The structure of the catalytic domain of the ATPsynthase from Mycobacterium smegmatis is a targetfor developing antitubercular drugsAlice Tianbu Zhanga,1, Martin G. Montgomerya,1, Andrew G. W. Leslieb, Gregory M. Cooka,c, and John E. Walkera,2

aThe Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge Biomedical Campus, CB2 0XY Cambridge, United Kingdom;bThe Medical Research Council Laboratory of Molecular Biology, Cambridge Biomedical Campus, CB2 0QH Cambridge, United Kingdom; and cDepartmentof Microbiology and Immunology, University of Otago, 9016 Dunedin, New Zealand

Contributed by John E. Walker, December 17, 2018 (sent for review October 12, 2018; reviewed by Thomas M. Duncan and Wayne D. Frasch)

The crystal structure of the F1-catalytic domain of the adeno-sine triphosphate (ATP) synthase has been determined from My-cobacterium smegmatis which hydrolyzes ATP very poorly. Thestructure of the α3β3-component of the catalytic domain is similarto those in active F1-ATPases in Escherichia coli and Geobacillusstearothermophilus. However, its e-subunit differs from those inthese two active bacterial F1-ATPases as an ATP molecule is notbound to the two α-helices forming its C-terminal domain, probablybecause they are shorter than those in active enzymes and they lackan amino acid that contributes to the ATP binding site in activeenzymes. In E. coli and G. stearothermophilus, the α-helices adoptan “up” state where the α-helices enter the α3β3-domain and pre-vent the rotor from turning. The mycobacterial F1-ATPase is mostsimilar to the F1-ATPase from Caldalkalibacillus thermarum, whichalso hydrolyzes ATP poorly. The βE-subunits in both enzymes are inthe usual “open” conformation but appear to be occupied uniquelyby the combination of an adenosine 5′-diphosphate molecule withno magnesium ion plus phosphate. This occupation is consistentwith the finding that their rotors have been arrested at the samepoint in their rotary catalytic cycles. These bound hydrolytic prod-ucts are probably the basis of the inhibition of ATP hydrolysis. It canbe envisaged that specific as yet unidentified small moleculesmight bind to the F1 domain in Mycobacterium tuberculosis, pre-vent ATP synthesis, and inhibit the growth of the pathogen.

Mycobacterium smegmatis | F1-ATPase | structure | inhibition |tuberculosis

In 2017, around 1.6 × 106 people died from tuberculosis (TB),and Mycobacterium tuberculosis, the causative bacterium, is

now the second greatest killer of mankind by a single infectiousagent, surpassed in its lethal impact only by human immunode-ficiency virus/acquired immunodeficiency syndrome (1). Of the10 × 106 people estimated to have developed TB in that singleyear, 4.6% were resistant to both rifampicin and isoniazid andare classed as multidrug resistant (MDR), and 8.5% of theMDR-TB cases were extensively drug resistant (XDR). Only55% of the MDR-TB and 30% of the XDR-TB cases weretreated successfully. These alarming statistics serve to emphasizethe urgent need to develop new drugs that are effective and fastacting against drug-resistant strains of M. tuberculosis. Prefera-bly, they should be effective also against latent M. tuberculosisinfections where the bacteria lie dormant in infected humans in anonreplicating state before emerging as an active infection. It hasbeen estimated that between a quarter and a third of the world’spopulation are latently infected (2). However, the impact of thislatency has been questioned recently as nearly everyone who fallsseriously ill with TB does so within 2 y of being infected, and la-tent infections rarely become active even in old age (3).In 2012, the U.S. Food and Drug Administration approved the

use of a novel oral drug bedaquiline (SIRTURO) for the treat-ment of MDR-TB (4, 5), and bedaquiline received fast-trackapproval as a component of a combination therapy for treating

