A molecule called adenosine triphosphate (ATP) is the energy currency in cells. Most of the ATP used by cells is made by the membrane-embedded enzyme ATP synthase. This enzyme is found in membranes inside specialized compartments known as mitochondria. ATP synthase is made up of many protein subunits that work together as a molecular machine. Hydrogen ions flow across the membrane through the ATP synthase, turning a rotor structure within the enzyme, which leads to the production of ATP.

It is not known how the transport of hydrogen ions causes rotation of the rotor. Some researchers have proposed that the enzyme works as a ratchet that is driven by the random Brownian motion of the rotor. That is, the rotational position of the rotor fluctuates randomly, but a ratchet mechanism ensures that there is a net rotation in one direction. However, there is currently little experimental evidence to back up this theory, which is known as the Brownian ratchet model.

Zhou, Rohou et al. used a technique called electron cryomicroscopy (or cryo-EM) to study ATP synthase from cows. The cryo-EM data made it possible to use computer software to construct a three-dimensional model of the enzyme that is more detailed than previous attempts. Zhou, Rohou et al. show that the structure of ATP synthase is flexible, with the different protein subunits bending, flexing, and rotating relative to each other. This variability in the position of the rotor is consistent with the Brownian ratchet model.

Together, these findings reveal important new details about the structure of ATP synthase and provide some of the first experimental evidence for the Brownian ratchet model. The new three-dimensional structure of ATP synthase will open the door to testing hypotheses of how the ATP synthase works.

In the mitochondria of eukaryotes, adenosine triphosphate (ATP) is produced by the ATP synthase, a ∼600 kDa membrane protein complex composed of a soluble catalytic F₁ region and a membrane-inserted F_O region. The ATP synthase is found in the inner membranes of mitochondria, with the F₁ region in the mitochondrial matrix and the F_O region accessible from the inter-membrane space between the mitochondrial outer and inner membranes. In the mammalian enzyme, the subunit composition is α₃β₃γδε for the F₁ region with subunits a, e, f, g, A6L, DAPIT, a 6.8 kDa proteolipid, two membrane-inserted α-helices of subunit b, and the c₈-ring forming the F_O region (Walker, 2013). The rotor subcomplex consists of subunits γ, δ, ε, and the c₈-ring. In addition to the rotor, the F₁ and F_O regions are linked by a peripheral stalk composed of subunits OSCP, d, F₆, and the hydrophilic portion of subunit b. Approximately 85% of the structure of the complex is known at high resolution from X-ray crystallography of constituent proteins, which have been assembled into a mosaic structure within the constraints of a cryo-EM map at 18 Å resolution (Walker, 2013; Baker et al., 2012).

The proton motive force, established by the electron transport chain during cellular respiration, drives protons across the F_O region through the interface between the a subunit and the c₈-ring, inducing rotation of the rotor (Boyer, 1997; Walker, 1998). While the mechanism by which ATP synthesis and hydrolysis are coupled to rotation of the γ subunit is now understood well (Walker, 2013), it is still unresolved how rotation of the central rotor is coupled to proton translocation through the F_O region. The most popular model suggests that proton translocation occurs through two offset half channels near the a subunit/c subunit interface (Junge et al., 1997, Junge, 2005). In this model, one half channel allows protons to move half-way across the lipid bilayer in order to protonate the conserved Glu58 residue of one of the c subunits. The other half channel allows deprotonation of the adjacent c subunit (Lau and Rubinstein, 2012), setting up the necessary condition for a net rotation of the entire c-ring. Rotation does not occur directly from the protonating half channel to the deprotonating half channel, but in the opposite direction so that the protonated, and therefore uncharged, Glu residues traverse through the lipid environment before reaching the deprotonating half channel. The deprotonated Glu residue prevents the ring from rotating in the opposite direction, which would place the charged residue in the hydrophobic environment of the lipid bilayer. Rotation of the c-ring occurs due to Brownian motion, making the enzyme a Brownian ratchet.

