A c Subunit with Four Transmembrane Helices and One Ion(Na�)-binding Site in an Archaeal ATP SynthaseIMPLICATIONS FOR c RING FUNCTION AND STRUCTURE*□S

Received for publication, August 17, 2012, and in revised form, September 15, 2012 Published, JBC Papers in Press, September 24, 2012, DOI 10.1074/jbc.M112.411223

Florian Mayer‡, Vanessa Leone§, Julian D. Langer¶, José D. Faraldo-Gómez§�1, and Volker Müller‡2

From the ‡Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe UniversityFrankfurt/Main, 60438 Frankfurt, Germany, the §Theoretical Molecular Biophysics Group and the ¶Department of MolecularMembrane Biology, Max Planck Institute of Biophysics, 60438 Frankfurt/Main, Germany, and the �Cluster of Excellence“Macromolecular Complexes,” 60438 Frankfurt/Main, Germany

Background: The ATP synthase of Pyrococcus has an unusual gene encoding rotor subunit c.Results: The c ring is made of protomers with one ion-binding site in four transmembrane helices and is highly Na�-specific.Conclusion: Unprecedented subunit c topology and ion configuration in an ATP synthase.Significance: Archaeal ATP synthases are a remnant of primordial bioenergetics.

The ion-driven membrane rotors of ATP synthases consist ofmultiple copies of subunit c, forming a closed ring. Subunit ctypically comprises two transmembrane helices, and the c ringfeatures an ion-binding site in between each pair of adjacentsubunits. Here, we use experimental and computational meth-ods to study the structure and specificity of an archaeal c subunitmore akin to those of V-type ATPases, namely that from Pyro-coccus furiosus. The c subunitwas purified by chloroform/meth-anol extraction and determined to be 15.8 kDa with four pre-dicted transmembranehelices.However, labelingwithDCCDaswell as Na�-DCCD competition experiments revealed only onebinding site for DCCD and Na�, indicating that the mature csubunit of this A1AO ATP synthase is indeed of the V-type. Astructural model generated computationally revealed oneNa�-binding site within each of the c subunits, mediated by aconserved glutamate side chain alongside other coordinatinggroups. An intriguing second glutamate located in-betweenadjacent c subunits was ruled out as a functional Na�-bindingsite. Molecular dynamics simulations indicate that the c ringof P. furiosus is highly Na�-specific under in vivo conditions,comparable with the Na�-dependent V1VO ATPase fromEnterococcus hirae. Interestingly, the same holds true for thec ring from the methanogenic archaeon Methanobrevibacterruminantium, whose c subunits also feature a V-type archi-tecture but carry two Na�-binding sites instead. These find-ings are discussed in light of their physiological relevance andwith respect to the mode of ion coupling in A1AO ATPsynthases.

Archaea produce ATP using an ATP synthase that is distinctfrom the well known F1FO ATP synthase found in bacteria,mitochondria, and chloroplasts (1). Archaeal A1AO ATP syn-thases are evolutionary more closely related to vacuolar V1VOATPases, notwithstanding the fact that these act as ATP-drivenion pumps and are therefore functionally different (2–4). LikeF-ATP synthases andV-ATPases, A-ATP synthases comprise amembrane motor, AO, which is driven by downhill transloca-tion of H� or Na�, and a soluble domain, A1, where ATP issynthesized fromADP and Pi. A1 andAO aremechanically cou-pled by three protein stalks: one central and two peripheral.Under suitable conditions A1 can also hydrolize ATP and func-tion as a motor for uphill ion translocation across AO (2, 5, 6).The membrane-bound AO motor contains subunits a and c

(2, 7). Subunit c consists at least of two transmembrane helicesand is expressed in multiple copies, which form a ring-likestructure that, like the FO motor (8), functions as a rotatingturbine driven by the movement of ions across the membrane.In most A-type ATP synthases, subunit c has a single-hairpintopology as seen in F-type ATP synthases. By contrast, inV-type ATPases (9, 10) the c subunit apparently underwentgene duplication, resulting in a protein with four transmem-brane helices (11).Moreover, one ion-binding site was lost dur-ing the duplication event leading to a rotor with only half thenumber of ion-binding sites. These missing binding sites havebeen seen as the reason for the inability of V-ATPases to act asATP synthases. Instead, the rotor favors generation of large iongradients, a function important for the cellular physiology ofeukaryotes (3, 4).In recent years, however, the determination of the genome

sequences of several archaea have revealed an unexpected fea-ture of A1AO ATP synthases: the gene encoding for subunit cunderwent duplication (12, 13), triplication (14), and evengreater multiplication, so far up to 13-fold (15–17). Moreover,in some species the sequence motif characteristic of the ion-binding site is absent in one hairpin, which would result in csubunits with one ion-binding site within four transmembranehelices or two within six transmembrane helices (10, 18). Inparticular, the DNAdata for Pyrococcus furiosus implies that its

* This work was supported by Grants SFB807 (to V. M.) and EXC115 (to J. D.F.-G.) from the Deutsche Forschungsgemeinschaft.

□S This article contains supplemental Figs. S1–S3.1 To whom correspondence may be addressed: Max Planck Inst. of Biophys-

ics, Max-von-Laue-Str. 3, 60438 Frankfurt/Main, Germany. Tel.: 49-69-63031500; Fax: 49-69-63031502; E-mail: jose.faraldo@biophys.mpg.de.