MDR-TB in adults. Its potential to shorten dramatically thetreatment time for MDR-TB is highlighted by two recent studies.In mouse models of TB, a combination of bedaquiline with PA-824, an antimycobacterial drug with a complex mode of action(6) and linezolid, a repurposed protein synthesis inhibitor, sig-nificantly improved efficacy and relapse rates compared with thefrontline regimen of rifampicin, isoniazid, and pyrazinamide (7,8). In the Nix-TB phase III clinical trial using this three-drugcombination, 74% of the patients with MDR-TB were culturenegative in 8 wk*. The most recent recommendations for thetreatment of MDR-TB, based on the balance of effectivenessand harm and a preference for oral administration, now includebedaquiline (9). Bedaquiline is effective against both activelygrowing and nonreplicating cells of M. tuberculosis and acts byinhibiting the ATP synthase (10, 11) thereby shutting off thesupply of cellular energy in the bacterium without noticeablyaffecting the human enzyme found in the inner membranes ofthe mitochondria. Thus, these observations provide proof ofprinciple that the mycobacterial ATP synthase is a suitable target

Significance

Last year, 1.6 × 106 people died from tuberculosis (TB), andabout 10 × 106 became infected with the causative bacte-rium Mycobacterium tuberculosis and 460,000 of them withmultidrug-resistant bacteria. Bedaquiline, a new anti-TB drug,developed to combat multidrug-resistant TB, kills M. tubercu-losis by preventing the operation of its molecular machine forgenerating adenosine triphosphate (ATP), the fuel of life,without affecting the equivalent human machine. Here, wedescribe the molecular structure of the module in the myco-bacterial machine where ATP is generated. Differences be-tween this module and the equivalent human module cannow be exploited to develop new anti-TB drugs, unrelated tobedaquiline, that also may help to prevent and cure TB byinhibiting the formation of ATP.

Author contributions: G.M.C. and J.E.W. designed research; A.T.Z. and M.G.M. performedresearch; A.T.Z., M.G.M., A.G.W.L., G.M.C., and J.E.W. analyzed data; J.E.W. wrote thepaper; and J.E.W. supervised project.

Reviewers: T.M.D., SUNY Upstate Medical University; andW.D.F., Arizona State University.

The authors declare no conflict of interest.

Published under the PNAS license.

Data deposition: The atomic coordinates and structure factors have been deposited in theProtein Data Bank, www.wwpdb.org (PDB ID code 6FOC).

See Commentary on page 3956.1A.T.Z. and M.G.M. contributed equally to this work.2To whom correspondence should be addressed. Email: walker@mrc-mbu.cam.ac.uk.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1817615116/-/DCSupplemental.

Published online January 25, 2019.

*Conradie F, et al. (2017) The NIX-TB trial of pretomanid, bedaquiline and linezoid totreat XDR-TB. Conference on RetroViruses and Opportunistic Infections 2017, Seattle,abstr 80LB.

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for developing new drugs to combat tuberculosis. A rationalapproach to the design of new drugs in addition to bedaquiline toinhibit the mycobacterial ATP synthase requires ideally that thestructures and mechanistic and regulatory mechanisms of thehuman and mycobacterial ATP synthases be understood. The hu-man enzyme is very similar in both respects to the well-studied bo-vine enzyme, which therefore provides an excellent surrogate.Mycobacterial ATP synthases have been less studied, and only thestructure of the c ring in the membrane domain of the enzyme’srotor in the enzyme from the nonpathogenic organism Mycobacte-rium phlei has been established (12). It is here that bedaquiline binds(12), presumably impeding the turning of the rotor in the intactenzyme. It has been proposed that it also binds at a secondary site inthe e-subunit (13, 14). Before, the work described here, the structureof its F1-catalytic domain was not known in any mycobacterial ATPsynthase, and there was no molecular understanding of why themycobacterial enzymes are barely capable of hydrolyzing ATP (15),whereas, for example, the enzymes from facultative anaerobes, suchas Escherichia coli can both synthesize and hydrolyze ATP. Here, wedescribe the structure of the inhibited state of the catalytic domain ofthe ATP synthase from another nonpathogenic Mycobacterium,Mycobacterium smegmatis. It is an excellent surrogate for the cata-lytic domain of the F1-ATPase fromM. tuberculosis as a comparisonof the sequences of the subunits from various mycobacterial speciesdemonstrates (SI Appendix, Fig. S1 and Table S1).

