Structure of a catalytic dimer of the α- and β-subunits of the F-ATPase from Paracoccus denitrificans at 2.3 Å resolution

The structure of the αβ heterodimer of the F-ATPase from the α-proteobacterium P. denitrificans has been determined at 2.3 Å resolution. It corresponds to the ‘open’ or ‘empty’ catalytic interface found in other F-ATPases.


Introduction
The structures and mechanisms of F-ATPases from eubacteria, chloroplasts and mitochondria have many common features in their structures and mechanisms. Our current knowledge of how they function by a rotary mechanism is based largely on the knowledge of the structures of mostly mitochondrial enzymes (Walker, 2013;Robinson et al., 2013;Bason et al., 2014Bason et al., , 2015 and 'single-molecule' experiments conducted almost entirely on enzymes from Escherichia coli and Bacillus stearothermophilus (or Geobacillus stearothermophilus) strain PS3 (Watanabe & Noji, 2013). For example, more than 25 high-resolution structures of the F 1 catalytic domain from bovine mitochondria with bound substrates, substrate analogues and inhibitors have been described (Walker, 2013;Robinson et al., 2013;Bason et al., 2014Bason et al., , 2015. In contrast, there are two structures of the F 1 catalytic domain of the E. coli enzyme (Cingolani & Duncan, 2011;Roy et al., 2012) and one of the same domain of the enzyme from B. stearothermophilus (Shirakihara et al., 2015), and another of the 3 3 subcomplex derived from the F 1 domain (Shirakihara et al., 1997), plus a structure of F 1 -ATPase from Caldalkalibacillus thermarum (Stocker et al., 2007). In addition, the structures of c-rings from the rotors of several eubacterial species have been determined at high resolution in isolation from the rest of the complex (Meier et al., 2005;Pogoryelov et al., 2009;Preiss et al., 2013Preiss et al., , 2014Matthies et al., 2014). There is also fragmentary structural information concerning the peripheral stalk region of the F-ATPase from E. coli determined by nuclear magnetic resonance in solution, the N-terminal domain of the -subunit and its mode of interaction with the N-terminal region of an -subunit (Wilkens et al., 2005), and for segments of the -subunit (Dmitriev et al., 1999;Del Rizzo et al., 2002;Priya et al., 2009). Part of the reason for this relative dearth of structural information on the catalytic domain of bacterial F-ATPases is that the F 1 domain of the enzyme from E. coli, for example, is rather unstable under the conditions that have been employed for crystallizing mitochondrial enzymes. Also, there is no generic method for purifying eubacterial F-ATPases, whereas it has been demonstrated that mitochondrial enzymes can be purified from a wide range of species by affinity chromatography with the inhibitory region of bovine IF 1 , the protein inhibitor of the bovine mitochondrial F-ATPase (Runswick et al., 2013;Walpole et al., 2015;Liu et al., 2015). Therefore, we have decided to explore the possibility of developing the F-ATPase from Paracoccus denitrificans as a subject for structural analysis. P. denitrificans is a member of the bacterial class -proteobacteria in the phylum Proteobacteria. The class includes the extinct protomitochondrion that became engulfed by the ancestor of eukaryotic cells, and the respiratory chain of P. denitrificans has been recognized as being especially similar to respiratory chains in mitochondria (John & Whatley, 1975).
Some eubacterial F-ATPases, exemplified by those from E. coli and B. stearothermophilus, can synthesize ATP from ADP and phosphate using the transmembrane proton motive force as a source of energy, and under anaerobic conditions can operate in reverse and use the energy released by the hydrolysis of ATP made by glycolysis to generate a transmembrane proton motive force. Other eubacterial F-ATPases, exemplified by those from C. thermarum (Cook et al., 2003) and P. denitrificans (Zharova & Vinogradov, 2012), can synthesize ATP in the presence of a proton motive force, but their ATP hydrolase activity is inhibited in its absence (Pacheco-Moisé s et al., 2000. The mechanism of inhibition in C. thermarum is not understood, but in P. denitrificans and other -proteobacteria the inhibition of ATP hydrolysis involves an inhibitor protein known as the inhibitor protein (Morales-Ríos et al., 2010). This inhibitor protein has not been detected in other classes of bacteria. The structure of the free inhibitor is known from studies employing nuclear magnetic resonance in solution (Serrano et al., 2014). It binds to the F 1 catalytic domain of the F-ATPase and can be cross-linked covalently to the -, -,and "-subunits (Zarco-Zavala et al., 2014). However, the cross-linked residues were not identified, and its mode of interaction with this domain is not known. Therefore, we have purified the F 1 -ATPase from P. denitrificans with the inhibitor protein bound to it, and a second complex devoid of the "-subunit, known as F 1 -and F 1 Á"-, respectively. As in other species where the subunit composition of the F 1 domain has been established experimentally, the F 1 domain in P. denitrificans is probably an assembly of three -subunits and three -subunits, where the catalytic sites are found, plus one copy of each of the -,and "-subunits, with theand "-subunits forming the central rotor of the enzyme penetrating along the central axis of the 3 3 domain, and the -subunit, a residual component of the peripheral stalk in the intact F-ATPase, sitting 'on top' of the 3 3 domain. In the bovine F 1 -ATPase, for example, the three catalytic sites are found at three of the six interfaces betweenand -subunits, known as the 'catalytic interfaces'. The asymmetry of the central stalk imposes different conformations on the three catalytic sites. In a ground-state structure of the catalytic domain (Abrahams et al., 1994;Bowler et al., 2007), two of them, the DP and the TP sites, have similar, but significantly different, closed conformations. Both bind nucleotides, but catalysis occurs at the DP site. The third, or E , site has a different open conformation with low nucleotide affinity. These three catalytic conformations correspond to 'tight', 'loose' and 'open' states in a binding-change mechanism of ATP hydrolysis and synthesis (Boyer, 1993).
As described here, we have attempted to crystallize the F 1and F 1 Á"complexes. Crystals were obtained for the F 1 Á"complex, but none were obtained for the F 1 -complex. However, as described below, the crystals with F 1 Á"as the starting material were found to contain a heterodimer of one -subunit and one -subunit, which had formed by dissociation of the complex under the conditions of crystallization. This heterodimer has no bound nucleotide, and it represents the 'open' or 'empty' E catalytic interface of the intact F-ATPase.

