Structure of a bacterial ATP synthase

ATP synthases produce ATP from ADP and inorganic phosphate with energy from a transmembrane proton motive force. Bacterial ATP synthases have been studied extensively because they are the simplest form of the enzyme and because of the relative ease of genetic manipulation of these complexes. We expressed the Bacillus PS3 ATP synthase in Eschericia coli, purified it, and imaged it by cryo-EM, allowing us to build atomic models of the complex in three rotational states. The position of subunit ε shows how it is able to inhibit ATP hydrolysis while allowing ATP synthesis. The architecture of the membrane region shows how the simple bacterial ATP synthase is able to perform the same core functions as the equivalent, but more complicated, mitochondrial complex. The structures reveal the path of transmembrane proton translocation and provide a model for understanding decades of biochemical analysis interrogating the roles of specific residues in the enzyme.


Introduction
Adenosine triphosphate (ATP) synthases are multi-subunit protein complexes that use an electrochemical proton motive force across a membrane to make the cell's supply of ATP from adenosine diphosphate (ADP) and inorganic phosphate (Pi). These enzymes are found in bacteria and chloroplasts as monomers, and in mitochondria as rows of dimers that bend the inner membrane to facilitate formation of the mitochondrial cristae 1,2 . Proton translocation across the membrane-embedded FO region of the complex occurs via two offset half-channels 3,4 . Studies with Bacillus PS3 ATP synthase in liposomes showed that proton translocation may be driven by ΔpH or ΔΨ alone 5 . The passage of protons causes rotation of a rotor subcomplex, inducing conformational change in the catalytic F1 region to produce ATP 6 while a peripheral stalk subcomplex holds the F1 region stationary relative to the spinning rotor during catalysis. For the mitochondrial enzyme, X-ray crystallography has been used to determine structures of the soluble F1 region 7 , partial structures of the peripheral stalk subcomplex alone 8 and with the F1 region 9 , and structures of the F1 region with the membrane-embedded ring of c-subunits attached 10,11 . Recent breakthroughs in electron cryomicroscopy (cryo-EM) allowed the suggests that the F1 regions of the maps, which are larger than the FO regions and appear to dominate the image alignment process, are mostly at between 2.5 and 3.5 Å resolution, whereas the FO regions were limited to lower resolution ( Fig. 1 -figure supplement 3). Focused refinement 33 of the FO region and peripheral stalk subunits ab2c10 and d (corresponding to the subunit OSCP in mitochondrial ATP synthase) improved the resolution of the FO regions considerably for all three classes but not enough to resolve density for most of the amino acid side chains. An improved map of the FO region was obtained by focused refinement of the membrane-embedded region only, excluding the soluble portion of subunit b with particle images from all three classes ( Fig. 1 -figure supplement 2). Overall, amino acid side chain detail can be seen for subunits a3, b3, g, d, e, a, c10-ring, and the transmembrane a-helices of b2 ( Fig.   1 -figure supplement 4). The soluble region of the two b-subunits was modeled as poly-alanine.
The general architecture of the enzyme resembles E. coli ATP synthase 25 and the more distantly related spinach chloroplast enzyme 15 but with striking differences. As observed previously in a Bacillus PS3 F1-ATPase crystal structure (PDB 4XD7) 19 , the three catalytic b subunits adopt "open", "closed", and "open" conformations, different from the "half-closed", "closed", and "open" conformations seen in the auto-inhibited E. coli F1-ATPase 17 , and the "closed", "closed", and "open" conformations seen in chloroplast ATP synthase 15 and most mitochondrial ATP synthase structures 7,10 . This difference, with the half-closed bDP of the E. coli enzyme appearing as open in the Bacillus PS3 enzyme, suggests species-specific differences in inhibition by subunit e (Fig. 1B, pink density), which inserts into the a/b interface and forces bDP into the open conformation.