2 To whom correspondence may be addressed: Molecular Microbiology &Bioenergetics, Inst. of Molecular Biosciences, Johann Wolfgang GoetheUniversity Frankfurt/Main, Max-von-Laue-Str. 9, 60438 Frankfurt/Main, Germany. Tel.: 49-69-79829507; Fax: 49-69-79829306; E-mail:vmueller@bio.uni-frankfurt.de.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 47, pp. 39327–39337, November 16, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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c subunit has a typical V-type topology with two hairpins butonly one Na�-binding site per subunit (10, 19). The implica-tion, according to common wisdom, would be that the enzymelost its function as an ATP synthase. However, the A1AO ATPsynthase from P. furiosus is the only ATP synthase encoded inthe genome and functions as an ATP synthase in vivo (19, 20).The structural rationale for the ATP synthesis activity of the

P. furiosus enzyme, despite the predicted V-type c subunit, isunknown. It could involve post-transcriptional modificationsas well as additional, yet hidden ion-binding sites in the matureprotein. Because the primary structure and number of ion-binding sites are assumed based on predictions from the DNAsequence, it was important to isolate the mature c subunit anddetermine experimentally its primary structure, molecularmass, and ion-binding sites. These studies culminate in a three-dimensional computationalmodel of the c ring fromP. furiosus,which provides a structural interpretation for our biochemicalexperiments, and enables us to assess the physiological ionspecificity of this and other archaeal A1AO ATP synthases.

EXPERIMENTAL PROCEDURES

Strain and Cultivation Condition—P. furiosus DSM 3638was obtained from the Deutsche Sammlung für Mikroorganis-men und Zellkulturen (Braunschweig, Germany) and wasgrown anaerobically in a 300 liter enamel-coated fermenter at98 °C in medium without sulfur, but with yeast extract, starch,and pepton as energy source and N2/CO2 (80:20, v/v) asdescribed before (21). The cells were harvested and stored at�80 °C until further use.Membrane Preparation and Protein Determination—20–40

g of P. furiosus cells (wet weight) were resuspended in buffer A(25mMTris, pH 7.5, 5mMMgCl2, 0.1mMPMSF) containing 0.1mg DNase I/ml. The cells were homogenized and disrupted bythree passages through a French pressure cell (Aminco) at3000 p.s.i. Cell debris and the thermosome, a cytoplasmaticheat shock protein present at high temperatures, were removedby four centrifugation steps (Beckman Avanti J-25, JA 14 rotor;7,500, 7,900, 8,200, and 8,500 rpm each for 20 min at 4 °C). Themembranes were sedimented from the crude extract by centrif-ugation (BeckmanOptima L90-K, 50.2 Ti rotor; 12,000 rpm for16h at 4 °C) andwerewashedwith buffer B (100mMHEPES, pH7.5, 5 mM MgCl2, 5% glycerol (v/v), 100 mM NaCl, 0.1 mM

PMSF). The washed membranes were collected by centrifuga-tion (Beckman Optima L90-K, 50.2 Ti rotor; 16,000 rpm, 5 h,4 °C) and resuspended in buffer C (100 mM HEPES, pH 7.5, 5mM MgCl2, 5% glycerol (v/v), 0.1 mM PMSF), and the proteinconcentration was determined as described (62).Purification of the A1AO ATP Synthase—Washed mem-

branes were resuspended in buffer C and used for membraneprotein solubilization. Triton X-100 was added to a concentra-tion of 3% (v/v) (1 g of Triton X-100/g of membrane protein),and membranes were incubated for 2 h at 40 °C and then over-night at room temperature under shaking. The membraneswere collected by ultracentrifugation (BeckmanOptima L90-K,TFT 65.13 rotor; 42,000 rpm for 2 h at 4 °C), and contaminatingproteins were precipitated with PEG 6000 (4.1%, w/w) for 30min at 4 °C. The precipitated proteins were removed by centrif-ugation (BeckmanOptima L90-K, TFT 65.13 rotor; 38,000 rpm

for 2 h at 4 °C), and the supernatant was loaded onto a sucrosegradient (20–66%) and centrifuged for 19 h in a vertical rotor(Beckman Optima L90-K, VTi50 rotor; 43,000 rpm at 4 °C).ATP hydrolysis activity of each sucrose gradient fraction wastested as described before (21). Fractions with the highestATPase activity were pooled and applied to anion exchangechromatography using DEAE-Sepharose, which was equili-brated with buffer D (50 mM Tris, pH 7.5, 5 mM MgCl2, 10%glycerol, 0.1mMPMSF, 0.1% (v/v) reducedTritonX-100). A saltgradient (0–1 M NaCl) in buffer D was used for protein elutionat a flow rate of 0.5 ml/min. Fractions with the highest ATPaseactivity were pooled, concentrated (molecular mass cutoff, 100kDa), and applied to gel filtration using a Superose 6 column(10/300 GL; GE Healthcare). Gel filtration was performed inbuffer E (50mMTris, pH 7.5, 5mMMgCl2, 10% glycerol, 0.1mM