A recent cryo-EM map of the Polytomella sp. ATP synthase dimer showed two long and tilted α-helices from the a subunit in contact with the c₁₀-ring of that species (Allegretti et al., 2015). This arrangement of α-helices from the a and c subunits was also seen in the Saccharomyces cerevisiae V-type ATPase (Zhao et al., 2015a). Cryo-EM of the S. cerevisiae V-ATPase demonstrated that images of rotary ATPases could be separated by 3D classification to reveal conformations of the complex that exist simultaneously in solution. In the work described here, we obtained and analyzed cryo-EM images of the bovine mitochondrial ATP synthase. 3D classification of the images resulted in seven distinct maps of the enzyme, each showing the complex in a different conformation. By averaging the density for the proton-translocating a subunit from the seven maps, we generated a map segment that shows α-helices clearly. Analysis of evolutionary covariance in the sequence of the a subunit (Göbel et al., 1994) allowed the entire a subunit polypeptide to be traced through the density map. The resulting atomic model for the a subunit was fitted into the maps for the different rotational states, suggesting a path for protons through the enzyme and supporting the Brownian ratchet mechanism for the generation of rotation (Junge et al., 1997; 2005), and thereby ATP synthesis, in ATP synthases.

Specimens of ATP synthase were isolated from bovine heart mitochondria and prepared for cryo-EM as described previously (Baker et al., 2012; Runswick et al., 2013) (Figure 1—figure supplement 1). Initial 3D classification produced three classes, each of which appear to show a ∼120° rotation of the central rotor within the F₁ region of the complex (Figure 1A, blue arrows), similar to what was seen previously with the S. cerevisiae V-ATPase (Zhao, et al., 2015a). Further classification of these three rotational states was able to separate state 1 into two sub-states, subsequently referred to as states 1a and 1b. State 2 could be divided into states 2a, 2b, and 2c, while state 3 could be separated into states 3a and 3b. Each of these 3D classes shows a different conformation of the enzyme (Figure 1—figure supplement 2 and Video 1). While the rotational states of the yeast V-ATPase were found to be populated unequally after 3D classification, bovine ATP synthase classes corresponding to different positions of the rotor had approximately equal populations. State 1 contained 43,039 particle images divided almost equally over its two sub-states, state 2 contained 48,053 particle images divided almost equally over its three sub-states, and state 3 contained 46,257 particle images divided almost equally over its two sub-states. The resolutions of the seven classes were between 6.4 and 7.4 Å (Figure 1—figure supplement 3).

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Video 1

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There is a distinct bend in the F_O region of the complex between the portion that is proximal to the c₈-ring and the portion that is distal to the c₈-ring (Figure 1B and Figure 1—figure supplement 2). This bent structure was seen previously in a lower-resolution cryo-EM map of the bovine mitochondrial ATP synthase (Baker et al., 2010). It is consistent with electron tomograms of ATP synthases in mitochondrial membranes (Strauss, 2008; Davies et al., 2011) and was also observed recently by electron tomography of membrane-reconstituted 2D crystals of the bovine enzyme (Jiko et al., 2015). The e and g subunits are expected to reside in the portion of the F_O region distal to the c₈-ring because cryo-EM maps of the mitochondrial ATP synthase from S. cerevisiae, where subunits e and g were removed by detergent, lacked this bent portion (Baker et al., 2010; Lau et al., 2008). The f subunit is also thought to be associated with the e and g subunits (Belogrudov et al., 1996). DAPIT and the 6.8 kDa proteolipid are not expected to be present in this preparation because the necessary lipids for maintaining their association were not added during purification (Chen et al., 2007; Carroll, 2009). While a detergent micelle can be seen around the entire F_O region, the portion of F_O distal to the c₈-ring also contains a feature with unusually low density (Figure 1C, white arrows). The content of this low-density region is uncertain. Low density in cryo-EM maps is often due to partial occupancy, flexibility, or disorder of a protein subunit. However, the low-density feature here is bounded on one side by unusually sharp density from the detergent micelle, suggesting more order than in the rest of the micelle, and on the other side by the a subunit, which also appears well ordered. Therefore, the low density region could be due to bound material with low density, possibly lipid, that remains after purification of the enzyme.

