ATP synthases (Werner Kühlbrandt, Thomas Meier)

Figure 2. Schematic structural organization of the F1Fo-ATP synthase.
© 2017 MPI of Biophysics

Membrane proteins and membrane protein complexes are more of a challenge for cryoEM than soluble complexes, partly because they are less rigid, but also because they are less stable and more difficult to pre­pare. Mitochondrial ATP synthases have been a long-standing research interest in the Department. ATP syn­thases produce virtually all the ATP in the cell by rotary catalysis. A trans-membrane proton gradient drives a rotor in the membrane, which powers ATP generation. The exact mechanism by which this happens remains elusive. In the review period, we have made significant progress towards fully elucidating this strictly con­served process, which is one of the most fundamental and ancient in biology.

All types of rotary ATPases conform to essentially the same building plan. F-type ATPases consist of a roughly globular, water-soluble F1 head and the Fo subcomplex in the membrane. The F1 head contains three catalytic β and three non-catalytic α subunits that alternate in a hexameric (αβ)3 ring. The Fo subcomplex consists of the c-ring rotor, the stator subunits a and b, and, in mitochondria, a number of other small hydrophobic subunits.  V-type and A-type ATPases correspondingly have a globular V1 (A1) catalytic head and a Vo (Ao) subcomplex with a rotor ring in the membrane. In all rotary ATPases, the central stalk links the catalytic head to the rotor ring. The rotor rings of F-type ATP synthases have 8 to 15 c-subunits (Figure 2). Most c-subunits are hairpins of two hydrophobic trans-membrane helices, whereas the ring subunits of V- and A-type ATPases often have two or more trans-membrane helix hairpins [5]. In addition to the central stalk, F-type ATPases have one peripheral stalk, which connects the catalytic head to the stator in the membrane to prevent idle rotation of the whole assembly. The A-type ATPases have two peripheral stalks and V-type ATPases three.

Figure 3. Schematic overview of rotary ATPases. (A) Mitochondrial F-type dimer; (B) Chloroplast or bacterial F-type monomer; (C) Vacuolar (V-type) ATPase; (D) Archaeal (A-type) ATPase. Colour scheme: red and orange, catalytic and non-catalytic α and β (or A and B) subunits; brown, central stalk; green, peripheral stalks; yellow, rotor ring; blue, subunit a. The two darker blue circles denote the two horizontal a-subunit helices adjacent to the rotor ring (from Kühlbrandt and Davies, TIBS 2016).

(a)  Mitochondrial ATP synthase from Polytomella (Werner Kühlbrandt)
We determined the structure of the 1.6 MDa ATP synthase dimer from mitochondria of the chloro­phyll-less green alga Polytomella sp. by single-particle cryoEM at 6.2 Å resolution. The Polytomella dimer is more rigid than that of yeasts (see below) or mammals and lends itself better to high-resolution cryoEM. The structure shows the arrangement of the two catalytic F1 heads, the central and peri­pheral stalks, and a pair of rotor rings in the dimer complex. We discovered that the a-subunit forms a bundle of four long membrane-intrinsic α-helices at right angles to the c-ring rotor. The long horizontal helices create a pair of proton half-channels at the a/c interface that enables protonation of the c-subunit glutamates from the lumenal side and proton release to the matrix, driving c-ring rotation and ATP syn­the­­sis (Allegretti et al., Nature 2015). Since our discovery, the long, quasi-hori­zontal helix hairpin of the a-subunit next to the c-ring rotor and the two half channels have proven to be fun­da­mental, highly conserved feature of all rotary ATPases (Kühlbrandt and Davies, TIBS 2016)

(b)  Mitochondrial ATP synthase from Yarrowia lipolytica (Werner Kühlbrandt, Thomas Meier)
ATP synthase dimers from yeasts resemble those from mammalian mitochondria but differ from those of plants and green algae in terms of structure and subunit composition. To gain insight into this more common and medically relevant ATP synthase, we performed single-particle cryoEM on dimers from the aerobic yeast Y. lipolytica, which we found to be more suitable for high-resolution cryoEM than the bovine complex. The Y. lipolytica dimer is an assembly of 17 different polypeptides in 62 individual protein subunits. The 6.2 Åresolution cryoEM map indicates a sharp ~90° bend in the detergent micelle surrounding the Fo part, reflecting the high local curvature at the ridges of the inner membrane cristae (Hahn et al., Mol Cell 2016). In the membrane region of the complex, we resolved and identi­fied seven different membrane subunits, including the dimer-specific subunits e and g, which we have shown previously to be essential for dimer stability and cristae formation (Davies et al., PNAS 2012).