PMSF, 0.05% n-dodecyl-�-D-maltoside) at a flow rate of 0.2ml/min. Again, fractions with the highest ATP hydrolysis activ-ity were pooled.Chloroform/Methanol Extraction of Subunit c of Membranes

from P. furiosus—The membranes resuspended in buffer Cwere mixed with 20 volumes of chloroform/methanol (2:1, v/v)for 20 h at 4 °C and filtered. 0.2 volume ofH2Owas added to thefiltrate and mixed for another 20 h at 4 °C. The organic phasewas separated from the aqueous and interphase using a separa-tion funnel and was washed twice with 0.5 volume of chloro-form/methanol/H2O (3:47:48, v/v/v). The washed organicphase was filled up with 1 volume of chloroform.Methanol wasadded until the turbid solution cleared up. The volume of thesolution was reduced to 1 ml using vacuum evaporation. Pro-tein was precipitated with 4 volumes of diethylether at �20 °Cfor 12 h and sedimented by centrifugation (Eppendorf 5417R,FA-45–24-11 rotor; 8,000 rpm at �8 °C). Sedimented proteinwas resolved in 1 ml of chloroform/methanol (2:1, v/v).N,N�-Dicyclohexylcarbodiimide Labeling Experiments—For

labeling experiments with N,N�-dicyclohexylcarbodiimide(DCCD, dissolved in ethanol),3 purified A1AO ATP synthasewas used. 1 ml of ATP synthase was dialyzed in a dialysis tube(molecular mass cutoff, 3.5 kDa) against 1000ml of buffer F (25mM Tris, 25 mM MES, 5 mM MgCl2, 10% glycerol) adjusted topH 5.5, 6.0, or 6.5 with HCl or KOH for 12 h at 4 °C. 20 �l ofATP synthase (9 �g of protein) was incubated with 250 or 500�M DCCD at pH levels of 5.5, 6.0, or 6.5 for 60 min at roomtemperature. For competition experiments between DCCDand NaCl or KCl, the salts were added to the ATP synthasesolution in concentrations of 1.25, 2.5, 5, 10, or 25 mM, directlybefore labeling. After labeling with DCCD, the ATP synthasewas purified using C4 Zip Tips to remove excessive DCCD andsalts. The C4 matrix (bed volume, 0.6 �l) of a 10-�l Zip Tip wasfirst equilibrated with 20 �l of 100% acetonitrile and 20 �l of0.1% trifluoroacetic acid. ATP synthase was coupled to theequilibrated matrix and washed with 30 �l of 0.1% trifluoro-acetic acid. The A1A0 ATP synthase was eluted with 10 �l of90% acetonitrile in 0.1% trifluoroacetic acid. To desintegratethe ATP synthase and the c ring of P. furiosus into cmonomers,10 �l of chloroform/methanol (2:1, v/v) was added and mixed.

3 The abbreviations used are: DCCD, N,N�-dicyclohexylcarbodiimide; FEP, freeenergy perturbation; CHES, 2-(cyclohexylamino)ethanesulfonic acid.

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The solution containing the cmonomers were dried by vacuumevaporation for 1.5 h at room temperature. The dried proteinpellet was mixed with 1 �l of 2,5-dihydroxyacetophenonematrix and applied to MALDI-TOF-MS as described below.MALDI-TOF-MS Measurements—Chloroform/methanol ex-

tracts for protein m/z determination were mixed in a 1:1 (v/v)ratio with matrix 2,5-dihydroxyacetophenone (15 mg/ml 2,5-dihydroxyacetophenone in 75% ethanol in 20 mM sodium cit-rate; Bruker Daltonics) or 2,5-dihydroxybenzoic acid (30 mg of2,5-dihydroxybenzoic acid/100 �l of TA solution (0.1% trifluo-roacetic acid/acetonitrile, 1:2 (v/v); Bruker Daltonics)) andspotted on ground steel target plates (Bruker Daltonics).MALDI mass spectra were recorded in a mass range of 5–20kDa using a BrukerAutoflex III Smartbeammass spectrometer.Detection was optimized form/z values between 5 and 20 kDaand calibrated using calibration standards (protein molecularweight calibration standard 1; Bruker Daltonics).Protein Identification and Quantification Using Mass Spec-

trometry (Peptide Mass Fingerprinting)—Chloroform/metha-nol extracts of P. furiosusmembranes were mixed in a 1:1 (v/v)ratio with 20 mM MES, pH 5.5, containing 0.5% n-octyl-�-D-glucopyranoside, and the organic phase was removed by a gen-tle N2 stream until the turbid solution was getting clear. Theprotein extract was then submitted to 12.5% SDS-PAGE (22)and stained with silver, suitable for mass spectrometry (23).Bands of interest were excised, reduced, alkylated, and digestedusing trypsin, chymotrypsin or both proteases according tostandard mass spectrometry protocols (24). Proteolytic digestswere applied to reverse phase columns (trapping column C18:particle size, 3 �M; length, 20 mm; and analytical column C18:particle size, 3 �M; length, 10 cm) (NanoSeparations, Nieuwk-oop, Netherlands) using a nano-HPLC (Proxeon easy nLC),eluted in gradients of water (0.1% formic acid, buffer A) andacetonitrile (0.1% formic acid, buffer B) in 50 min at flow ratesof 300 nl/min and ramped from 5 to 65% buffer B. Eluted pep-tides were ionized using a Bruker Apollo electrospray ioniza-tion source with a nanoSprayer emitter and analyzed in a qua-drupole time-of-flight mass spectrometer (Bruker maxis). Theproteins were identified bymatching themass lists on aMascotserver (version 2.2.2; Matrix Science) against NCBInr database.Modeling of the P. furiosus c Ring Structure—Homologous

sequences of the P. furiosus target c subunit sequence wereobtained after five PSI-BLAST iterations (25) on the nonredun-dant database, using 0.001 as the E-value cutoff. For scoring weused the BLOSUM62 matrix (26), a gap open penalty of 11 anda gap extension penalty of 1. The results were then clustered at65% sequence identity using CD-HIT (27, 28). Representativesequences of each cluster, plus target and template sequences,were used as input for a multiple alignment, using T-Coffee(29). The pairwise target-template alignment used for homol-ogy modeling was derived from this multiple alignment. Two-thousand structural models of the c10 ring of P. furiosus wereconstructed using the structure of the c ring from EnterococcushiraeV-type ATPase (30) as template (Protein Data Bank entry2BL2; 36% sequence identity with P. furiosus c subunit). Mod-eler 9v8 was employed to generate these models. The scoringfunctions GA341 (31) and DOPE (discrete optimized potentialenergy) (32) were used to select the best three models. The

