672

Since the chemiosmotic theory was proposed by Peter Mitchellin the 1960s, a major objective has been to elucidate themechanism of coupling of the transmembrane proton motiveforce, created by respiration or photosynthesis, to thesynthesis of ATP from ADP and inorganic phosphate. Recently,significant progress has been made towards establishing thecomplete structure of ATP synthase and revealing itsmechanism. The X-ray structure of the F1 catalytic domain hasbeen completed and an electron density map of the F1–c10subcomplex has provided a glimpse of the motor in themembrane domain. Direct microscopic observation of rotationhas been extended to F1-ATPase and F1Fo-ATPase complexes.

Addresses*The Medical Research Council Dunn Human Nutrition Unit,Hills Road, Cambridge CB2 2XY, UK†The Medical Research Council Laboratory of Molecular Biology,Hills Road, Cambridge CB2 2QH, UK‡e-mail: walker@mrc-dunn.cam.ac.uk

Current Opinion in Structural Biology 2000, 10:672–679

0959-440X/00/$ — see front matterPublished by Elsevier Science Ltd.

AbbreviationsEM electron microscopyF1 factor 1Fo factor oligomycinOSCP oligomycin sensitivity conferring proteinPi inorganic phosphatepmf proton motive force

IntroductionATP synthase contains a rotary motor involved in biologi-cal energy conversion. Respiratory complexes inmitochondria and eubacteria, and photosynthetic complexesin chloroplasts and photosynthetic eubacteria use energyderived from the oxidation of nutrients and from light,respectively, to generate a transmembrane proton motiveforce (pmf) [1–3]. ATP synthase uses the pmf to makeATP from ADP and inorganic phosphate (Pi). As sum-marised in Figure 1a,b, the enzyme has two majorstructural domains, known as F1 (factor 1) and Fo (factoroligomycin). The globular F1 catalytic domain in the mito-chondrial enzyme is an assembly of five subunits with thestoichiometry α3β3γ1δ1ε1. Subunits γ, δ and ε form a centralstalk linking the (αβ)3 subcomplex of F1 to the membranedomain, Fo. The (αβ)3 subcomplex and Fo are also linkedby a peripheral stalk, sometimes called the stator [2].

In the F1 domain, the three α subunits and the three β sub-units are arranged alternately around a central α-helicalcoiled coil in the γ subunit [4]. This arrangement suggestedthat the enzyme works by a mechanism involving the cyclic

modulation of nucleotide affinity in catalytic β subunits, asrequired by the binding-change mechanism [1], by rotationof the asymmetrical γ subunit. During ATP synthesis, therotation would be generated in Fo and fuelled by the pmf.During ATP hydrolysis in F1Fo (or in F1 alone), the energyreleased by hydrolysis would drive rotation in the oppositedirection and reverse the direction of proton translocation.Subsequently, the rotation of the γ subunit in an (αβ)3γcomplex was observed directly by microscopy and wasshown to depend on ATP hydrolysis [5].

Recent structural results have provided additional insightinto the nature of the central stalk [63••]. This feature linksthe F1 and Fo domains, and forms part of the rotor in theATP synthase molecular motor. The way in which the cen-tral stalk is linked to a ring of c subunits in the Fo domain hasbeen suggested from a low-resolution electron density mapof a subcomplex of the yeast enzyme. As yet, no structuralinformation is available on other key subunits in the Fodomain, but a number of models have been proposed fortorque generation. A much clearer picture of the molecularmechanism of the motor in ATP synthase is slowly emerging.

The central stalkUntil recently, the protruding part of the central stalk wasdisordered in crystals of bovine F1-ATPase [4], althoughthe (αβ)3 domain and the penetrating α-helical coiled-coilpart of the central stalk were resolved in the same crystals.By modification of the cryoprotection conditions, the crys-tal lattice of bovine F1-ATPase (covalently inhibited withdicyclohexylcarbodiimide) has been shrunk, therebyordering the protruding central stalk region and allowingthe entire structure to be resolved to 2.4 Å (Figure 1c)(C Gibbons, MG Montgomery, AGW Leslie, JE Walker,unpublished data; see [63••]). This analysis has revealed anew α/β domain in the γ subunit, containing a Rossmannfold, that does not bind nucleotides. It appears to be a but-tress, stabilising the lower section of the coiled-coil shaft.There is little agreement between the structure of thebovine γ subunit in the Rossmann fold region of the cen-tral stalk and a model of the same region of the Escherichiacoli γ subunit, deduced from a 4.4 Å resolution electrondensity map of bacterial F1-ATPase [6].