Results and DiscussionCharacterization of F1-ATPase fromM. smegmatis. The nine subunitsof the ATP synthase in M. smegmatis are encoded by the atpoperon, which includes the cluster of genes atpAGDC encodingthe constituent α-, γ-, β-, and e-subunits, respectively, of the F1-ATPase complex. This cluster was amplified by the polymerasechain reaction, modified to encode the N terminus of theβ-subunit fused to a hexahistidine tag with an interveningprotease cleavage site, and the vector containing the fourgenes was introduced into M. smegmatis. Attempts to over-express the M. tuberculosis orthologs in M. smegmatis in thesame way failed as the genes from the two mycobacterialspecies recombined. The overexpressed purified F1-ATPase(SI Appendix, Fig. S2) was a single complex with a mass of 380kDa, composed of the expected complement of α-, β-, γ-, ande-subunits with their characteristic molecular masses (SI

Appendix, Fig. S3 and Table S2). It had a very low ATP hy-drolase activity (0.07 U/mg), and, in contrast to some otherlatent F1-ATPases, for example, the enzyme from Caldalka-libacillus thermarum (16), this low activity could not be stim-ulated by lauryldimethylamine oxide (LDAO). However, whenthe mycobacterial enzyme was treated briefly with trypsin, thespecific activity increased by 100-fold to 7 U/mg. Althoughcharacterization of the proteolytic fragments (SI Appendix,Table S3) did not provide a clear indication of the mechanismof activation, it is worth noting that the e-subunit had beendegraded almost completely after 2 min with a correspondingsignificant increase in activity. In E. coli F1-ATPase andF1Fo-ATPase from M. smegmatis, activation by trypsinolysishas been attributed to removal of the e-subunit (17). The activityof F1-ATPase from M. smegmatis uncovered by trypsinolysis wasdoubled by the addition of LDAO (SI Appendix, Fig. S4).

Structure Determination. Hexagonal crystals of the complex con-taining all four subunits (SI Appendix, Fig. S5) have the unit-cellparameters a = b = 105.2, c = 628.6 Å with α = β = 90.0° and γ =120.0° and belong to space group P3121 with one F1-ATPase inthe asymmetric unit (see SI Appendix, Table S4 for a summary ofdata collection and refinement statistics). The quality of theelectron density map is indicated in SI Appendix, Fig. S6 whererepresentative segments and their interpretation are shown. Thestructure (Fig. 1A) contains the following residues: αE, 31–190,202–511, and 1512–1522 (corresponding to the C-terminal ex-tension where the register is unclear. Here, the residue numbershave been increased by 1,000 to indicate uncertainty as requiredby the PDB); αTP, 30–190, 202–406, and 414–511; αDP, 30–190,202–409, and 412–511; βE, 9–41, 47–108, 116–132, and 136–471;βTP, 8–41, 47–108, and 114–471; βDP, 9–41, 47–108, and 116–471;γ, 4–57, 84–108, 119–129, 139–163, 188–198, and 238–304; e, 3–115. Overall, the structure is similar to those of F1-ATPasesdetermined previously in other species (SI Appendix, Fig. S7 andTable S5) and especially to the F1-ATPase from C. thermarum(18) (Fig. 1B). Although the structures of the αE-, αTP-, and αDP-subunits terminate at residue 511, the sequences of the subunitsextend to residue 548. This C-terminal extension is characteristicof α-subunits in mycobacteria and is not found in other eubac-terial, chloroplast, or mitochondrial sequences (SI Appendix, Fig.S8). By two independent programs, this extension is predicted to

A B C

Fig. 1. The structure of the F1-ATPase from M. smegmatis. (A) Side view in ribbon representation with the α-, β-, γ-, and e-subunits in red, yellow, blue, andgreen, respectively, and bound nucleotides in black. (B) Comparison of the F1-ATPase complexes from M. smegmatis (sky blue) and C. thermarum (18) (or-ange). The structures were superimposed via their α3β3-domains. (C) The C-terminal extension in the αE-subunit of F1-ATPase from M. smegmatis. Densityextending from the C-terminal helix (residues 494–511) was modeled from residues 1512–1522 (red), but the register is uncertain, and the following 27residues are unresolved. An adenosine 5′-diphosphate (ADP) molecule (black) is shown bound to the nucleotide binding site.