coordinates of the bound Na� were translated from the tem-plate to the target structure. Secondary structure and trans-membrane predictions for the P. furiosus c ring, obtained withPsipred v2.5 (33) and TopCons (34), respectively, were com-paredwith the actual secondary structure (determinedwith theDSSP algorithm (35)) and transmembrane spans (estimatedwith OPM (36)) of the template.Modeling of a c3 Subconstruct of the c Ring of Methanobre-

vibacter ruminantium—A pairwise alignment of M. ruminan-tium target sequence with the c subunit from E. hirae (33%sequence identity) was generated as for the P. furiosussequence. We then generated and selected the best model of ac3 subconstruct of M. ruminantium in the same manner asexplained previously for the P. furiosus c10 ring (the stoichiom-etry of M. ruminantium c ring is unknown). Secondary struc-ture and transmembrane predictions for the target were com-pared with the secondary structure and transmembraneregions of the template as mentioned above.Molecular Dynamics Simulations and Calculations of the Ion

Selectivity of the c Ring Binding Sites—The c10 rings of E. hiraeand P. furiosus and the c3 construct of M. ruminantiumwere inserted in a hydrated palmitoyloleoylphosphatidylcho-line membrane (540, 542, and 189 lipid molecules and 38499,38319, and 11763 water molecules, respectively), using GRIF-FIN (37). The c10 rings have a single Na� in each c subunit,coordinated by Glu-139/Glu-142. The c3 construct ofM. rumi-nantium, however, carries one Na� coordinated by Glu-140 ineach c subunit and another coordinated by Glu-59 in betweenadjacent c subunits. The protein/membrane systems wereequilibrated using constrained all-atom molecular dynamicssimulations. The strength of the constraints on the proteinwere graduallyweakened over 12 ns for the c10 rings and 7 ns forthe c3 construct. Subsequently, unconstrained simulationswere carried out for 40 and 10 ns, respectively. The conforma-tions obtained after the unconstrained equilibrationswere usedas input of all-atom free energy perturbation (FEP) calculationsof the exchange between Na� and H� in each binding site andvice versa. The FEP calculations were performed in the forwardand backward direction, in 32 intermediate steps; each of thesesteps consists of 500 ps of sampling time, including 100 ps ofequilibration. Both molecular dynamics and FEP calculationswere carried out with NAMD2.7 (38) using the CHARMM27force field for proteins and lipids (39, 40). All simulations wereat constant pressure (1 atmosphere) and temperature (298 K),and with periodic boundary conditions in all directions. Thedimensions of the simulation box in the plane of themembrane(150 � 150 Å for the c10 rings and 72 � 96 Å for the c3 con-struct) were kept constant. The particle mesh Ewald methodwas used to compute the electrostatic interactions, with a realspace cutoff of 12 Å. A cutoff of 12 Å was also used for van derWaals interactions, computed with a 6–12 Lennard-Jonespotential. During the molecular dynamics and FEP simulationsof the M. ruminantium c3 construct, the conformations of thefirst (residues 6–77) and last hairpins (residues 86–161) werepreserved using a weak harmonic restraint on the root meansquare deviation of the backbone, relative to the initial model.

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RESULTS

Purification of Subunit c and Mass Determination—Subunitc of ATP synthases/ATPases is a very hydrophobic protein thatcan be isolated frommembranes using organic solvents such aschloroform/methanol (12, 41, 42). Membranes of P. furiosuswere thus extracted by chlorofom/methanol, and the extractwas applied to an SDS gel. As can be seen in Fig. 1, this proce-dure yielded two bands with apparent molecular masses of 16and 10 kDa. To identify these proteins, peptide mass finger-printingwas used, and theirmolecularmasseswere determinedbyMALDI-TOF-MS. Analysis of the 10-kDa band showed thatit actually consists of two proteins. One is the subunit K of theRNA polymerase (PF1642) with an apparent molecular mass of6,269 Da, and the other is subunit F of a putative monoca-tion/H� antiporter (PF1452) with an apparent mass of 9,076Da. The 16-kDa band in the SDS gel was identical to subunit c ofthe A1AO ATP synthase (PF0178). Its apparent molecular mass

was 15,853Da, whichmatches almost exactly themass deducedfrom the genome sequence (Mr� 15,806). The difference in themolecularmass, of around 50Da, is likely caused by a low signalintensity of the peak and multiple nonresolved oxidations ofsubunit c. Nevertheless, this is evidence that the mature c sub-unit ofP. furiosus is indeed a duplication of the “classical” 8-kDac subunit of F-type ATP synthases.Validation of the Amino Acid Sequence Predicted from DNA

Data—Peptide mass fingerprinting was used to verify the pre-dicted sequence of the P. furiosus subunit c (Fig. 2). Subunit cwas digested by trypsin, chymotrypsin, and a combination ofboth, and the fragments were analyzed by electrospray ioniza-tion-MS. The sequence coverage was 78.6%, and only one largefragment, from Ser-109 to Phe-131, was not resolved. Theexperimental data not only verified the predicted start codonbut also unequivocally confirmed the predicted amino acidsequence. As will be discussed later, the absence of a glutamineat position 26 (replaced by valine) and a glutamate at position55 (replaced by methionine) are particularly noteworthy. Ofspecial interest is the presence of a second glutamate at position51.Quantitative DCCD Labeling Indicates That Each c Subunit