Subunit a, b and A6L in the F_O region

In order to improve the signal-to-noise ratio for the F_O region of the complex, the membrane regions from the seven different maps were aligned and averaged. Averaging maps increases the signal-to-noise ratio where the structures are similar, but blurs regions where the maps differ. In principle, this method could also be applied to other map regions of the ATP synthase or other heterogeneous protein complexes by applying an appropriate transform before averaging. Averaging the F_O region provides a clear view of the portion of the F_O region adjacent to the rotor, allowing the trans-membrane α-helices from the a, b, and A6L subunits to be identified reliably (Figure 1C and D, green arrows, and Figure 2). The c-ring has a lower density in the averaged membrane region than in the original maps, suggesting that its position differs between maps (Figure 1C and D, purple arrows). The averaged density for the F_O region revealed the a subunit to have five membrane-inserted α-helices and an additional α-helix along the plane of the membrane surface (Figure 2). Three additional trans-membrane α-helices are also apparent, presumably two from the b subunit (Walker et al., 1987) and one from the A6L subunit (Fearnley and Walker, 1986). The mammalian mitochondrial a subunit possesses the two highly tilted α-helices in contact with the c-ring that were seen previously for the Polytomella sp. F-type ATP synthase (Allegretti et al., 2015) and S. cerevisiae V-ATPase (Zhao et al., 2015a) (Figure 2A).

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A model for the a subunit was built into the cryo-EM density map using constraints from analysis of evolutionary covariance in sequences of the a subunit from different species. Analysis of covariance in evolutionarily related protein sequences can identify pairs of residues in a protein structure that are likely to interact physically with each other (Göbel et al., 1994; Cronet et al., 1993; Hopf et al., 2012; Ovchinnikov et al., 2014). Spatial constraints from covariance analysis were sufficient not only to identify tentatively trans-membrane α-helices of the a subunit that are adjacent to each other, but also suggest which face each α-helix presents to the other α-helices (Figure 2B and Video 2, red lines). The constraints show patterns of interaction consistent with the predicted α-helical structure of the a subunit (Figure 2—figure supplement 1A), as well as interactions between the a subunit and the outer α-helix of a c subunit in the c₈-ring (Figure 2—figure supplement 1B). As a result, we were able to trace the path of the a subunit polypeptide through the cryo-EM density map. The fit of the α-helices in the a subunit density was improved by molecular dynamics flexible fitting (MDFF) (Trabuco et al., 2008) and the 34 residue long connecting loop from residues 115 to 148 was built with Rosetta (Rohl et al., 2004) (Figure 2B and Video 2). This connecting loop was built to be physically reasonable, but because its structure is not derived from experimental data it is not included in the discussion below. The final model places the α carbons of the residues in the co-varying pairs within 15 Å of each other in 94% of the top 90 identified pairs, with an average C_α to C_α distance of 10.3 Å. The 6% of constraints that are violated by the model are consistent with the false positive rate observed when testing covariance analysis approaches with proteins of known structure (Marks et al., 2011).