The bovine structure confirms the structural homologybetween the mitochondrial δ and bacterial (and chloro-plast) ε subunits. Similar to the bacterial ε subunit [7], thebovine δ subunit has two domains, an N-terminal β sand-wich with 10 strands (residues 15–98) and a C-terminalα-helical hairpin (residues 105–145). The 50 amino acidbovine ε subunit has no counterpart in bacteria or chloro-plasts. It has a helix-loop-helix structure and appears to

The rotary mechanism of ATP synthaseDaniela Stock*, Clyde Gibbons*, Ignacio Arechaga*,Andrew GW Leslie† and John E Walker*‡

The rotary mechanism of ATP synthase Stock et al. 673

stabilise the foot of the central stalk, where the γ, δ andε subunits all interact extensively. It is probable that allthree subunits contact the Fo domain.

In E. coli F1-ATPase, interactions between and within sub-units have been examined by the introduction of cysteineresidues at specific sites and formation of disulfide cross-links by oxidation. Cross-links observed within thebacterial ε subunit [8•] and ε–γ cross-links [9,10] are consis-tent with the bovine model, but the β–ε and α–ε cross-links[11–13] are not, as they are between 40 and 60 Å apart inthe bovine structure. One possible interpretation is that the

bacterial ε subunit detaches wholly or partially from thefoot during the catalytic cycle, so that it can interact withthe lower surface of the (αβ)3 domain. However, the func-tional significance of such a rearrangement is obscure. Acritical re-examination of the formation of the α–ε andβ–ε cross-links is warranted.

The peripheral stalkThere is general agreement that the F1 and Fo domains arealso connected by a second, peripheral, stalk [2]. This hasbeen observed by single-particle analysis using electronmicroscopy (EM) in negative stain of bacterial [14,15•],

Figure 1

Structure of ATP synthase. (a,b) Summary ofcurrent knowledge of the structure of ATPsynthase from mitochondria and eubacteria.(a) Mitochondrial ATP synthase. The model isbased on EM studies of single particles [17•]. Itincorporates the structure of bovine F1-ATPase[4,62•] and information from the electrondensity map of the F1–c10 complex fromS. cerevisiae [36••]. The composition,stoichiometry and arrangement of the subunitsin the peripheral stalk (subunits OSCP, F6,b and d) come from biochemical andreconstitution studies [20,33]. The position ofsubunit a relative to the c10 ring was deducedfrom studies of the bacterial enzyme [28].Minor subunits (e, f, g, A6L) in the Fo domainare not shown. They have no known functionsin the enzyme’s mechanism. (b) EubacterialATP synthase. The overall model is also basedon EM studies [14,15•]. The core structure ofthe central F1–c ring was deduced byhomology with the mitochondrial enzyme.However, the c ring may contain 12 c subunits,not 10 [45]. The positions of the subunits in theperipheral stalk (subunits b and δ) aresupported by biochemical and EM studies[14,18•,26•]. The δ subunit (structuredetermined by NMR studies [27]) appears,from EM work, to sit on top of the (αβ)3 domain[26•]. The structure of the E. coli ε subunit wasalso determined independently [7,11]. Thegeneral structure of ATP synthase fromchloroplasts is very similar to that of thebacterial enzyme. The main differences are thatthe c ring may contain 14 c protomers [46••]and that the two identical b subunits in someeubacterial enzymes are replaced byhomologous, but not identical, subunits b andb′. A similar arrangement of b and b′ subunits isalso found in other eubacterial species. (c) Thecomplete structure of bovine F1-ATPase shownin stereo (C Gibbons, MG Montgomery,AGW Leslie, JE Walker, unpublished data; see[63••]). The α and β subunits (red and yellow,respectively) are arranged alternately around anα-helical coiled coil in the γ subunit (blue).Regions of the γ subunit present in the originalF1 structure [4] are shown in sky blue, thoseregions determined in the latest structure [63••]are in dark blue. The central stalk consists ofthe γ, δ and ε subunits (blue, green andmagenta, respectively).