In the FO region, one copy of subunit b is positioned at a location equivalent to that of the mitochondrial subunit b, while the second copy occupies the position of yeast subunit 8 (mammalian A6L) on the other side of subunit a (Fig. 1B). Despite the different c-ring sizes (10 c-subunits in Bacillus PS3 versus 14 in spinach chloroplasts), the backbone positions of subunits ab2 from Bacillus PS3 overlap with subunits abb¢ from spinach chloroplast ATP synthase 15 ( Fig.  1 -figure supplement 5A). Comparison of the atomic model of the FO region from Bacillus PS3 and the backbone model of the E. coli complex from cryo-EM at ~7 Å resolution (PDB 5T4O) 25 showed significant structural differences in transmembrane a-helices of subunit b relative to subunit a ( Fig. 1 -figure supplement 5B). Rather than reflecting true differences between E. coli and Bacillus PS3 ATP synthase structures, these deviations likely suggest that the 6 to 8 Å resolution E. coli maps were not at sufficient resolution to allow accurate backbone tracing of FO subunits.

Flexibility in the peripheral and central stalks
As expected, the most striking difference between the three rotational states of the Bacillus PS3 structure is the angular position of the rotor (subunits gec10) ( Fig. 2A, Video 1). The structure of the ATP synthase, with three ab pairs in the F1 region and ten c-subunits in the FO region, results in symmetry mismatch between the 120° steps of the F1 motor and 36° steps of the FO motor. The 120° steps of the F1 motor gives an average rotational step of 3.3 c-subunits, with the closest integer steps being 3, 4 and 3 c-subunits. By comparing the positions of equivalent c-subunits in different rotational states, the observed rotational step sizes in the three rotational states of the ATP synthase appear to be almost exactly 3, 4 and 3 c-subunits (Fig. 2B). At the present resolution, the structures of subunit a and the c-ring do not appear to differ between rotary states. Similar integer step sizes were found in yeast ATP synthase 34 and V-ATPase 35 , which also contain 10 c-subunits. However, non-integer steps were seen in the chloroplast (14 c-subunits) 15 and bovine (8 c-subunits) 36 ATP synthases, indicating that the c-subunit steps between the rotational states of rotary ATPases likely depends on the number of c-subunits.
Flexibility is thought to be important for the smooth transmission of power between the F1 and FO regions, which often have mismatched symmetries [37][38][39] . Earlier studies suggested that the central stalk (subunits g and e in bacteria) is the main region responsible for the transient storage of torsional energy in rotary ATPases 40,41 . Comparison of the three rotational states of the Bacillus PS3 enzyme also shows that C-terminal water-soluble part of subunit b displays the most significant conformational variability between states, while the subunits in the F1 region show little flexibility beyond the catalytic states of the ab pairs ( Fig. 2C; Video 1). The structure of the yeast ATP synthase FO dimer 12 , which lacked the the F1 region and an intact peripheral stalk, showed that the c-ring and subunit a are held together by hydrophobic interactions rather than by the peripheral stalk. In Bacillus PS3 ATP synthase, the peripheral stalk is structurally simpler and more flexible than in yeast mitochondria 14 , suggesting that the bacterial subunits a and the c-ring are also held together by hydrophobic interactions and not the peripheral stalk. Given that these structures represent resting states of the bacterial ATP synthase, additional subunits, such as those in the central stalk, may show flexibility while under strain during rotation.

Nucleotide binding in the F1 region and inhibition by subunit e
The structure of the F1 region of the intact Bacillus PS3 ATP synthase and the earlier crystal structure of the dissociated F1-ATPase (PDB 4XD7) 19 both show that the three catalytic bsubunits (bE, bTP, and bDP) adopt "open", "closed", and "open" conformations, respectively (Fig.   3A). In the crystal structure, which was prepared in the presence of CyDTA (trans-1,2-Diaminocyclohexane-N, N, N¢, N¢-tetraacetic acid monohydrate) as a chelating agent, there was no nucleotide in the three noncatalytic sites of the three a-subunits and the only nucleotide in a catalytic site was an ADP molecule without a Mg 2+ ion in the bTP site. In contrast, all three noncatalytic sites in the cryo-EM map are occupied by Mg-ATP, while a Mg-ADP molecule and a weak density tentatively assigned to Pi are found in the bTP site and by the p-loop of bE, respectively. The presence of physiological Mg 2+ ions and nucleotide occupancy 42 in the cryo-EM map suggest that it shows a snapshot of the enzyme in the middle of its physiological catalytic cycle.