Carries a Single Na� Site—DCCD inhibits ATP synthases/ATPases by covalently binding to a key carboxylate side chainfound in the ion-binding sites in the c subunit. In H�-drivenATP synthases, this carboxylate is the site of H� binding,through protonation (43). In Na�-coupled c subunits, this sidechain can also be protonated in the absence of Na�, but other-wise it is deprotonated and coordinates theNa�directly (30, 44,45). DCCD reacts with this carboxylate side chain only in itsprotonated state; therefore, in Na�-driven c subunits, DCCDand Na� compete for this common binding site, in a mannerthat is pH-dependent (46, 47). Thus, a DCCD labeling assay canin principle be used to quantify the number of ion-binding sitesin the c subunit, as well as to reveal whether or not they bindNa�.We first measured DCCD labeling to individual c subunits

extracted with chloroform/methanol from P. furiosus mem-branes. The c subunits were transferred from the organic phaseto a water phase with different pH levels of 5.5, 7.0, and 10.0 (25mM MES, pH 5.5, Tris, pH 7.0, CHES, pH 10.0, containing 1%n-octyl-�-D-glucopyranoside) by mixing both phases andremoving the chloroform/methanol by a N2 stream, becausethe DCCD labeling reaction does not proceed readily in thisorganic solvent. After the addition of 500�MDCCD, samples (1

FIGURE 1. Protein composition of the chloroform/methanol extract. Theextract was subjected to SDS-PAGE on 12.5% gels and stained with silver. Onthe left, molecular mass markers are provided.

FIGURE 2. Analysis of amino acid sequence of subunit c of P. furiosus. To analyze the amino acid sequence of subunit c, the protein was excised from 12.5%silver-stained gels, destained, reduced, alkylated, and digested using trypsin, chymotrypsin, or both proteases. The amino acids whose identities weredetermined are marked in bold type. Those potentially involved in ion-binding sites are shaded in grey.

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�l of sample mixed with 1 �l of 2,5-dihydroxybenzoic acid)were taken at 0, 30, 60, 90, and 150 min and examined withMALDI-TOF-MS. This analysis revealed one DCCD moleculebound to each c subunit, as is evident from the increase inmolecular mass by 206 Da, which corresponds to one moleculeof DCCD. DCCD labeling was time-dependent (34% after 30min, 46% after 60min, and 53% after 90min) and dependent onpH. Labeling was only observed at pH 5.5 and not at pH 7.0 or10.0.Unfortunately, DCCD labeling of c subunits isolated by chlo-

roform/methanol was not protected by NaCl, even whensmaller amounts of DCCD (50 or 100 �M) were used. This islikely due to the partial unfolding of subunit c as a result of theharsh purification procedure in chloroform/methanol. Instead,we labeled the purified A1AO ATP synthase with DCCD, andthe c subunit was isolated by chloroform/methanol afterwards.Upon incubation of the enzyme with DCCD, the molecularmass of subunit c increased from 15,803 to 16,010 Da (for theunoxidized protein), from 15,818 to 16,026Da (for the one timeoxidized protein), and from 15,835 to 16,042 Da (for the twotimes oxidized protein), indicating again that one c subunit hadbound oneDCCDmolecule (Fig. 3). The extent of DCCD label-ing was clearly dependent on the DCCD concentration and thepH used (Fig. 4). The labeling efficiency with 500 �M DCCDafter 60 min was roughly twice that observed with 250 �M, forthe same pH. For the same DCCD concentration, the labelingefficiency at pH6.5was one-fourth of that at pH5.5. Again, onlyone DCCD-reactive site was identified. Crucially, DCCD label-ing was prevented by Na�, but not K� (Fig. 5). The competingeffect of Na� on DCCD modification was clearly pH-depen-dent: the higher the pH, the less Na� was required to preventDCCD labeling.

Structural Model of the c Ring of P. furiosus with Its Na�-binding Sites—After the primary structure predicted from theDNA sequence had been verified, we generated a structuralmodel of the c ring of P. furiosus. The model is based on thestructure of the c ring from E. hirae, which also consists ofV-type c subunits. Supplemental Fig. S1 shows the alignment ofthe c subunit sequences from the V-type ATPase from E. hiraeand the A-type ATP synthase from P. furiosus. The known sec-ondary structure and the transmembrane spans (TM1 to TM4)of the E. hirae c subunit match well those predicted for theP. furiosus sequence. Furthermore, given that laser-induced liq-uid beam ion desorption-mass spectroscopy (LILBID-MS) datasuggest that the c ring of P. furiosus assembles as decamer (7), itis reasonable to employ the crystallographic structure of the c10rotor from E. hirae (30) as a template to model the archaealring.

FIGURE 3. Subunit c of P. furiosus binds one DCCD. Purified A1AO ATP synthase/ATPase of P. furiosus was incubated with 250 �M DCCD at room temperaturefor 60 min. Subunit c was extracted by chloroform/methanol from unlabeled (A) and DCCD-labeled (B) ATP synthase/ATPase. The molecular mass of both csubunits was determined by MALDI-TOF-MS.

FIGURE 4. pH- and dosis-dependent DCCD labeling of subunit c. The puri-fied A1AO ATP synthase of P. furiosus was incubated with 250 �M DCCD (lightgray bars) or 500 �M DCCD (dark gray bars) at room temperature and at pH 5.5,6.0, or 6.5 for 60 min. Subunit c was extracted by chloroform/methanol andexamined with MALDI-TOF-MS.

