Arrangement of subunits in intact mammalian mitochondrial ATP synthase determined by cryo-EM

Abstract

Mitochondrial ATP synthase is responsible for the synthesis of ATP, a universal energy currency in cells. Whereas X-ray crystallography has revealed the structure of the soluble region of the complex and the membrane-intrinsic c-subunits, little is known about the structure of the six other proteins (a, b, f, A6L, e, and g) that comprise the membrane-bound region of the complex in animal mitochondria. Here, we present the structure of intact bovine mitochondrial ATP synthase at ∼18 Å resolution by electron cryomicroscopy of single particles in amorphous ice. The map reveals that the a-subunit and c(8)-ring of the complex interact with a small contact area and that the b-subunit spans the membrane without contacting the c(8)-ring. The e- and g-subunits extend from the a-subunit density distal to the c(8)-ring. The map was calculated from images of a preparation of the enzyme solubilized with the detergent dodecyl maltoside, which is visible in electron cryomicroscopy maps. The structure shows that the micelle surrounding the complex is curved. The observed bend in the micelle of the detergent-solubilized complex is consistent with previous electron tomography experiments and suggests that monomers of ATP synthase are sufficient to produce curvature in lipid bilayers.

Fig. 1.

Structure of the intact F-type ATP synthase. (A) Three different views of an isosurface representation of the map. The soluble F₁ region is at the top of the image, and the membrane-bound F_O region is at the bottom in i and iii. A view from the F₁ region to the F_O region is shown in ii. (B) Views of the map after segmenting. Segments are shown as colored surfaces, and the subunits or subcomplexes represented by each segment are labeled. The intact map is shown as a transparent gray surface. (C) Slices through the intact map and map segments. Lower shows slices through the map, whereas Upper shows the map segments truncated at the same height as the slice. The colors of the boxes outlining the panels correspond to the horizontal lines in i. (ii) In the F₁ region of the complex, the three α- and three β-subunits can be seen surrounding the γ-subunit, with the peripheral stalk running along an α/β-interface. (iii) Near the middle of the membrane region, the density that contains subunit a contacts the c₈-ring. (iv) Further towards the intermembrane space, the segment that contains the a-subunit is separate from the c₈-ring. (v) At the intermembrane space side of the membrane region, a density that contains the e- and g-subunits can be seen running along the detergent micelle. (Scale bars: 50 Å.) The scale bar in A, ii applies to all map segments, and the scale bar in C, ii applies to all of the map slices.

Fig. 2.

The peripheral stalk region of the complex. (A) At the top of the F₁ region, the peripheral stalk in the EM map (green mesh) follows the same path observed in the crystal structure of the membrane extrinsic region of the ATP synthase (i; green atomic model), whereas the path of the peripheral stalk is different in the two structures along the side of F₁ (ii–iv). (B) Towards the F_O region, the curvature of the peripheral stalk in the EM map differs from the curvature of the crystal structure of an isolated b, d, and F₆ subcomplex, supporting the proposal that there are two flexible regions (blue and pink arrows) in the crystal structure of the peripheral stalk (9, 10). (i) An abrupt change in the trajectory of the peripheral stalk is observed where the stalk enters the lipid bilayer (orange arrow). The atomic model of the isolated peripheral stalk subcomplex was docked into the EM map by alignment of regions that overlap with the F₁ peripheral stalk atomic model shown in A. (ii) The curvature of this extrapolated peripheral stalk structure from crystallography would place the peripheral stalk in contact with the c₈-ring. (Scale bar: 50 Å.)

Fig. 3.

The membrane region of the complex. (A and B) The location of subunits e and g in the bovine enzyme was confirmed by comparison with a previously published map of the complex from the yeast S. cerevisiae (7), from which subunits e and g are lost on being solubilized with detergent. The intact yeast map is shown in white in A, with the positive and negative differences from the current bovine map shown in green and red, respectively. Positive densities show regions present in the bovine complex but absent in the yeast enzyme, and can be attributed to the e- and g-subunits. The large positive difference density in the membrane is overlaid as a pale purple transparency on the segments from the bovine map in B. (C–F) Segments were obtained for the detergent-lipid micelle, c₈-ring, N-terminal region of subunit b, and a subcomplex that contains subunit a and the remaining subunits of the F_O region (e, f, g, and A6L). The segment containing subunit a can be considered to have two portions. One portion spans the membrane next to the c₈-ring. The second portion overlaps with the positive difference density from A and is thought to contain subunits e and g. This portion extends from the rest of the membrane region and maintains a large contact with the detergent-lipid micelle, which strongly deviates from the planar structure thought to exist for most membrane proteins. The c₈-ring and the segment that contains subunit a make only limited contacts above the middle of the lipid bilayer (C), with no density above or below the contact point between the a- and c-subunits (shown in F with the c₈-ring removed for clarity). (Scale bar: 50 Å.)

Fig. 4.

Models of proton translocation in monomers and dimer formation in curved membranes. (A, i) From the known direction of rotation of the c₈-ring during ATP synthesis (15), the tentative locations of proton-conducting half-channels from the intermembrane space (IMS) to the middle of the lipid bilayer and from the middle of the lipid bilayer to the mitochondrial matrix are indicated through the segment that contains the a-subunit. Only the outer helix of each c-subunit has been shown for clarity (ribbon diagram). The location of this region of the complex is shown in ii. (B and C) Side and top views of a model of dimers of ATP synthase based on the observed arrangement of protein and micelle in the cryo-EM map as well as the overall size and shape of dimers observed in mitochondrial membranes (blue outline in B; traced from ref. 20). The angle between the two monomers in the dimer would be ∼86°, which is in good agreement with the ∼80° observed by electron tomography. (Scale bar: 25 Å.)