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Docking of atomic models into the cryo-EM maps

To analyze the different enzyme conformations detected by 3D classification, the maps were segmented and available crystal structures for the F₁:IF₁ complex (Gledhill et al., 2007), F₁ peripheral stalk complex (Rees et al., 2009), peripheral stalk alone (Dickson et al., 2006), and F₁-c₈ complex (Watt et al., 2010) were combined into each of the maps by MDFF (Trabuco et al., 2008). Residues for the b subunit were extended from the N terminus of the b subunit crystal structure into the membrane region based on trans-membrane α-helix prediction. While MDFF with maps in this resolution range cannot be used to determine the locations or conformations of amino acid side chains, loops, or random-coil segments of models, it can show the positioning of α-helices in the structures. Figures 3A and B compare the fitting for state 1a (Figure 3A) and state 1b (Figure 3B), illustrating the accuracy with which α-helical segments could be resolved in the maps of different sub-states. The atomic model alone for state 1a is shown in Figure 3C, with the c₈-ring removed for clarity in Figure 3D. Transitions between the different states were illustrated by linear interpolation (Video 3). As seen previously for the S. cerevisiae V-ATPase, almost all of the subunits in the enzyme undergo conformational changes on transition between states (Zhao, et al., 2015a). Because there were two sub-states identified for states 1 and 3 there is only a single sub-state to sub-state transition for these two states. In comparison, three different sub-states were identified for state 2 and consequently there are three sub-state to sub-state transitions that are possible for this state. All of the sub-state to sub-state transitions include a slight rotation of the c₈-ring against the a subunit. It is possible that this movement is due to partial disruption of the subunit a/c₈-ring interface. However, the structural differences within the F_O regions of different classes are significantly smaller than the structural differences seen elsewhere in the enzyme, suggesting that these changes do not originate from disruption within the membrane region of the complex and instead reflect flexibility in the enzyme.

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The largest change between the sub-states of each state can be approximated by rotations of the α₃β₃ hexamer relative to the rest of the complex by angles ranging from 10 to 16°. A comparison of the maps for the different sub-states and the axes of these rotations are shown in Figure 3—figure supplement 1. The resulting conformational changes can be seen most clearly in Videos 4 and 5. The state 1a to 1b transition reveals a bending of the peripheral stalk towards the top of the F₁ region near the OSCP and F₆ subunits (Videos 4 and 5, panel A). In comparison, the state 3a to 3b transition reveals bending of the peripheral stalk near where the b subunit enters the membrane (Videos 4 and 5, panel D). Transitions between the three sub-states of state 2 show both motions: the transition between 2a and 2b shows mostly bending of the peripheral stalk near OSCP and F₆ subunits, while the 2b and 2c transition shows mostly bending near the membrane-inserted portion of the peripheral stalk (Videos 4 and 5, panels B and C, respectively). The transition from state 2a to 2c shows a combined bending at both of these positions. It is most likely that the different modes of bending exist in all of the states and further classification of larger datasets would be expected to reveal these complex motions. The different sub-states do not appear to have a specific sequence or represent specific intermediates in the catalytic rotation sequence. Instead, the differences in conformation between sub-states when taken together illustrate the flexibility of the enzyme, a property that has been linked to its rapid rate of enzymatic activity (Zhao, et al., 2015a; Zhou et al., 2014). The functional significance of the sub-states may also be determined by the orientation of the c₈-ring with respect to the a subunit, as discussed below.

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Predicting the path of protons through membrane protein complexes has proven difficult, even in cases where high-resolution atomic models including bound water molecules are available from X-ray crystallography (Hosler et al., 2006). Nonetheless, features in the structure of the bovine mitochondrial F_O region suggest a possible path for proton translocation similar to a model put forward based on the structure of the Polytomella sp. ATP synthase (Allegretti et al., 2015). The arrangement of α-helices in the F_O region is remarkably similar to the arrangement of α-helices in the V_O region of the yeast V-ATPase (Zhao, et al., 2015a), even though the V-ATPase a subunit has eight α-helices and little detectable sequence similarity with the F-type ATP synthase a subunit. The conserved general architecture of the membrane-inserted regions in F-type ATP synthases and V-type ATPases suggests that the observed arrangement of α-helices is functionally important and likely involved in proton translocation (Figure 4A and B). The matrix half channel of the ATP synthase is likely to be formed by the cavity between the c₈-ring and the matrix ends of tilted α-helices #5 and #6 of the a subunit. The lumenal half channel in the V-ATPase is probably formed entirely from α-helices from the a subunit, whereas the corresponding inter-membrane space half channel in the ATP synthase is likely composed of the inter-membrane space ends of α-helices #5 and #6 and one or both of the two trans-membrane α-helices of the b subunit. Defining the exact placement of half channels will likely require higher-resolution maps from cryo-EM or X-ray crystallography that reveal amino acid side chain density and bound water molecules.