Protein methods
The protein compositions of various samples were analysed by SDS-PAGE in 12-22% polyacrylamide gradient gels (Laemmli, 1970). Proteins were stained with 0.2% Coomassie Blue dye or with silver. Protein concentrations were measured by the bicinchoninic acid method (Life Technologies, Paisley, Scotland). The latent ATP hydrolase activities of the F 1 -ATPase and of the enzyme lacking the "-subunit (F 1 Á") from P. denitrificans were activated with 0.1% lauryldimethylamine oxide (LDAO) and 4 mM sodium sulfite, and their activities were measured by coupling them to the oxidation of NADH monitored using the absorbance of ultraviolet light at 340 nm (Pullman et al., 1960).

Cell growth
A starter culture of P. denitrificans (strain PD1222, Rif r , Spe r , enhanced conjugation frequencies, m + , or host-specific modification) was grown at 30 C for 18 h in 1 l Luria-Bertani medium (Miller, 1987) containing 100 mg ml À1 spectinomycin. It was inoculated into 70 l succinate medium consisting of research communications 1%(w/v) sodium succinate, 50 mM disodium hydrogen phosphate, 1.25 mM magnesium chloride, 1 mM citric acid, 100 mM calcium chloride, 90 mM ferric chloride, 50 mM manganese chloride, 25 mM zinc chloride, 10 mM cobalt chloride and 10 mM boric acid. The culture was grown at 30 C for 16 h in an Applikon ADI 1075 fermenter (100 l maximum capacity). The yield of wet cells was 2 kg. Inside-out vesicles were prepared by osmotic shock (Pacheco-Moisé s et al., 2000).