Bacillus PS3 ATP synthase is found in a conformation where ATP synthesis is permitted but ATP hydrolysis is auto-inhibited. In this state subunit e maintains an up conformation and inserts into the aDPbDP interface, forcing bDP to adopt an open conformation (Fig. 3A, lower, dashed box) 19 . In the crystal structure (PDB 4XD7) 19 , the C-terminal sequence of subunit e was modeled as two a-helical segments broken at Ser 106, while the cryo-EM structures show the Cterminal part is in fact entirely a-helical. In comparison, subunit e from the auto-inhibited E. coli F1-ATPase structure (PDB 3OAA) 17 maintains its two C-terminal a-helices (Fig. 3B), with its bDP adopting a half-closed conformation that binds to Mg-ADP. The C-terminal a-helix of the E. coli subunit e inserts slightly deeper into the aDPbDP interface but overall in a manner similar to that of the Bacillus PS3 subunit e. However, the second a-helix in E. coli is offset by a ten-residue loop that allows it to interact with subunit g. This interaction (Fig. 3B, lower, dashed box) may stabilize the up conformation of subunit e in E. coli, explaining why auto-inhibition in E. coli does not depend on ATP concentration 29,30 while in Bacillus PS3 it does. Interestingly, during ATP synthesis, Bacillus PS3 subunit e maintains the up conformation 27 , suggesting that it only blocks ATP hydrolysis but not ATP synthesis. For a canonical ATP synthase, the substrates ADP and Pi bind to an open bE. The bE subsequently transitions to become the closed bDP and then bTP, driven by rotation of the central rotor, producing an ATP molecule that is ultimately released when the closed bTP converts back to an open bE 7 . For the Bacillus PS3 ATP synthase to produce ATP with subunit e in the up conformation, substrate would need to bind to the bDP site instead of the usual bE site, with an ATP molecule produced on transition to a closed bTP. The cryo-EM maps show that a clash between subunit e and bTP blocks the central rotor turning in the direction of ATP hydrolysis while it is still free to turn in the direction of ATP synthesis ( Fig. 3C), explaining the ability of subunit e to selectively inhibit ATP hydrolysis 27 .

Subunit organization in the FO region
In the bacterial ATP synthase structure, the FO subunits ab2 display an organization similar to the yeast FO complex (PDB 6B2Z, Fig. 4A) 12 . Subunit a and the first copy of subunit b occupy the same positions as their yeast counterparts, while the second copy of subunit b is found at a position equivalent to subunit 8 in the yeast enzyme, which is known as A6L in mammals. Atomic models for ATP synthase from mitochondria [12][13][14] and chloroplasts 15 support the idea that transmembrane proton translocation in ATP synthases occurs via two offset half-channels formed by subunit a 3,4 . Subunit a from Bacillus PS3 shares 21.0% and 29.1% sequence identity with its yeast and chloroplast homologs, respectively, and the atomic model shows that the folding of these homologs is mostly conserved ( . The sequence for this loop varies significantly among species, suggesting that it is unlikely to be involved in the core function of proton translocation, despite being proximal to the cytosolic proton half-channel. Yeast and mammalian mitochondrial ATP synthases contain subunit f, which has a transmembrane a-helix adjacent to the transmembrane a-helix 1 of subunit a (Fig. 4A, right), anchoring subunit b between a-helices 5 and 6 of subunit a. The location of the loop between a-helices 3 and 4 of the Bacillus PS3 subunit a suggests that it serves a similar structural role, compensating for the lack of subunit f in bacteria. The loop forms an additional interface with subunit b near the periplasmic side of the membrane region and may interact with the N terminus of subunit b in the periplasm as well. Two interfaces are also present between the second copy of subunit b and subunit a, one with the first transmembrane a-helix, and the other with the hairpin of a-helices 3 and 4 (Fig. 4A). The structure suggests that two interfaces are necessary for subunits a and b to maintain a stable interaction.