Figure 4. Single-particle cryoEM of mitochondrial and chloroplast F1-Fo ATP synthases (A) 6.2 Å map of the ATP synthase dimer from Polytomella sp. mitochondria. The massive, rigid peripheral stalks (local resolution currently 5.6 Å) make this dimer particularly suitable for cryoEM at higher resolution. (B) The conserved assembly of long horizontal a-subunit helices (blue) at right angles to the c-ring rotor (yellow) creates two proton half channels in the membrane (grey). From (Allegretti et al., Nature 2015). (C) 6.2 Å map of the mitochondrial ATP synthase dimer from Y. lipolytica with fitted atomic model. (D) Detailed view of Fo subunits (c-ring, yellow; a-subunit, blue; subunit f, pink; subunit 8, green; subunit b, purple; subunit i, orange) surrounded by the detergent micelle (grey) seen from the matrix (left) and from the membrane (right). From (Hahn et al., Mol Cell 2016).

Figure 5. Cryo-EM map of the Polytomella mitochondrial ATP synthase dimer, highlighting the helix-turn helix pair (blue) and c-ring rotor (yellow).

Figure 6. Morph between the three different conformational states observed in the Y. lipolytica F1c10 crystal structure. Subunit representations and coloring as follows: α, dark green or dark grey ribbon; β, light green or light grey ribbon; γ, blue surface; δ, cyan surface; ε, white surface. MgADP is represented as stick-ball model with C, N, O, P, and Mg colored in yellow, blue, red, orange, and magenta, respectively. (From Hahn et al, Mol. Cell 2016).
Figure 7. Structures of ATPase rotor rings. Top row from left: c8 ring of bovine mitochondrial ATP synthase (dark blue); c9 ring of Mycobacterium phlei (blue); c10 ring of Saccharomyces cerevisiae mitochondrial ATP synthase (light blue); c11 ring of Ilyobacter tartaricus (turquoise); c12 ring of Bacillus pseudofirmus OF4 mutant (green). Bottom row from right: c13 ring of Bacillus pseudofirmus OF4 (lime green); c14 ring of Pisum sativum chloroplast ATP synthase (ochre); c15 ring of Spirulina platense cyanobacterial ATP synthase (orange); K10 ring of Enterococcus hirae A-type ATPase (red). The A-type K subunit has two helix hairpins. One helix in each K subunit (pink) contains the ion-conducting glutamate. All other rings are of F-type ATP synthases with one helix hairpin per c-subunit. Solid black arrowheads indicate the 11-13 Å distance between ad-jacent ion-binding sites in the c-rings. In the larger K-ring of the A-type ATPase this distance is 21.5 Å (open arrowheads). All ring structures are viewed from the direction of the catalytic head (from Kühlbrandt and Davies, TIBS 2016).
Figure 8. The different c-ring rotor sizes work like bicycle gears. Small rings are better for a shallow pH gradient, as in mitochondria. Larger rings are more efficient with a steep gradient, as in cyanobacteria or chloroplasts. Note that the ring size is species specific. Gear changes are not possible in ATP synthases or ATPases in any given species.
Figure 9. Proposed mechanism of proton translocation and c-ring rotation. Protons (‘plus’ sign within a red circle) enter the Fo subcomplex via the conserved histidine (light blue) in the long-horizontal a-subunit helix (blue) that is exposed to the lumenal aqueous half-channel. Red arrows mark the proposed proton path. Protons bind to the open conformation of the deprotonated, negatively charged glutamate (orange) of the c-subunit (curved yellow cylinders). The glutamate side chain changes to a closed conformation, which locks the proton and can enter the hydrophobic environment of the lipid bilayer. After an almost full revolution of the c-ring (grey arrow), the glutamate encounters the high pH8 environment of the matrix half-channel, where it changes back to the open conformation and is de-protonated. The process of protonation in the lumenal half channel and de-protonation in the matrix half-channel causes the ring to rotate in anti-clockwise direction as seen from the matrix. Backward rotation is prevented by the positive charge of the strictly conserved arginine (light blue). The arginine is located 2.5 helix turns (13.5 Å) downstream of the conserved histi-dine/glutamate. This distance matches the spacing between adjacent protonation sites in c-rings of any known subunit stoichiometry. The functionally important arginine and histidine residues in the long a-subunit helix can thus interact with two adjacent c-subunits in the ring simultaneously, as required for proton translocation, c-ring rotation and ATP synthesis by rotary catalysis (from Kühlbrandt and Davies, TIBS 2016).