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be intrinsically disordered (SI Appendix, Fig. S9), and it was pos-sible to build a segment of 10 residues of extended structure im-mediately following the C-terminal α-helix of the αE-subunit (Fig.1C). However, the sequence register could not be determinedunambiguously, and so this segment is modeled as unknown andnumbered 1512–1522. In a peptide representing residues 521–540,it has been shown by solution nuclear magnetic resonance (NMR)that residues 526–539 are α-helical (but no coordinates areavailable) and on the basis of structure prediction that thisα-helical structure prevailed in the intact protein (19). However,the current prediction of intrinsic disorder in the entire C-terminalregion from residues 512–548 (SI Appendix, Fig. S9) and thesecondary structures of the subunit in seven mycobacterialspecies predicted with the program PSIPRED (20) (SI Ap-pendix, Fig. S10) are not in accord with this proposal. Anotherprogram, Predator (21), used previously (19), gives an am-biguous answer, predicting an α-helix in the region of residues530–540 in four out of the seven species including M. tuber-culosis but not M. smegmatis (SI Appendix, Fig. S10), M. phlei,or Mycobacterium ulcerans. In conclusion, on the basis of thecurrent structure predictions (and acknowledging that NMRstudies show that residues 526–539 in the isolated segmentfrom residues 521–540 are α-helical), it appears on balance tobe unlikely that an α-helix forms in this region of the intactα-subunit. Nonetheless, it remains possible that this additionalregion of the mycobacterial subunits could play a role in theregulation of the enzyme (19).

The α3β3-Domain. In the nucleotide binding sites of the threeα-subunits and the βTP-subunit, additional electron density iscompatible with them being occupied by an ADP molecule plus amagnesium ion (Fig. 2). There is also additional electron densityassociated with the nucleotide binding sites in both the βDP- andthe βE-subunits. Although the additional density is discontinuousin the former, it can be interpreted plausibly as ADP plus amagnesium ion. In the latter site, the additional density is asso-ciated with the region of the phosphate-binding loop (P-loop)where the α- and β-phosphates of a nucleotide would be boundas, for example, in the βE-subunit of the F1-ATPase from C.thermarum (18) (where an ADP molecule with no magnesiumion is bound at 50–100% occupancy when crystals are grown inthe presence of 500 μM ADP). Both the ADP and a singlephosphate ion were tested near the P-loop at various occupan-cies, but neither refined well into this site. However, there is, alsoadditional density above the P-loop where a phosphate ion has

been modeled as in the βE-subunit of the C. thermarum enzymewhere a phosphate ion sits above the ADP. Although thisdensity has been modeled as a phosphate, it could possibly bea sulfate introduced from the crystallization buffer. Superimposi-tion of the structures of the F1-ATPases from M. smegmatis andC. thermarum via their α3β3-domains showed that the two struc-tures are very similar [root-mean-square deviation (rmsd) valueof 0.91 Å]. The occupancy of nucleotide binding sites in theC. thermarum enzyme is ADP and a magnesium ion in the threeα-subunits and in the βTP- and βDP-subunits and ADP and aphosphate without a magnesium ion in the βE-subunit. There-fore, although the additional density in the P-loop region of theβE-subunit in the M. smegmatis enzyme remains uninterpreted,the close similarity of the structure to the structure of the inhibitedcomplex in C. thermarum suggests that the site is probably occu-pied by an ADP molecule (with no accompanying magnesium ion)at low occupancy.