2.3.
Purification of the complex of the F 1 -ATPase and the f inhibitor protein from P. denitrificans Using modification of an earlier method (Morales-Ríos et al., 2010), the F 1 -inhibitor complex was released from a suspension of membranes from P. denitrificans (30 ml) by the addition of chloroform (15 ml). The two phases were mixed for 30 s and then centrifuged (2939g, 25 C). The upper aqueous phase was centrifuged again (50 min, 224 468g, 4 C), and the supernatant was applied to a HiTrap Q HP column (5 ml; GE Healthcare) equilibrated with purification buffer consisting of 50 mM Tris-HCl pH 7.5, 10%(v/v) glycerol, 0.5 mM ATP, 2 mM MgCl 2 and protease-inhibitor tablets (cOmplete, EDTA-free; Roche; one tablet per 100 ml). The column was eluted with buffer containing a gradient of sodium chloride with steps of 50, 100,150,200,225,250,275, 300 and 325 mM. The fractions (15 ml) were analysed by SDS-PAGE, and those containing the purest enzyme-inhibitor complex were pooled and concentrated (final volume 500 ml; protein concentration 15 mg ml À1 ) with a Vivaspin ultrafiltration concentrator (molecular-weight cutoff 50 kDa; 2939g, 15 C). The two separate concentrates of the F 1 -and the F 1 Á"complexes (see below) were applied individually to a column of Superdex 200 (10 Â 300 mm; GE Healthcare) equilibrated with purification buffer and eluted at a flow rate of 0.5 ml min À1 . The peak fractions (3 ml) were pooled and concentrated as above (final volume 150 ml; protein concentration 10 ml min À1 ).   The crystals were grown at 25 C by the microbatch method under oil in 72-well Nunc plates. Drops (2 ml) were formed by mixing the solution of purified F 1 Á"-(protein concentration 10 ml min À1 ) with an equal volume of buffer consisting of 50 mM Tris-HCl pH 7.8, 12%(w/v) polyethylene glycol 10 000, 1%(w/v) cadaverine, 10%(v/v) glycerol, 1 mM ATP. They were harvested with micro-mounts (MiTeGen) and vitrified in liquid nitrogen in the presence of cryoprotection buffer consisting of 25 mM Tris-HCl pH 7.8, 15%(v/v) glycerol, 10%(w/v) polyethylene glycol 10 000, 1%(w/v) cadaverine. 25 crystals were washed three times in buffer with the same composition as the mother liquor and analyzed by SDS-PAGE. Similar, but unsuccessful, attempts were made to grow crystals of F 1 -.

Data collection, structure solution and refinement
X-ray diffraction data were collected from the cooled cryoprotected crystals using a Pilatus 6M-F detector (Dectris) on beamline I03 (wavelength 0.9763 Å ; beam size 90 Â 35 mm) at the Diamond Light Source, Harwell, Oxfordshire, England. The data were processed with programs from the CCP4 suite . Diffraction images were integrated with iMosflm (Battye et al., 2011) and the data were reduced with AIMLESS (Evans & Murshudov, 2013). Molecular replacement was carried out with Phaser (McCoy et al., 2007) with the E -and E -subunits of the currently most accurate structure of bovine F 1 -ATPase (Bowler et al., 2007; PDB entry 2jdi) as a template. The model was built with Coot (Emsley et al., 2010) and refined with REFMAC5 (Murshudov et al., 2011). The stereochemistry of the model following each round of refinement was assessed with Coot and MolProbity

Results and discussion
3.1. Characterization of the complex of the F 1 -ATPase and the f inhibitor protein from P. denitrificans Three peaks (j, k and l in Fig. 1a) containing subunits of the P. denitrificans F 1 -ATPase complex were eluted from the Q Sepharose column. Analysis by SDS-PAGE revealed that peak j contained a complex of the -, -,and -subunits from the F 1 domain of the F-ATPase plus the inhibitor protein (the F 1 Á"complex), and the two subsequent peaks k and l contained a complex of the intact F 1 -ATPase with the protein (the F 1 -complex). The ATP hydrolase activities of the F 1 -and F 1 Á"complexes were 0.01 AE 0.002 and 0.02 AE 0.001 U per milligram of protein, respectively, and after relief of the inhibitory activity of the inhibitor protein they were 3.5 AE 0.1 and 4 AE 0.1 U per milligram of protein, respectively. These values are comparable with those of other inhibited bacterial F-ATPases where no inhibitor protein is involved. For example, the values for the F 1 -ATPase from the cyanobacterium Thermosynechococcus elongatus are 0.2 and 9.0 U per milligram of protein before and after activation with LDAO (Sunamura et al., 2012). For C. thermarum they are 0.9 and 28.5 U per milligram of protein before and after activation (Keis et al., 2006), and for the chloroplast F 1 -ATPase from Spinacia oleracea they are 4.4 and 39.7 U per milligram of protein before and after activation (Groth & Schirwitz, 1999). Enzymes that are not inhibited in ATP hydrolysis have higher recorded values than those of the activated inhibited enzymes. Values in the range 60-130 U per milligram of protein have been reported for the F 1 -ATPase from E. coli (Dunn et al., 1990). With the bovine F 1 -ATPase, activities in excess of 120 U per milligram of protein have been recorded routinely (van Raaij et al., 1996).
The concentrated F 1 Á"complex was subjected to gelfiltration chromatography (Fig. 1). This experiment removed minor contaminants, and confirmed that the -, -,and -subunits from the F 1 domain of the F-ATPase, plus the inhibitor protein, form an integral F 1 Á"complex that is stable under the conditions of chromatography (Figs. 1c and  1d). Other experiments (not shown) were conducted with the F 1 -complex, with similar conclusions.