In addition to bending and twisting of the peripheral stalk and central rotor of the enzyme, the differences between the sub-states of each state show variability in the rotational position of the c₈-ring in relation to the a subunit (Figure 4C and D), even in the nucleotide-depleted conditions in which cryo-EM grids were frozen for this analysis. This lack of a rigid interaction between the c₈-ring and a subunit is consistent with the Brownian ratchet model of proton translocation (Junge et al., 1997). In the Brownian ratchet model, the rotational position of the ring fluctuates due to Brownian motion, but the ring cannot turn to place the Glu58 residue of a c subunit into the hydrophobic environment of the lipid bilayer until the Glu58 is protonated at the inter-membrane space half channel. Therefore, with this model, the different sub-states would correspond to energetically equivalent or nearly-equivalent conformations that occur due to Brownian motion. Video 6 illustrates the extent of rotational oscillation predicted from the transition between states 2a and 2c. It is most likely that this oscillation occurs as each c subunit passes the interface with the a subunit, with 8/3 c subunits on average contributing to the synthesis of one ATP molecule. The rotational flexibility of the c₈-ring that exists even when the γ subunit is locked within the α₃β₃ hexamer suggests that flexing and bending of the components of the ATP synthase smooths the coupling of the 8-step rotation of the c₈-ring with the 3-step rotation of the F₁ region. This model suggests that the observed flexibility in the enzyme, which apparently complicates determination of atomic resolution structures directly from cryo-EM data, is also essential to the mechanism of ATP synthesis.

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Author details

1. Anna Zhou

   1. The Hospital for Sick Children Research Institute, Toronto, Canada
   2. Department of Medical Biophysics, The University of Toronto, Ontario, Canada

   Contribution

   AZ, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Alexis Rohou

   Competing interests

   The authors declare that no competing interests exist.

2. Alexis Rohou

   Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States

   Contribution

   AR, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Anna Zhou

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-3343-9621

3. Daniel G Schep

   1. The Hospital for Sick Children Research Institute, Toronto, Canada
   2. Department of Medical Biophysics, The University of Toronto, Ontario, Canada

   Contribution

   DGS, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

4. John V Bason

   MRC Mitochondrial Biology Unit, Cambridge, United Kingdom

   Contribution

   JVB, Acquisition of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

5. Martin G Montgomery

   MRC Mitochondrial Biology Unit, Cambridge, United Kingdom

   Contribution

   MGM, Acquisition of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

6. John E Walker

   MRC Mitochondrial Biology Unit, Cambridge, United Kingdom

   Contribution

   JEW, Conception and design, Drafting or revising the article, Contributed unpublished essential data or reagents

   For correspondence

   walker@mrc-mbu.cam.ac.uk

   Competing interests

   The authors declare that no competing interests exist.

7. Nikolaus Grigorieff

   Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States

   Contribution

   NG, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   niko@grigorieff.org

   Competing interests

   The authors declare that no competing interests exist.

8. John L Rubinstein

   1. The Hospital for Sick Children Research Institute, Toronto, Canada
   2. Department of Medical Biophysics, The University of Toronto, Ontario, Canada
   3. Department of Biochemistry, The University of Toronto, Ontario, Canada

   Contribution

   JLR, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   john.rubinstein@utoronto.ca

   Competing interests

   The authors declare that no competing interests exist.

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