Proton translocation through the FO region
The Bacillus PS3 ATP synthase structure implies a path for proton translocation through the bacterial complex involving two half-channels and similar to the paths described for the mitochondrial and chloroplast enzymes. The cytoplasmic half-channel consists of an aqueous cavity at the interface of subunit a and the c-ring (Fig. 4B, left). The periplasmic half-channel is formed from a cavity between a-helices 1, 3, 4 and 5 of subunit a, and reaches the c-ring via a gap between a-helices 5 and 6 ( Fig. 4B, right). In the atomic model, both channels are visible when modelling the surface with a 1.4 Å sphere that mimics a water molecule 43 (Fig. 4B). The channels are wide and hydrophilic, suggesting that water molecules could pass freely through each of the channels before accessing the conserved Glu 56 of the c-subunits. During ATP synthesis, protons travel to the middle of the c-ring via the periplasmic half-channel and bind to the Glu 56 residue of a subunit c (Fig. 4C). Protonation of the glutamate allows rotation of the ring counter-clockwise, when viewed from F1 towards FO, delivering the subunit c into the hydrophobic lipid bilayer. Protonation of the remaining nine subunits in the c-ring returns the first glutamate to subunit a, now into the cytoplasmic half-channel, where it releases its proton to the cytoplasm due to interaction with the positively charged Arg 169 of subunit a.
In eukaryotes, subunit a is encoded by the mitochondrial genome, limiting genetic interrogation of the roles of different residues. In contrast, numerous mutagenesis studies have been performed on bacterial subunits a and b, with E. coli ATP synthase being the most frequently studied 44,45 . A single G9D mutation in the E. coli subunit b (positionally equivalent to Y13D in Bacillus PS3), results in assembled but non-functional ATP synthase 46 , while multiple N-terminal mutations in subunit b can either disrupt enzyme assembly or ATP hydrolysis 47 . In Bacillus PS3, Tyr 13 is part of the transmembrane a-helix of subunit b and is adjacent to Gly 188 of subunit a (Fig. 4 -figure supplement 3, dashed box). In E. coli subunit a, Gly 188 is replaced by a leucine (Leu 229). Therefore, the G9D mutation in E. coli not only introduces a charged residue into a hydrophobic transmembrane a-helix, but also creates a steric clash with Leu 229 of subunit a, explaining why the mutation leads to an inactive enzyme. Remarkably, the single N-terminal membrane-embedded a-helix in each of the two copies of subunit b in the Bacillus PS3 ATP synthase forms different interactions with subunit a (Fig. 4A). One surface interacts with transmembrane a-helices 1, 2, 3, and 4 of subunit a while the other interacts with a-helices 5 and 6 and the loop between a-helices 3 and 4 of subunit a. Given that the N-terminal a-helix of subunit b makes interactions with different regions of subunit a, it is not surprising that mutations in this region are often detrimental to the assembly and activity of the complex. Crosslinking experiments suggested that the N terminus of the two copies of subunit b are in close proximity with each other 48 . However, the atomic model shows that the transmembrane a-helix of the b-subunits are on opposite sides of subunit a, suggesting that the cross-linking results may be due to non-specific interactions of b-subunits from neighboring ATP synthases.