Crystallization of the dimer of the aand b-subunits of the F-ATPase from P. denitrificans
Attempts were made to crystallize both the F 1 -and F 1 Á"inhibited complexes. No crystals were obtained for the former, but the latter yielded crystals with two different Structure of the catalytic dimer of theand -subunits of the F-ATPase from P. denitrificans. Theand -subunits are shown in red and yellow, respectively, and a bound phosphate ion is denoted by cyan spheres. (a) View from the front, looking inwards towards the central stalk in the rotor of the intact enzyme. The arrangement of subunits, with the -subunit on the left and the -subunit on the right, corresponds to a catalytic interface in the intact F 1 -ATPase. (b) View from the inside of the intact complex, looking outwards. morphologies: needles and rhomboids (Fig. 2). The rhombic crystals reached their maximum size (approximately 200 Â 40 Â 5 mm) after 25 d of growth at 25 C and only these crystals gave useful X-ray diffraction data. The dimensions of the unit cell calculated from the X-ray diffraction data were a = 72.6, b = 102.9, c = 89.2 Å , and the space group was determined as P2 1 . The asymmetric unit of this cell is too small to accommodate an F 1 -ATPase complex. Therefore, it seemed likely that a subcomplex of the enzyme had formed under the conditions of crystallization and the subcomplex had crystallized. This conclusion was confirmed by analysis of the rhombic crystals by SDS-PAGE, which showed that the crystals contained onlyand -subunits (Fig. 2c); presumably this subcomplex had formed by loss of theand -subunits and dissociation of the 3 3 subcomplex during the crystallization process. At this stage, the precise composition of the subcomplex was unclear, as it could conceivably have contained one, two or three copies of each of theand -subunits. Again, the size of the, 3 3 subcomplex was incompatible with the unit-cell parameters and, given that the 2 2 subcomplex has never been observed from any F-ATPase, it was most likely that the crystals were formed from one of two possible hetereodimers, containing either a catalytic or a noncatalytic interface of the F 1 -ATPase.
3.3. Structure of the dimer of the aand b-subunits of the F-ATPase from P. denitrificans The structure of the P. denitrificans complex (Fig. 3) was solved by molecular replacement with data to 2.3 Å resolution. Both the catalytic and noncatalytic dimers of bovine F 1 -ATPase were tried, but it was clear that the former was appropriate and the latter was not. The packing of the protein complexes in the crystal lattice provided additional confirmation that the crystal lattice consisted of dimers and not 3 3 hexamers (Fig. 2d). Data-processing and refinement Alignment of the structures of the catalytic dimer ofand -subunits of the F-ATPase from P. denitrificans with theand -subunits forming the open (or empty) catalytic interface in the ground-state structure of bovine F 1 -ATPase (grey). (a) and (b) show alignments via theand -subunits, respectively. Table 1 Crystallographic data-collection and refinement statistics.
Values in parentheses are for the highest resolution bin.  (hkl)i is the mean weighted intensity after the rejection of outliers. ‡ R factor = P hkl jF obs j À jF calc j = P hkl jF obs j, where F obs and F calc are the observed and calculated structure-factor amplitudes, respectively. § R free = P hkl&T jF obs j À jF calc j = P hkl&T jF obs j, where F obs and F calc are the observed and the calculated structure-factor amplitudes, respectively, and T is the test set of data omitted from refinement. statistics are summarized in Table 1. The final model contains residues 24-511 of the -subunit and residues 4-273, 279-314 and 320-469 of the -subunit. Associated with the structure are eight molecules of glycerol, 79 molecules of water and one phosphate ion. As in other structures of F 1 -ATPases, theand -subunits of the F-ATPase from P. denitrificans have very similar folds (r.m.s.d. of 5.1 Å ). Both are composed of three domains. The N-terminal domains (residues 24-95 in the -subunit and residues 4-77 in the -subunit) consist of six -strands. In the intact enzyme in other species, alternating N-terminal domains from each of the threeand -subunits are hydrogen-bonded together in the stable circular 'crown' structure of the F 1 -ATPase. The N-terminal domains of the and -subunits in the dimer from P. denitrificans are followed by the central nucleotide-binding domains (residues 96-381 in the -subunit and residues 78-355 in the -subunit). They consist of ten -strands and eight -helices and seven -strands and five -helices, respectively. The remainder of theand -subunits, residues 382-511 in the -subunit and residues 356-469 in the -subunit, are folded into a bundle of six and seven -helices that form the C-terminal domains of the subunits.