The γ-Subunit. The structure of the γ-subunit is the least wellresolved of the eight constituent subunits of the enzyme, prob-ably because it is not constrained by any contacts with other F1-ATPase complexes in the crystal lattice. In C. thermarum, forexample, where the subunit is constrained in the crystal lattice, itis resolved entirely apart from residues 1–3. The C. thermarumsubunit is folded into two α-helices in its N- and C-terminal re-gions with an intervening Rossmann fold as in other species thathave been studied (Fig. 3) (18, 22–25). The N- and C-terminalα-helices make an antiparallel coiled coil occupying the centralaxis of the α3β3-domain, and the Rossmann fold has fiveβ-strands with α-helices among strands 1 and 2, 2 and 3, and 3and 4. In the mycobacterial enzyme, the N- and C-terminalα-helices are well resolved except for approximately threeα-helical turns at the N terminus of the C-terminal α-helix. Theintervening α-helices 2, 3, and 4 were also resolved, but none ofthe five β-strands and connecting loops in the Rossmann foldcould be built. However, superposition of the fragmentarystructure of the mycobacterial γ-subunit upon the C. thermarumγ-subunit is consistent with the structures of the two orthologsbeing closely similar. This structural similarity extends to theγ-subunits from E. coli (22), P. denitrificans (23), and spinachchloroplasts (24) (Fig. 3). The overall fold of these γ-subunitsis also similar to the γ-subunits from the enzymes from bovine(25) (Fig. 3) and yeast (26) mitochondria except that the N-terminal α-helices of the bacterial γ-subunits extend further inthe C-terminal direction and they are less curved. However, the

A B C

D E F

Fig. 2. Occupancy of nucleotide binding sites in theα- and β-subunits of the F1-ATPase from M. smegma-tis. A Fo-Fc difference density map was calculated forthe complex with the nucleotides, phosphate, Mg2+,and water molecules at zero occupancy. The differ-ence density is shown as green mesh contoured to 2.5σ. (A–C) The αDP-, αTP-, and αE-subunits; (D–F) the βDP-,βTP-, and βE-subunits. In (A–E), the sites are occupiedby ADP and an accompanying magnesium ion (blacksphere) with four water ligands (red crosses); the fifthand sixth ligands are provided by O2B of the ADP andthe hydroxyl of either αThr-179 or βThr-167. In (F), theupper region of the catalytic site is occupied by aphosphate (or sulfate) ion (orange and red). Althoughthe electron density beneath it in the vicinity of the P-loop cannot be interpreted with confidence, it prob-ably can be accounted for by an ADP molecule(without a magnesium ion) at partial occupancy.

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γ-subunits of the M. smegmatis and C. thermarum enzymes havean additional highly significant similarity that distinguishes themfrom the γ-subunits in the other F1-ATPases and supports theview that the two determined structures represent the sameinhibited state of the enzyme. In the M. smegmatis and C.thermarum γ-subunits, residues 22–33, the “rigid-body” re-gions (see Materials and Methods) are rotated approximatelyto the same extent (10.5° in M. smegmatis, and 9° and 13° in thetwo copies in the asymmetric unit of the crystals of the C.thermarum enzyme, respectively), whereas the rotation anglesin the E. coli, P. denitrificans, and spinach chloroplast en-zymes, which have different nucleotide occupancies in theircatalytic sites to the C. thermarum enzyme, are 50°, 27°, and22°, respectively.A feature, discussed before, that distinguishes mycobacterial

γ-subunits from nonmycobacterial species that might be a targetfor drug design, is that 12–14 amino acids are inserted in theregion from residues 165–169 in the aligned sequences (SI Ap-pendix, Fig. S11). This insertion is at the C terminus of α-helix 4(Fig. 3A) and is predicted to form a random coil (SI Appendix,Fig. S12) that might extend to the bacterial membrane surface(27). As this region is unresolved, it is not known whether thissuggestion is correct.