In E. coli, Arg 210 of subunit a (Arg 169 in Bacillus PS3) tolerates the fewest mutations [49][50][51][52] . Recent structures of rotary ATPases suggest that the importance of this residue derives from its role in releasing protons bound to the Glu residues of the c-subunits as they enter the cytoplasmic half-channel, as well as preventing short-circuiting of the proton path by protons flowing between half-channels without rotation of the c-ring 18,35,36,53,54 . Other residues in the E. coli subunit a identified by mutation as being functionally important include Glu 196 (Glu 159 in Bacillus PS3) 55 49 , and Gln 252 (Gln 217) 57,61 (Fig. 4D). When mapped to the Bacillus PS3 structure, only Glu 196 (Glu 159 in Bacillus PS3) is close to the cytoplasmic half-channel. Extensive mutations of E. coli Glu 196 showed that enzyme activity depends on the charge and polarity of the residue with Glu > Asp > Gln = Ser = His > Asn > Ala > Lys 55 . Therefore, the negative surface charge from Glu 196 (Glu 159) near the cytoplasmic half-channel facilitates proton transport across the lipid bilayer. The atomic model of subunit a also suggests that other residues such as Bacillus PS3 Thr 165, Asn 162, Glu 158, Tyr 228, and His 231, which are close to the cytoplasmic half-channel, may contribute to channel formation. Many functional residues identified by mutagenesis are clustered around the periplasmic half-channel. In the atomic model of the Bacillus PS3 subunit a, Asp 19 and Glu 178 are close to the periplasm, while Ser 210, Asn 173, and Gln 217 are deeper inside the membrane. Among these residues, Glu 178 and Ser 210 are considered to be more important to enzyme function than Asn 173 and Gln 217, as mutations of corresponding residues in E. coli are more likely to abolish the proton translocation by the complex 44 . Interestingly, although many of these functional residues appear important, their mutation to amino acids that cannot be protonated or deprotonated often does not completely abolish proton translocation 44 . The atomic model of Bacillus PS3 subunit a shows that the proton half-channels are wide enough for water molecule to pass through freely. This observation suggests that the function of these conserved polar and charged residues is not the direct transfer of protons during translocation. Rather, their presence may help maintain a hydrophilic environment for water-filled proton channels. This role allows different species to use unique sets of polar and charged residues forming their proton half-channels. For instance, the function of the Glu 219/His 245 pair in E. coli 59

Material and Methods
Protein expression and purification E. coli strain DK8, in which the genes encoding endogenous ATP synthase subunits were deleted 31 , was transformed with plasmid pTR-ASDS 32 encoding Bacillus PS3 ATP synthase with a 10´ His tag at the N terminus of subunit b. Transformed E. coli cells were grown in 2´TY medium at 37 ºC for 20 hours before being harvested by centrifugation at 5,400 g. Cell pellets were resuspended in lysis buffer (50 mM Tris-HCl pH 7.4, 5 mM MgCl2, 10 % [w/v] glycerol, 5 mM 6-aminocaproic acid, 5 mM benzamidine, 1 mM PMSF) and lysed with three passes through an EmulsiFlex-C3 homogenizer (Avestin) at 15 to 20 kbar. All protein preparation steps were performed at 4 ºC unless otherwise stated. Cell debris was removed at 12,250 g for 20 min and the cell membrane fraction was collected by centrifugation at 184,000 g for 1 h. Membranes were washed twice with lysis buffer before being resuspended in solubilization buffer (50 mM Tris-HCl pH 7.4, 10 % [w/v] glycerol, 250 mM sucrose, 5 mM 6-aminocaproic acid, 5 mM benzamidine, 1 mM PMSF) and solubilized by the addition of glycol-diosgenin (GDN) to 1 % (w/v) and mixing for 1 h at room temperature. Insoluble material was removed by centrifugation at 184,000 g for 45 min and solubilized membranes were loaded onto a 5 ml HisTrap HP column (GE Healthcare) equilibrated with buffer A (solubilization buffer with 20 mM imidazole, 300 mM sodium chloride, and 0.02 % [w/v] GDN). The column was washed with 5 column volumes of buffer A, and ATP synthase was eluted with 3 column volumes of buffer B (buffer A with 200 mM imidazole). Fractions containing ATP synthase were pooled and concentrated prior to being loaded onto a Superose 6 increase 10/300 column (GE Healthcare) equilibrated with gel filtration buffer (20 mM Tris-HCl pH 7.4, 5 mM MgCl2, 10 % [w/v] glycerol, 150 mM sodium chloride, 5 mM 6-aminocaproic acid, 5 mM benzamidine, 0.02 % [w/v] GDN). The peak corresponding to Bacillus PS3 ATP synthase was pooled and concentrated to ~10 mg/ml prior to storage at -80 ºC.