ββ

c9–12

b2

α α

γ

a

ε

αδ

ββ

c10

F6

d

b

α α

γ

a

δ ε

OSCPα

(c)

(a) (b)

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674 Proteins

chloroplast [16] and mitochondrial [17•] F1Fo-ATPases. Itsfunction has not been demonstrated, but it may act as a sta-tor to counter the tendency of the (αβ)3 domain to followthe rotation of the central stalk [2]. In E. coli, it containsthe δ subunit and the extrinsic membrane domains of twoidentical b subunits that form a parallel α-helical coiledcoil [18•] (see Figure 1b). The membrane domains of theb subunits (one transmembrane α helix each) also interact

and form part of Fo [19•]. In some other bacterial speciesand in chloroplasts, the two identical b subunits arereplaced by single copies of homologous subunits b and b′.The bovine peripheral stalk contains one copy each of theOSCP (oligomycin sensitivity conferring protein) subunit(the equivalent of bacterial δ), the extrinsic domain of sub-unit b and the d and F6 subunits [2] (see Figure 1a). It hasbeen assembled in vitro and interacting regions have been

Figure 2

55 Å

58 Å

50 Å

83 Å

α αβ β

δγ

C

(a)

(b)

123

45 6 7

89

10δ

Current Opinion in Structural Biology

Stereo views of an electron density map of the F1–c10 complex from S. cerevisiae at 3.9 Å resolution [36••]. (a) Side view. (b) End-on view, rotated 90°with respect to (a). Two rings, an inner ring and an outer ring, composed of 10 c protomers are visible. The inserts indicate the locations of subunits.

defined [20]. In Saccharomyces cerevisiae, cross-links havebeen observed between the b subunit and subunits β,OSCP and d (in agreement with the bovine findings), andalso to the membrane subunit a (and other minor Fo sub-units) [21,22]. The peripheral stalk subunits are poorlyconserved (relative to F1 components, for example) andsubunits b can be shortened and lengthened without havinga major effect on the enzyme’s activity [23,24].

For many years, it has been known that the δ and OSCPsubunits in the E. coli and bovine enzymes, respectively,interact with the N-terminal regions of the α subunits,which protrude from the ‘crown’ at the top of F1. Thisarrangement has been confirmed by cross-linking experi-ments [25] and EM [26•]. The structure of the N-terminaldomain of the E. coli δ subunit has been established byNMR studies [27].

The Fo domainIn E. coli, the Fo domain is composed of three subunitswith the stoichiometry a1b2c9–12 (Figure 1b). The a andc subunits are in contact and protons are thought to betranslocated through the interface between them [28,29].Both subunits are conserved in all F-ATPases. The E. colia subunit is hydrophobic and is probably folded into fivetransmembrane α helices [30,31]. It contains basic andacidic residues (Arg210, His245, Glu196, Glu219) that areessential for proton translocation. The c subunit is alsohydrophobic. The protomer structure, determined by

NMR spectroscopy in organic solvents, has two trans-membrane α helices linked by a polar loop [32]. TheC-terminal α helix contains a carboxyl group (Asp61) thatis also essential for proton translocation. The conservationand arrangement of the b subunits was discussed above.The Fo domains of mitochondrial enzymes contain a num-ber of small subunits that appear to have no direct role incatalysis [33–35]. They are absent from bacterial andchloroplast enzymes.

The first view of the structure of the Fo domain came froman electron density map of F1-ATPase associated with aring of 10 c subunits from S. cerevisiae [36••] (see Figure 2).This F1–c10 complex was formed from ATP synthase dur-ing the crystallisation process, when other subunitsdissociated. The electron density map contains a numberof important features. First, the 10 c protomers appear tohave secondary structure similar to the c protomer struc-ture determined by NMR. The map also shows that theC-terminal α helices form an outer ring, with the N-termi-nal α helices in a second inner ring. Second, the map showsthat the extensive footprint of the central stalk sits asym-metrically on the polar loop regions of six c subunits. Thisarrangement is consistent with the rotation of the centralstalk and the c ring as an ensemble, as are covalent cross-links between the E. coli ε and c subunits that do not affectthe enzyme’s activity [37•,38,39•]. Third, 10 c subunits arefound in the ring and not 12, as was widely anticipated.Therefore, there is a symmetry mismatch between the

The rotary mechanism of ATP synthase Stock et al. 675

Figure 3

Observations of rotation in ATP synthase. The direct observation of rotationusing fluorescently labelled actin filaments attached to (a) the γ subunit inthe (αβ)3γ complex [5,50•,52•], (b) F1-ATPase [51,53•] and (c) F1Fo-ATP

synthase [55••,56••]. The N termini of α subunits in the (αβ)3 domain areassociated with a nickel-coated glass surface. Counterclockwise rotationdependent on ATP hydrolysis was observed in a fluorescence microscope.

Actin filamentActin filament

Actin filament

ATP

ADP + Pi

ATP

ADP + Pi

ATP

ADP + Pi

c ring

γε

Fo

F1

(a)(b)

(c)

Current Opinion in Structural Biology

F1 and Fo domains, which may help to facilitate rotation byavoiding the deeper energy minima that would accompanymatching symmetries. Symmetry mismatch has been dis-cussed in relation to other macromolecular assemblies thatcontain rotating elements [40–43].