The e-Subunit. As in ATP synthases from other eubacteria, chlo-roplasts, and mitochondria (where the orthologous protein isknown as the δ-subunit) (28), the M. smegmatis e-subunit has twodomains (Fig. 4). The N-terminal domain is folded into aneight-stranded β-sandwich and is very similar to those inother species. For example, the rmsd values for the com-parisons of the N-terminal domain of the e-subunit from M.smegmatis with those from E. coli and C. thermarum are 1.2and 1.0 Å, respectively. In contrast, the C-terminal domaindiffers substantially from those in the orthologs. In E. coli, C.thermarum, G. stearothermophilus, and in bovine and yeastmitochondria, this region is folded into two α-helices, ∼23and 30 Å long. In E. coli (22, 29–32) and G. stear-othermophilus (33–35), the α-helices adopt two differentstates, referred to as “down” and “up.” In the down state ofthe F-ATPase from G. stearothermophilus (35), the α-helicesof the e-subunit bind an ATP molecule and are associatedwith the β-sandwich. In the absence of bound ATP, theα-helices assume the up position where they lie alongside theγ-subunit and interact with the α3β3-domain, inhibiting ATPhydrolysis. Up positions have been captured in structures ofthe F1-domain from E. coli (22) and in the intact E. coli ATPsynthase complex (32), but the isolated E. coli e-subunitadopts a down conformation, although ATP is not bound toit suggesting that ATP does not influence the position of thee-subunit (29–31). In the F1-ATPase from C. thermarum, evenin the absence of a bound ATP molecule, the α-helices re-main in the down position, and the up state has not beenobserved (18). In mitochondria, the two C-terminal α-helicesof the orthologous δ-subunit are also permanently down, andthe site where the ATP molecule is bound in E. coli, G.stearothermophilus, and C. thermarum and is occupied by thesingle α-helix of a small protein not found in bacteria and chloro-plasts, known confusingly as the e-subunit (25). In the mycobacteriale-subunits, the sequences of their C-terminal regions are shorter thanin the other species where the structure of the subunit is known, andin the M. smegmatis e-subunit, a C-terminal α-helical hairpin alsoforms next to the N-terminal domain in the down state, but theα-helices are truncated relative to E. coli, G. stearothermophilus, andC. thermarum. However, despite the shorter α-helical hairpin in theM. smegmatis e-subunit, the general appearance of the interaction ofthe truncated C-terminal α-helix with the N-terminal domain of theprotein is conserved (Fig. 4B) as are the number of interactions (eightin each case) and their approximate positions in the N- and C-terminal domains, although none of the side chains of these resi-dues are conserved significantly in M. smegmatis (SI Appendix, Fig.S13). Furthermore, there is no evidence of an ATP molecule bound

A B C

D E F

Fig. 3. Comparison of the structure of the γ-subunits of the F-ATPases fromM. smegmatis with those of the orthologs. (A) M. smegmatis with the fiveα-helices numbered 1–5 from the N to the C terminus; (B) E. coli (22); (C) C.thermarum (18); (D) Paracoccus denitrificans (23); (E) chloroplast fromspinach (24); (F) bovine mitochondria (25).

A B

Fig. 4. The structure of the e-subunit of the F1-ATPase from M. smegmatiscompared with the orthologs. (A) The mycobacterial e-subunit showing theN-terminal β-sandwich and the C-terminal α-helical domains; (B) superim-position of the e-subunits from M. smegmatis (green), C. thermarum (18)(gray), E. coli (29) (pink), and the bovine δ-subunit (25) (slate blue). The N-terminal domains are very similar, but both C-terminal helices of the M.smegmatis protein are shorter than in the other examples.

