 Å³ at this contour level, which is ∼68% of the full molecular volume, but shows the underlying structure more clearly. (C) The 2D angular plot of the orientation angles, θ and φ, resulting from FREALIGN for the class-sum shown in Figure 3A(I). The orientation was searched and refined by randomization for 20 000 cycles. The radial axis defines θ from 0° (centre) to 90° (outer shell) in projection. The angle, φ, is represented by a counterclockwise rotation from 0° to 360°. The orientations in which the particle gives rise to the phase residuals within 5° of the best value are shown by crosses in the plot (θ = 0°). (D) The 2D angular plot examined for the class-sum shown in Figure 3A(II). The minima for the phase residual were found at θ = 90° and φ = 30°, 90°, 150°, 210°, 270° or 330°.

Fig. 4. The 3D map of the unliganded H⁺-ATPase hexamers calculated by FREALIGN. Surface-shaded map presentation. Scale bar = 50 Å. (A) A view from the top along the 6-fold symmetry axis. The cytoplasmic domain is nearest to the viewer. (B) The bottom view rotated by 180° from that shown in (A). The protrusions (membrane domain) are arranged around the 6-fold axis with a centre-to-centre distance of ∼88 Å across the diameter. The membrane domains are nearest to the viewer. (C) A side view rotated by 90° from that shown in (B). The cytoplasmic domain is at the top. (D) A view rotated by a further 20° from that shown in (C). The density that belongs to the tentative monomer is indicated by a dotted line.

Fig. 5. The 3D map of the H⁺-ATPase in the Mg²⁺/ADP-bound state determined by FREALIGN. Shaded-surface presentation. Scale bar = 50 Å. (A) A view from the top along the 6-fold axis. The cytoplasmic domain is nearest to the viewer. (B) The bottom view rotated by 180° from that shown in (A). The membrane domains are nearest to the viewer. The centre-to-centre distance between membrane domains is ∼96 Å across the diameter. (C) A side view rotated by 90° from that shown in (B). The cytoplasmic domain is at the top. (D) A view rotated by a further 20° from that shown in (C).

Fig. 6. The difference map obtained by subtracting the structure of the H⁺-ATPase in the unliganded state from that of the Mg²⁺/ADP-bound state, showing the positive (red) and the negative (yellow) density peaks. The structures in both ligand-free (blue) and the Mg²⁺/ADP-bound (green) states are superimposed on the difference map. The sections are ordered (A–H) from the membrane domain to the cytoplasmic head, with each slice being ∼15 Å thick. (I) Superposition of the highest difference peaks on a surface-rendered map.

Fig. 7. Determination of absolute hand of the H⁺-ATPase hexamer deduced by single-particle analysis (A), and by comparison to the electron crystallographic map (B and C). (A) Phase residuals plotted out as a function of the relative tilt around the X- and Y-axis between particles from a pair of micrographs are contoured in red for lower phase residuals and in blue for higher phase residuals. A clear minimum was found in the phase residual at (7, –19) degrees, which agrees with the 20° tilt angle and the direction of the tilt axis between the pair of micrographs, as recorded on the microscope goniometer. Thus, the absolute hand of the structure reconstructed by single-particle analysis was correctly determined as shown in Figure 4. (B and C) The blue contours represent the same sections of the transmembrane domain as shown in Figure 6C and D. These structures, viewed with the cytoplasmic side behind, were superimposed on the corresponding regions of the 3D map (Auer et al., 1998) of the 2D crystals (magenta) truncated to a resolution of 14 Å. In (B), six arrowhead-shaped features within the membrane domains of both maps point clockwise tangentially, indicating that both structures have the same hand.