(c)  Electron cryo-tomography of mitochondrial ATP synthases in situ (Werner Kühlbrandt)
Unlike the bacterial and chloroplast ATP synthase, or the V and A-type ATPases, all known mitochondrial ATP synthases form dimers in the membrane. as we discovered by cryoET of intact mitochondria or cristae vesicles (Strauss et al., EMBO J 2008; Davies et al., PNAS 2011; Daum et al., PNAS 2013). The dimers arrange in long rows along the tightly curved ridges of the mitochondrial inner membrane cristae. The dimer rows have a profound effect on respiratory activity, growth rates and mitochondrial morphology.  The rows are instrumental for the formation of the inner membrane cristae, which increase the membrane area available for respiratory chain complexes. The cristae lumen can be considered as a mitochondrial sub-compart­ment that may assist ATP production through local variations in proton concentration.

Figure 10. Tomographic volume of a small fungal mitochondrion, showing rows of ATP synthase dimers (yellow) along tightly curved ridges of the inner membrane cristae (light blue). The outer membrane is grey.
Figure 11. Low-resolution subtomogram averages of mitochondrial ATP synthase dimers in the membrane of seven different species. Mitochondrial ATP synthases of all organisms investigated are dimers that associate into long rows (up to 1 µm) along the cristae ridges. Yellow arrowheads point to the 10 nm globular densities of the catalytic F1 heads. Insets show cross-sections through the rows on the tightly curved cristae membranes.
Figure 12. Cristae. The electron transport complexes of the respiratory chain, which are excluded from the tightly curved cristae ridges, reside in the membrane regions at either side of the dimer rows. This arrangement suggests that the cristae work as proton conduits or traps, in which the protons are efficiently guided from their source at the respiratory chain supercomplexes to the proton sinks at the dimer rows at the cristae ridges (Figure 12). We are now investigating the link between mitochondrial membrane organization and aging, which may be important for understanding mitochondria-related diseases such as Parkinson's.

Cristae vesicles from protozoan mito­chondria often look very different from the standard mor­pho­logy. Subtomogram averaging revealed that the dimers themselves can be very different, especially with respect to the angle included by the central stalks, which is ~90° in fungi and mammals but ranges from around 60° (trypano­so­mes, Polytomella) to 0° (Paramecium). Differences are striking in Paramecium, where U-shaped ATP synthase dimers assemble into regular helical arrays, which impose a helical structure and thereby membrane curvature on the tubular cristae that are characteristic of this and similar species. Remarkably, the dimer rows induce high, uniform membrane curvature, even though the dimer angle is 0° and therefore an individual dimer does not bend the membrane (Mühleip et al., PNAS 2016b). Dif­feren­ces are even more striking in trypanosomes and Euglena, where the structure of the F1 subcomplex that was thought to be universally conser­ved in all F-type ATPases deviates from the consensus structure. We discovered that in the Euglenacea, which include dangerous human parasites, the α and β-subunits form a trigonal pyramid , rather than the usual near-sixfold barrel. This unusual architecture is due to a split in the α-subunit into separate N and C-terminal domains, which disrupts the catalytic site of ATP production in the F1 head. The unique structure of these ATP synthases, on which the organisms depend for survival, opens a new route for drug develop­ment to combat sleeping sickness that is caused by trypanosomes (Mühleip et al., PNAS 2016a).

Figure 13. Electron cryo-tomography of ATP synthase dimers in situ. (A) Club-shaped crista vesicle with dimer ribbons and (B) subtomogram average volume of ATP synthase dimer from Polytomella sp. mitochondria. The dimer volume in (B) is colour-coded by local map resolution from ~16 Å (blue) to ~30 Å (orange). (C) Tubular crista vesicle with helical dimer ribbon (yellow) and (D) subtomogram average of ATP synthase dimer from the ciliate Paramecium tetraurelia with fitted F1 head and c-ring rotor (purple). (E) Lamellar crista vesicle with short dimer ribbons (yellow and orange) at the edge of lamellar cristae and (F) subtomogram average of ATP synthase dimer from Euglena gracilis.


Max Planck Institute of Biophysics

Prof. Dr. Werner Kühlbrandt, Director
Department of Structural Biology
Secretary: Monika Hobrack

Phone: +49 (0) 69 6303-3001
Fax: +49 (0) 69 6303-3002
E-mail: monika.hobrack(at)