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to the subunit. This was anticipated as Arg-94, one of the key residuesinvolved in binding the nucleotide in E. coli, G. stearothermophilus,and C. thermarum is substituted in the equivalent site by an alanineresidue inM. smegmatis (SI Appendix, Fig. S13), and superposition ofthe M. smegmatis and E. coli F1-ATPases demonstrated that theformer e-subunit cannot assume the up position in the structure ofthe inhibited enzyme as its C-terminal α-helix would clash with theDELSEED region in the C-terminal domain of the βDP-subunit (SIAppendix, Fig. S14). Moreover, there is no evidence for the presenceof the up state in the current electron density map, and in an NMRstructure of the isolated e-subunit fromM. smegmatis the protein is inthe down position (14). This structure resembles the e-subunit de-scribed here, but as the deposited coordinates have not been released,a precise comparison with the current structure is not possible. Thus,there is currently no structural evidence that the e-subunit plays a rolein the regulation of the hydrolytic activity of the M. smegmatis ATPsynthase complex.

Regulation of ATP Hydrolysis. It is becoming clear that a variety ofmechanisms operates to regulate the ATP hydrolytic activity ofATP synthases in eubacteria, mitochondria, and chloroplasts. Inα-proteobacteria, exemplified by P. denitrificans, ATP hydrolysisappears to be inhibited by a protein called the ζ-subunit (23, 36)where the N-terminal inhibitory region binds to a catalytic in-terface under hydrolytic conditions in a closely related fashion tothe inhibitory action of the orthologous mitochondrial inhibitoryprotein IF1 on the mitochondrial ATP synthase (37–39). In thecase of mammalian IF1, inhibition of ATP hydrolysis of theprotein is activated by a fall in the pH (40), such as would occurin the mitochondrial matrix accompanying an increased relianceby cells on the provision of ATP by glycolysis. In the chloroplastsof green plants and algae, during the hours of darkness when theproton motive force is low, ADP-Mg remains bound to one ofthe three catalytic sites of the enzyme forming an inactiveADP inhibited state of the enzyme (41, 42). This inhibitedstate is stabilized by the formation of an intermoleculardisulfide bond in the γ-subunit of the enzyme. The formationof this disulfide is proposed to stabilize a β-hairpin structureformed by a unique additional sequence in the γ-subunit(residues 198–233 in SI Appendix, Fig. S11; see SI Appendix,Fig. S15) that wedges between the β-subunit and the centralstalk thereby blocking the rotation of the γ-subunit and pre-venting futile ATP hydrolysis (43). With daylight and a risingproton motive force, the synthetic activity of the enzyme isrestored by reduction of the disulfide bond by thioredoxin.The γ-subunits in cyanobacterial ATP synthases also contain arelated insertion (44), but it lacks the nine residue sequencecontaining the two cysteine residues (SI Appendix, Fig. S11),and, although the residual additional loop appears to inhibitATP hydrolysis, it is not regulated by the redox mechanismfound in chloroplasts (45).The ATPases in the aerobic bacterium G. stearothermophilus

(35) and in the facultative anaerobe E. coli (22, 32, 46) appear tobe regulated by their e-subunit. For G. stearothermophilus, it hasbeen proposed that, when the proton-motive force and ATPconcentration are low, this ATP molecule is released from thee-subunit, allowing its two C-terminal α-helices to assume the upinhibitory position where they penetrate into a catalytic sitealongside the rotary γ-subunit and impede the turning of therotor (33, 47, 48). However, there is no evidence for the oper-ation of a similar inhibitory mechanism in the thermoalkaliphileC. thermarum where it appears that ATP hydrolysis is preventedeither by the failure to release the products of ATP hydrolysisfrom one catalytic site or, less likely, for those products to bereleased and rebound (18).A characteristic feature of bacterial F-ATPases with latent

hydrolytic activity is that ATP hydrolysis can be activated artifi-cially in vitro. For example, LDAO activates the hydrolytic ac-tivity of F1-ATPase from C. thermarum 30-fold, and maximumactivation was achieved by the removal of the C-terminal do-main of the e-subunit (49). The hydrolytic activity of this enzyme