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As explained under “Experimental Procedures,” we pro-duced a large ensemble of tentative models and ranked themaccording to two independent scoring functions, namelyDOPEand GA341. Among these, we selected the twomodels with topranks according to either score, plus a third one that was alsohighly ranked in both scoring schemes. For all of them, theGA341 score was �0.8 (the closer to 1, the better is the model).All these three models are highly similar in their transmem-brane region (C�-trace root mean square deviation, �1.2 Å)but differ elsewhere (root mean square deviation, �9 Å).Mostly they vary in the long loop that connects the second andthird transmembrane spans, where there is a gap in the target-template alignment. Importantly, the ion-binding sites, clearlylocated within themembrane domain, do not vary significantly inthe different models. In sum, given the high confidence in thesequence alignment, the quality of the template structure, and theconvergence in the calculations toward a unique prediction, we

expect these models of the P. furiosus c ring to be very realistic,particularly in the transmembrane domain.One of these equivalent models is depicted in Fig. 6. The

model is perfectly consistent with the notion that ATP synthe-sis in this archaeon is driven byNa� gradients. The ion-bindingsites are located within each c subunit, flanked by TM2 andTM4 (Fig. 6B). The Na� is coordinated by the side chains ofGlu-142 (TM4), Gln-113 (TM3), Thr-56 (TM2), and Gln-57(TM2) and by the backbone of Leu-53 (TM2). In addition, theside chain of Tyr-60 (TM2) forms a hydrogen bond with Glu-142 and contributes to stabilize the geometry of the ion coor-dination shell. This network of interactions is identical to thatrevealed by the crystal structure of the c10 rotor from theE. hiraeV-type ATPase, which has been established to functionas a Na� pump under physiological conditions (48).As mentioned, the c subunit from the P. furiosus ATP syn-

thase also resembles that from the E. hirae ATPase in that itconsists of four transmembrane helices. It is therefore reason-able to ask whether Na�-binding sites may be found not onlywithin each c subunit, but also in between them, as occurs inrotor rings whose c subunits have a two-helix topology (49).Our structural model suggests that this is highly unlikely,because this region is markedly hydrophobic, namely Val-26(TM1�), Leu-48 (TM2�), Met-55 (TM2�), and Met-140 (TM4)(Fig. 6C). Such an environment could not possibly counter thecost of dehydration incurred upon Na� binding within themembrane domain. Consistently, the analogous location inthe crystal structure of the E. hirae rotor lacks a bound Na�; inthat structure, all of these hydrophobic residues are conserved,except for Met-55, which is substituted by Gly-63.Molecular Dynamics Simulations of the P. furiosus c Ring in

the Membrane—To further assess the verisimilitude of the c10model of the P. furiosus c ring, we carried out a moleculardynamics simulation of thismodel embedded in a phospholipidmembrane and compared the outcome with an analogous sim-ulation of the c ring from the E. hirae ATPase (Fig. 7A). Therationale here is that if themodel is a realistic approximation of

FIGURE 5. DCCD labeling of subunit c is Na�-dependent. The purified A1AOATP synthase of P. furiosus was incubated with different concentrations ofNaCl or KCl and labeled with 250 �M DCCD at room temperature and at pH 5.0for 60 min. Then subunit c was extracted by chloroform/methanol and exam-ined with MALDI-TOF-MS.

FIGURE 6. Structural model of the c10 rotor from the A1AO ATP synthase from P. furiosus. A, view of the complete c10 ring from P. furiosus, from theperiplasmic side. The bound sodium ions are shown as yellow spheres; alternate colorings (orange and green) indicate different c subunits. B, close-up view ofthe Na�-binding site, flanked by TM4 and TM2 within each c subunit. Residues involved in ion coordination are highlighted. Hydrogen bonds are indicated withdashed lines. C, close-up view of the interface between TM2� and TM4 in adjacent c subunits, at the level of the Na�-binding sites in B. Hydrophobic side chainsin this region are indicated. Also, note the protonated Glu-51 side chain, one helix turn away, toward the cytoplasmic side. This side chain hydrogen bonds toa carbonyl group in the backbone of TM4.

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the actual structure, its behavior in simulation ought to be com-parable with that of an experimentally determined structure.What we observe is that the dynamical range of the individual csubunits is essentially identical when comparing the modelfrom P. furiosus and the structure from E. hirae (Fig. 7B). Like-wise, the structure and dynamics of the Na�-binding sites inboth c rings are largely undistinguishable (Fig. 7C). Theseresults indicate that the internal structure of the c subunits inthe P. furiosusmodel is indeed very plausible. The relative ori-entation of the c subunits in the initial model of the ring, how-ever, seems to be somewhat suboptimal. In the first half of thesimulation, the structure of the ring as a whole departs from thestarting model more than the c ring from E. hirae does, i.e.,more than themagnitude of the natural room temperature fluc-

tuations. Nevertheless, also this overall structural arrangementbecomes stable in the second half of the simulation (Fig. 7B).A Second, Constitutively Protonated Glutamate within the

Membrane Domain—A noteworthy feature of the P. furiosussequence is the presence of a second glutamate side chain inTM2� (Glu-51), one helix turn toward the cytoplasmic side ofthe rotor (Fig. 6C). Could this be a second ion-binding site?Ourmodel suggests that this side chain is constitutively protonatedand that it contributes to the stability of the interface betweenadjacent c subunits in the assembled ring, by forming a hydro-gen bondwith the carbonyl group of residue Phe-137 (in TM4).Consistently, this nonconserved side chain is replaced by glu-tamine in homologous sequences, for example in E. hirae.Indeed, in the crystal structure of the E. hirae rotor, this gluta-mine side chain is seen to form the same interaction, acrossfrom TM2� to TM4 of the adjacent c subunit. Therefore wehypothesize that protonation of Glu-51 is structurally impor-tant, but not functionally relevant.In support of this view, DCCD labeling of the assembled

rotor results in one residue modification per subunit (Fig. 3).Based on our structuralmodel, we interpret this result to reflectthe reaction of DCCD with Glu-142, which can be expected to