Despite the presence of ATP and magnesium ions in the mother liquor during the formation of crystals, in the structure of the dimer no nucleotide was found to be associated with either of the subunits. The nucleotide-binding and C-terminal domains of the -subunit are in a conformation similar to the open or empty conformations in E -subunits in almost all of the known structures of F 1 -ATPase, and therefore the interface appears to correspond to the empty or open catalytic interface of the P. denitrificans F-ATPase. However, the E E interface is more open than in bovine F 1 -ATPase because of contacts in the crystal lattice. Thus, the global r.m.s.d. of the dimer from P. denitrificans compared with the E E dimer from the bovine ground-state F 1 -ATPase is 3.0 Å (Fig. 4). The values for theand -subunits alone are 2.1 and 2.0 Å , respectively.
Although there are no nucleotides associated with the P. denitrificans E E catalytic dimer, electron density interpreted as a phosphate ion is associated with the phosphatebinding loop or P-loop region (residues 169-176) in the nucleotide-binding domain of the -subunit. It is bound via interactions with residues Thr173, Gly174, Lys175 and Thr176 (Fig. 5). The P-loop is a feature of many NTPases, and is so named because it interacts with phosphate moieties of bound NTP or NDP molecules (Walker et al., 1982). Neither phosphate nor sulfate was present in any of the buffers employed in the purification and crystallization processes, and it probably arises from hydrolysis of ATP in the purification and crystallization buffers.
Phosphate has not been observed bound in the vicinity of the P-loop regions of -subunits in other structures of F 1 -ATPase. However, electron density in the E -subunit adjacent to the P-loop has been interpreted as either a phosphate or a sulfate ion in the structures of bovine F 1 -ATPase in the ground state (Abrahams et al., 1994;PDB entry 1bmf), in complexes inhibited with beryllium fluoride (Kagawa et al., 2004; PDB entry 1w0j) or azide (Bowler et al., 2006;PDB entry 2ck3) and in the complex of F 1 -ATPase and the peripheral stalk subcomplex (Rees et al., 2009; PDB entry 2wss). However, the anion-binding site in the E P-loop is about 8 Å from where the -phosphate of the substrate ATP is bound in the catalytically active DP -subunit and from where presumably phosphate is released following scission of the bond between the Association of a phosphate ion with the phosphate-binding or P-loops of the -subunit from P. denitrificans and of the E -subunit from bovine F 1 -ATPase inhibited with the ATP analogue AMP-PNP (adenylylimidodiphosphate; PDB entry 1h8h; Menz et al., 2001). (a) The P-loops of the -subunit from P. denitrificans (residues 169-176) shown in deep red and (b) the P-loops of the the bovine -subunit (residues 157-163) shown in yellow. The bound phosphate ions are shown in orange and red. In (c), for reference, ATP is shown bound to the nucleotide-binding site of the E -subunit of bovine F 1 -ATPase (PDB entry 1h8h). involvement of a phosphate ion bound in the vicinity of the E P-loop of F 1 -ATPase in the catalytic mechanism of the enzyme.

Significance of the structure
The F-ATPase from P. denitrificans is an attractive target for further structural and functional study, especially because the mechanism of the regulation of its ATP hydrolase activity involving the inhibitor protein is not understood. The intact enzyme has been crystallized and diffraction data have been collected (Morales-Ríos et al., 2015). The current structure should be helpful in the interpretation of the structural data for the intact F-ATPase.