is not activated by proteolysis, and its e-subunit is resistantto such treatment (49). In contrast, the F1-ATPase from M.smegmatis is activated by trypsinolysis, and its activity is stim-ulated further by the addition of LDAO. However, it is notactivated by LDAO before trypsinolysis has taken place (SIAppendix, Fig. S4). In other bacterial species, the activation ofhydrolytic activity by LDAO activation has been attributed toeither release of an ADP molecule from a catalytic site (50, 51)or perturbation of the interaction between the e-subunit andthe α3β3-domain (52). However, the molecular basis of theactivation of the hydrolytic activity of F1-ATPases by trypsinolysisand/or LDAO, including the enzyme fromM. smegmatis, remainsunclear.The current structure of the F1-ATPase from M. smegmatis,

albeit at modest resolution, is very similar to that of the inhibitedcomplex from C. thermarum in terms of the protein structureitself (apart from the C-terminal extension of the α-subunits,which could also be involved in regulation of ATP hydrolysis).Especially, the rotational state of the γ-subunit suggests that theATP hydrolytic activities of the two enzymes have been arrestedat the same point in the rotary cycle. In C. thermarum, phosphateand ADP (at 50–70% occupancy) without a magnesium ion arebound to the site (18), whereas the occupancy of the βE-subunitin the M. smegmatis enzyme is likely to be similar. The order ofrelease of the products of ATP hydrolysis by F-ATPases has notbeen established firmly, although it appears that the magnesiumion leaves first as the catalytic site opens (18, 26, 53, 54). Thedata about whether the subsequent release of ADP precedes thatof phosphate are conflicting (26, 53–58), although in otherNTPases, phosphate leaves first (59–61). The current structureof the F1-ATPase from M. smegmatis can be interpreted as beingconsistent with such an order.Mycobacteria are obligate aerobes with an extraordinary

ability to survive for prolonged periods of hypoxia. A key ele-ment of their survival is their ability to keep their respiratorychain energized and thereby to maintain their energy require-ments by continuing to make ATP (62, 63). The membranepotential used by mycobacteria to drive ATP synthesis underhypoxia is low (−65 to −75 mV) (62, 63), and they are faced withthe thermodynamic challenge of inhibiting ATPase activity whileat the same time remaining competent for ATP synthesis. If theATP synthase were freely reversible, the cells would becomedepleted of ATP rapidly to reestablish the membrane poten-tial, and they would die. Thus, the extreme latency of theenzyme in the direction of ATP hydrolysis is a characteristicfeature of ATP synthases from fast and slow growing myco-bacteria (15), and the mechanism of ATP inhibition is an intrinsicfeature of the F1-domain. The structure of the mycobacterialF1-domain reported here is a big step toward uncovering themolecular basis of this inhibitory mechanism, and it providesa framework for the structure-based design of a small mole-cule that might activate ATP hydrolysis or inhibit ATP syn-thesis specifically in the pathogen.

Materials and MethodsThe F1-ATPase from M. smegmatis (subunits α, β, γ, and e) was overexpressedfrom a plasmid in the M. smegmatis strain mc2 4517, purified by nickel af-finity chromatography via a His6-tag attached to the β-subunit, size exclusionchromatography, crystallized by vapor diffusion, and its structure solvedfrom X-ray diffraction data by molecular replacement with the F1-ATPasefrom C. thermarum (PDB5hkk). Images of the structures and electron den-sity maps were prepared with PyMOL (64). For further details, see theSI Appendix.

ACKNOWLEDGMENTS. This work was supported by the Medical ResearchCouncil, U.K. by Grants MC_U105663150 and MR/M009858/1 (to J.E.W.) andMC_U105184325 (to A.G.W.L.) and by the European Drug Initiative on Chan-nels and Transporters (EDICT) via Contract HEALTH-F4-2007-201924 (toJ.E.W.). G.M.C. was supported by a James Cook Fellowship from The RoyalSociety of New Zealand. We thank the beamline staff at the Swiss LightSource, the European Synchrotron Radiation Facility and at beamline I04,the Diamond Light Source for their help, and Dr. I. M. Fearnley and Dr. S. Dingfor recording mass spectra.

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