FIGURE 8. DCCD accessibility to Glu-142 and Glu-51. A, two libraries of 5832possible rotamers of DCCD-modified Glu-142 and Glu-51 were created byrotation of �1, �2, and �3 angles (in 18° increments). For each rotamer, thecontact distance between DCCD and the rest of the protein was computed.DCCD modification can occur only if the resulting contact distance is largerthan �2.5 Å. B and C, two snapshots of the rotamer libraries generated forGlu-142 and Glu-51. The conformation in B is very similar to that found incrystal structures of DCCD-modified rotor rings; in C, the DCCD label clasheswith the outer helices TM2� and TM4.

FIGURE 7. Molecular dynamics simulations of the c rings from P. furiosusand E. hirae in a lipid membrane. A, simulation systems for the c rings fromthe E. hirae ATPase (left panel) and the P. furiosus ATP synthase (right panel),each embedded in a phospholipid membrane (gray). The individual c sub-units are colored alternately (orange and green). Water molecules and otherdetails are omitted for clarity. The view is from the cytoplasmic side.B, variability in the structure of the c rings during the simulation, in terms ofthe root mean square (RMS) difference relative to the starting structure. Thedata are shown for the rings evaluated as a whole and for the c subunitsanalyzed individually and then averaged. C, ion-protein coordination dis-tances in the Na�-binding site, shown as probability distributions. The distri-butions derive from the complete time span of the simulation, i.e., they reflectnot only the variability among different binding sites in the ring but also thestructural dynamics of each site.

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be transiently protonated at low Na� concentrations, ratherthan the modification of Glu-51. Indeed, as shown in Fig. 8,DCCDmodification is structurally viable in the case ofGlu-142,upon an outwards rotation of the carboxylate group. Thisminorbut necessary rearrangement is essentially identical to that seen incrystal structures ofDCCD-modified c rings (50, 51). In the case ofGlu-51, however,we find thatDCCDmodificationwould be steri-cally impossible in all rotamers of the side chain (in �1, �2, and �3).Thus, Glu-51 cannot be modified in the context of the assembledrotor. Consistent with this interpretation, increasing concentra-tions of Na� inhibit DCCD labeling of the rotor (Fig. 5), becauseNa� binding precludes protonation of Glu-142 (but not Glu-51).P. furiosus Is Not the Only Archaeon That Has a 16-kDa c

Subunit with Only One Na�-binding Site—An alignment of allc subunit sequences available for archaea (supplemental Fig. S2)indicates that c subunits with four transmembrane helices arefound in Crenarchaeota and Euryarchaeota, but not in Korar-chaeota and Thaumarchaeota. Pyrococci and Thermococci arethe only archaea of the Euryarchaeotawith a c subunit contain-ing four transmembrane helices and a single Na�-binding sitebetween TM2 and TM4 of the same subunit, like P. furiosus. Inthe Crenarchaeota phylum, the Desulfurococci and Staphylo-thermus species, as well as Ignisphaera aggregans, also feature aduplicated c subunit with a single Na�-binding site. Interest-ingly, among the archaeal c subunits with four transmembranehelices, only those from methanogens contain two Na�-bind-ing sites per c subunit. Methanobrevibacter, Methanothermo-bacter, and Methanobacterium species, as well as Methano-sphaera stadtmanae, feature a binding site analogous to that inP. furiosus, i.e., formed within the c subunit, and a second one,identical in its amino acid composition, which would appearbetween adjacent c subunits in the assembled ring, i.e., medi-ated by a glutamate in TM2, a glutamine in TM1 and the pro-totypic set of additional coordinating groups in TM3� andTM4�. A close-up view of the structure of these two bindingsites, derived from simulations of a homology model of theM. ruminantium c ring is shown in Fig. 9 (A and B) (see alsosupplemental Fig. S3).The Rings of E. hirae, P. furiosus, and M. ruminantium Have

Equivalent Na� Specificity—Althoughmost ATP synthases aredriven by transmembrane gradients of either protons or Na�,recent studies of the methanogenic archaeon Methanosarcina

acetivorans have revealed that its c ring is coupled to both gra-dients, i.e., its c subunit is effectively nonspecific under typicalin vivo concentrations of H� and Na� (52). This is to say thatthe ion-binding sites in theM. acetivorans c ring are sufficientlyH� selective to counter the large physiological excess of Na�

over H�, but not so much as to preclude Na� binding alto-gether. Because methanogenesis in this cytochrome-contain-ing organism is coupled to primary Na� and H� translocation,the ability ofM. acetivoransATP synthase to use both seems tobe a very efficient bioenergetic adaptation. However, the gen-erality of this solution is unclear. It has been suggested that thec ring from M. ruminantium might also be able to utilize bothgradients (53), but thismethanogen does not have cytochromesand therefore does not have a primary proton but only a Na�

gradient generated by themethyltetrahydromethanopterin-co-enzymeMmethyltransferase, and thus its ATP synthase shouldbe Na�-specific.To clarify this question, we used molecular dynamics simu-

lations to compute the free energy of selectivity for H� overNa� of the binding sites in the c rings of P. furiosus andM. ruminantium, relative to the selectivity of the c ring of theNa�-pumping ATPase from E. hirae (Fig. 9C). From this anal-ysis, we conclude that the ion specificity of the c rings in thesethree species is largely identical, consistent with the similarityin the amino acid make-up of their ion-binding sites. That is,theM. ruminantium ATP synthase is very likely to be coupledexclusively by Na� under in vivo conditions. TheH� selectivityof the M. acetivorans c ring is, by contrast, much more pro-nounced. As mentioned, this enables this ATP synthase to uti-lize the proton gradient even under conditions of Na� excess.Organisms such as the cyanobacterium Spirulina platensis andthe alkaliphilic bacterium Bacillus pseudofirmus have c ringswith an even greater H� selectivity (43, 54), so much so thatNa� binding is no longer viable, despite its excess, and there-fore these ATP synthases are exclusively coupled to H�.