The number of c subunits in the c ringBased on metabolic labelling and mechanistic models ofthe generation of rotation, the notion has grown up thatE. coli Fo contains 12 c subunits arranged in a ring and, byimplication, that mitochondrial and chloroplast Fo domainsalso contain 12 c subunits similarly arranged. Cross-linkingexperiments and genetic fusions [44,45] have been inter-preted as supporting this view. This notion has beenchallenged by the F1–c10 structure (above) [36••] and bythe observation of 14-fold symmetry in rings of c subunitsfrom spinach chloroplasts [46••]. At the present time, thepossibility that subunits were lost from the S. cerevisiaec ring during crystallisation cannot be excluded, unlikelyas this proposal seems. However, there are now clear indi-cations that the c-ring symmetry may differ among species.The c-ring symmetry may also vary within a single speciesaccording to physiological conditions [47]. If the concept ofsymmetry mismatch is an important general feature ofATP synthases, it would argue against c-ring stoichiome-tries divisible by three. It also implies that the number ofprotons that transverse the membrane for each ATP syn-thesised is nonintegral, possibly between three and four inmitochondria. As the generation of each ATP requires a120° rotation of the central stalk, an elastic element, possi-bly in the γ subunit, may be needed to store energy andrelease it in quanta, as required by a stepping motormechanism [48•,49•] (see below).

Direct observation of rotationBy attachment of fluorescent actin filaments to either theγ or ε subunit, rotation of the central stalk driven by ATPhydrolysis has been observed by microscopy of tetheredα3β3γ [5,50•] complexes and of F1 itself [51,52•,53•] (seeFigure 3a,b). The main characteristics of this rotation arethat it is highly efficient in energy usage, that it proceedsin 120° steps [54] and that the rotation is counterclockwiseas viewed from the tip of the central stalk protrusion.

Attempts have also been made to observe the rotation inF1Fo-ATPase preparations by attaching actin filaments tothe c ring on the surface distal from F1 [55••,56••](Figure 3c). Although technical objections have beenvoiced concerning these experiments [57•], they can bereasonably interpreted as showing that the F1–c ring rotatesas an ensemble in response to ATP hydrolysis in F1.However, because the detergents used to isolate the com-plex destabilise interactions of the c ring with the a subunit,these experiments should not be taken as definitive proofof the rotation of the F1–c ring in an intact F1Fo complexthat is capable of synthesising, as well as hydrolysing, ATP.Definitive proof may require rotation to be observed underconditions in which ATP is being synthesised.

Generation of torqueA hypothetical model of how rotation might be generatedwas developed by Junge et al. [58], based upon models ofbacterial flagellar rotation (see [3,49•,59] for a detaileddescription and further discussion of this model)(Figure 4a). A related model has been described to explainthe generation of rotation by the Na+-motive F1Fo-ATPasefrom the bacterium Propionigenium modestum [60•]

676 Proteins

Figure 4

a subunit a subunitc subunits c subunits

ac

140°

Na+

Na+

H+

H+(a) (b) (c)

Current Opinion in Structural Biology

Models of the generation of rotation by movement of ions through theFo domain of ATP synthase. (a) A two-channel model proposed byJunge [2,58]. Two half channels across the interface between thea subunit and the c ring are linked by rotation of the c ring. (b) Asingle-channel model [60•] for the Na+-motive ATP synthase inP. modestum. Sodium ions enter via a channel in the interface betweenthe a subunit and the c ring, and bind to c protomers near to thecytoplasmic surface where they are released. (c) A model based on

pH-induced structural changes observed by NMR of the c protomer inorganic solvents [61••]. Deprotonation of Asp61 and release of theproton triggers a 140° rotation of the c protomer C-terminal (outer)helix and concomitant movement of the c ring. The observed directionof rotation in Figure 3 is counterclockwise, as viewed from themembrane towards F1, and driven by ATP hydrolysis. In Figure 4, thedirection of rotation during ATP synthesis is counterclockwise, asviewed from F1 towards the membrane.

(Figure 4b). In this model, the carboxyl sidechains of theessential residue Glu65 in subunit c are negatively chargedwhen they enter the interface between the c ring and sub-unit a. The positive charge of Arg227 in subunit a attractsthe negative charge of the essential carboxylate in subunitc and also prevents ion leakage. Once this carboxylate hasbeen neutralised by a Na+ ion from the periplasm, it willmove by thermal vibrations, bringing the next negativelycharged carboxylate into the channel. Electrostatic forcesstrongly bias the rotation, making it effectively unidirec-tional. As in the Junge model, the central stalk is attachedto the c ring, which drives its rotation directly.