Cryo-EM and image analysis
Prior to grid freezing, glycerol was removed from samples with a Zeba spin desalting column (Thermo Fisher Scientific). Purified ATP synthase (2.5 μL) was applied to homemade nanofabricated EM grids 62 consisting of a holey layer of gold 63,64 that had been glowdischarged in air for 2 min. Grids were then blotted on both sides in a FEI Vitrobot mark III for 26 s at 4 °C and ~100 % RH before freezing in a liquid ethane/propane mixture 65 . Cryo-EM data were collected with a Titan Krios G3 electron microscope (Thermo Fisher Scientific) operated at 300 kV equipped with a Falcon 3EC direct detector device camera automated with EPU software. Data were recorded as 60 s movies at 2 seconds per frame with an exposure rate of 0.8 electron/pixel/second, and a calibrated pixel size of 1.06 Å.
All image processing steps were performed in cryoSPARC v2 66 unless otherwise stated. 10,940 movies were collected. Movie frames were aligned with an implementation of alignframes_lmbfgs within cryoSPARC v2 67 and CTF parameters were estimated from the average of aligned frames with CTFFIND4 68 . 1,866,804 single particle images were selected from the aligned frames with Relion 2.1 69 and beam-induced motion of individual particles corrected with an improved implementation of alignparts_lmbfgs within cryoSPARC v2 67 . A subset of 1,238,140 particle images were selected by 2D classification in cryoSPARC v2. After initial rounds of ab-initio 3D classification and heterogeneous refinement, three classes corresponding to three main rotational states of the enzyme were identified, containing 405,432, 314,448, and 175,694 particles images (Figure 1 -figure supplement 2). These 3D classes were refined with non-uniform refinement to overall resolutions of 2.9 Å, 2.9 Å and 3.1 Å, respectively, with the F1 region reaching higher resolution than the FO region of the complex as seen from estimation of local resolution (Figure 1 -figure supplement 3). Masked refinement with signal subtraction (focused refinement) 33 around subunits ab2c10d excluding the detergent micelle improved the map quality of the membrane-embedded region as well as the peripheral stalk for all three classes. The membrane-embedded region (subunits ac10 and transmembrane ahelices of the b-subunits) was improved further by focused refinement with particle images from all three classes, yielding a map at 3.3 Å resolution. All Fourier shell correlation (FSC) curves were calculated with independently refined half-maps and resolution was assessed at the 0.143 criterion with correction for the effects of masking maps. For illustration purposes, composite maps for each of the three rotational states were generated by combining the F1 region of the maps from non-uniform refinement, the peripheral stalk region from the maps obtained with focused refinement of subunits ab2c10d, and the map from focused refinement of the membraneembedded region. Specifically, each map was multiplied by a mask surrounding the region of interest and the resulting maps were adjusted to similar absolute grey scale by multiplying with a constant with relion_image_handler before being merged with the maximum function volume operation in UCSF Chimera 70 . These composite maps were not used for model refinement.

Model building and refinement
Atomic models for subunits a3b3ged from all three rotational states were built with Coot 71 into the maps of the intact complex from non-uniform refinement using PDB 4XD7 19 and PDB 6FKF 15 as initial models for subunits a3b3ge and subunit d, respectively. Subunits ac10 and the membrane-embedded regions of subunits b2 were built de novo in the 3.3 Å map of the membrane-embedded region of the complex from focused refinement. Backbone models of the soluble region of subunits b2 for all three conformations were built with the maps from focused refinement of the peripheral stalk. Models were refined into their respective maps with phenix.real_space_refine 72 using secondary structure and geometric restraints followed by manual adjustments in Coot (Table. S1). The quality of the models was evaluated by MolProbity 73 and EMRinger 74 . To generate full models for all three rotational states, the model of subunits ac10 and the membrane region of subunit b2 were fit into the full maps of each conformation as three rigid bodies (a, c10, and b2 membrane region) with phenix.real_space_refine. For class 1 and 3, the backbone models of the soluble region of subunit b2 did not fit the full maps well, and thus the fit was improved by molecular dynamics flexible fitting (MDFF) 75               @KL?>68