DISCUSSION

Archaea not only inhabit environments with extreme tem-peratures, pH, and/or salinity, but some can also liveautotrophically. They are believed to be early life forms (55),implying that also their bioenergetics is ancient. Methanogen-esis (and acetogenesis), processes in which carbon dioxide is

FIGURE 9. Ion selectivity of the c ring from P. furiosus and other representative cases. A and B, close-up views of the two Na�-binding sites in a model ofa c3 construct from the M. ruminantium ATP synthase. One is flanked by TM4 and TM2 within a single c subunit, and the second is formed in between adjacentc subunits in the context of the ring. Residues involved in ion coordination are highlighted. C, ion selectivity scale of representative c ring rotors, relative to thec ring from the E. hirae ATPase, including those from P. furiosus and M. ruminantium. The data derive from free energy calculations based on all-atom moleculardynamics simulations of complete c rings (E. hirae (E.h.), P. furiosus (P.f.), and S. platensis (S.p.)) or c3/4 constructs (M. ruminantium (M.r.), M. acetivorans (M.a.), andB. pseudofirmus (B.p.) OF4) in phospholipid membranes.

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reduced to acetyl-CoA via the Wood-Ljungdahl pathway, areseen as ancient pathways in which carbon dioxide formation iscoupled to the synthesis of ATP via a transmembrane sodiumion gradient (56). In the simplestmethanogens that do not con-tain cytochromes, the sodium-motivemethyltetrahydrometha-nopterin-coenzyme M methyltransferase is the only energeticcoupling site, and the ��Na

� established the only driving forcefor ATP synthesis (57, 58). This is consistent with our view thatthe ATP synthase from M. ruminatium is highly Na�-specificunder in vivo conditions (because it is only weakly H� selec-tive). With the advent of additional proton bioenergetics inmethanogens because of the evolution of methanophenazineand cytochromes (59), the advantages of evolving an ATP syn-thase that can couple to both Na� and H� gradients arose. Asexemplified inM. acetivorans, its ATP synthase is concurrentlydriven by Na� and H� (52).In contrast to the autotrophic methanogens, P. furiosus is

heterotrophic and grows by fermentation. However, glycolysisis coupled to the reduction of the low potential electron carrierferredoxin (E0� � �480 mV) (60). Oxidation of reduced ferre-doxin with subsequent reduction of protons to hydrogen gaswas experimentally shown to be coupled to the generation of atransmembrane electrochemical ion gradient able to drive thesynthesis of ATP (20). The nature of the ion translocated hasnot been determined yet, but it was assumed to be H�. How-ever, in light of the finding that its ATP synthase is Na�-depen-dent, the gradient energizing themembrane ought to be ofNa�.Anyway, this experiment clearly demonstrated that the enzymeis capable of ATP synthesis.Here we have demonstrated that the rotor subunit c of

P. furiosus is indeed a protein with four transmembrane helicesbut only one ion-binding site. Therefore, the solution to theenigma of how this enzyme synthesizes ATP is neither a post-transcriptional/post-translational modification of the mature csubunit, nor the presence of an unexpected second ion-bindingsite. The explanation may be the number of c subunits in the cring, which LILBID-MSandEMdata indicate to be 10 (7). If thisinterpretationwas correct, theV1VOATPase ofE. hiraewith its10 c subunits in the ring should also be able to synthesize ATP.An additional piece that could contribute to the solution of theenigma is a lower phosphorylation potential in these archaea.Indeed, calculation of the �Gp in Methanothrix soehngeniibased onmeasurement of the nucleotides revealed a value of 45kJ/mol for�Gp (61). Indeed, thiswould drop the number of ionsrequired for ATP synthesis from 3.4 at 60 kJ/mol down to 2.5.The data presented here are not only consistent with our

previous hypothesis that the A1AO ATP synthase of P. furiosusis Na�-motive (21) but also predict the presence of V-typeNa�-dependent c subunits in a number of archaea. Only inmethanogens are two ion-binding sites found in the four trans-membrane helices of subunit c. This most likely reflects theirautotrophic life style at the thermodynamic limit of life. Theother archaea with V-type c subunits are metabolicallymore versatile, and the prominent function of the enzyme maybe that of an ATP-driven ion pump. In P. furiosus, for example,the amount of ATP synthesized by chemiosmosis is probablymuch less than that by substrate level phosphorylation linked to

sugar degradation. Thus, in vivo, such a c ring may be an adap-tation to growth at thermodynamic equilibrium.

Acknowledgments—We thank Michael Thomm and Harald Huber(University of Regensburg) for supplying cells of P. furiosus.

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MüllerFlorian Mayer, Vanessa Leone, Julian D. Langer, José D. Faraldo-Gómez and Volker

STRUCTUREan Archaeal ATP Synthase: IMPLICATIONS FOR c RING FUNCTION AND

)-binding Site in+ Subunit with Four Transmembrane Helices and One Ion (NacA

doi: 10.1074/jbc.M112.411223 originally published online September 24, 20122012, 287:39327-39337.J. Biol. Chem. 

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