A radically different model for the generation of rotation ofthe central stalk has been advanced on the basis of NMRstudies in organic solvents of the c protomer from E. coli, inwhich reduction in pH and protonation of Asp61 cause theC-terminal α helix to rotate by 140° about its helix axis. Itis proposed that this rotation either drives the rotation ofthe c ring (Figure 4c) or, alternatively, generates rotation ofthe central stalk without the c ring itself turning [61••].

ConclusionsThe rather extensive current knowledge of how ATP syn-thase works is based largely upon accurate and novelstructures of subcomplexes of the enzyme [4,36••,62•,63••];striking progress had been made using this approach in thepast six years. However, current models for explaining thegeneration of rotation in Fo are tentative and require fur-ther experimental validation. It is unlikely that themechanism of rotation in ATP synthase will be understoodfully until accurate molecular models of the entire enzymecomplex in different conformational states have beenestablished. Determination of these structures requireseither the crystallisation of the intact ATP synthase com-plex or the establishment of an accurate low-resolutionmodel by EM of single complexes, which can then be usedas a framework for building a molecular model fromstructures of subcomplexes and individual subunits.

References and recommended readingPapers of particular interest, published within the annual period of review,have been highlighted as:

• of special interest•• of outstanding interest

1. Boyer PD: The ATP synthase — a splendid molecular machine.Annu Rev Biochem 1997, 66:717-749.

2. Walker JE: ATP synthesis by rotary catalysis (Nobel Lecture).Angew Chem Int Ed Engl 1998, 37:2309-2319.

3. Nakamoto RK, Ketchum CJ, Alshawi MK: Rotational coupling in theFoF1 ATP synthase. Annu Rev Biophys Biomol Struct 1999,28:205-234.

4. Abrahams JP, Leslie AGW, Lutter R, Walker JE: Structure at 2.8 Åresolution of F1-ATPase from bovine heart mitochondria. Nature1994, 370:621-628.

5. Noji H, Yasuda R, Yoshida M, Kinosita K: Direct observation of therotation of F1-ATPase. Nature 1997, 386:299-302.

6. Hausrath AC, Gruber G, Matthews BW, Capaldi RA: Structuralfeatures of the gamma subunit of the Escherichia coli F1-ATPase revealed by a 4.4 Å resolution map obtained by X-ray

crystallography. Proc Natl Acad Sci USA 1999,96:13697-13702.

7. Uhlin U, Cox GB, Guss JM: Crystal structure of the epsilon subunitof the proton-translocating ATP synthase from Escherichia coli.Structure 1997, 5:1219-1230.

8. Schulenberg B, Capaldi RA: The epsilon subunit of the F1Fo• complex of Escherichia coli — cross-linking studies show the

same structure in situ as when isolated. J Biol Chem 1999,274:28351-28355.

The authors provide evidence that the isolated ε subunit has a similar conformation [11] as when it is associated with F1-ATPase.

9. Watts SD, Tang CL, Capaldi RA: The stalk region of the Escherichiacoli ATP synthase — tyrosine 205 of the gamma-subunit is in theinterface between the F1 and Fo parts and can interact with boththe epsilon and c oligomer. J Biol Chem 1996, 271:28341-28347.

10. Tang CL, Capaldi RA: Characterization of the interface betweengamma and epsilon subunits of Escherichia coli F1-ATPase. J BiolChem 1996, 271:3018-3024.

11. Wilkens S, Capaldi RA: Solution structure of the epsilon subunit ofthe F1-ATPase from Escherichia coli and interactions of thissubunit with beta subunits in the complex. J Biol Chem 1998,273:26645-26651.

12. Aggeler R, Haughton MA, Capaldi RA: Disulfide bond formationbetween the COOH-terminal domain of the beta subunits and thegamma and epsilon subunits of the Escherichia coli F1-ATPase.Structural implications and functional consequences. J Biol Chem1995, 270:9185-9191.

13. Aggeler R, Weinreich F, Capaldi RA: Arrangement of the epsilonsubunit in the Escherichia coli ATP synthase from the reactivity ofcysteine residues introduced at different positions in this subunit.Biochim Biophys Acta 1995, 1230:62-68.

14. Wilkens S, Capaldi RA: ATP synthase’s second stalk comes intofocus. Nature 1998, 393:29.

15. Bottcher B, Bertsche I, Reuter R, Graber P: Direct visualisation of • conformational changes in EFoF1 by electron microscopy. J Mol

Biol 2000, 296:449-457.The authors describe the first three-dimensional reconstruction of E. coliATP synthase.

16. Bottcher B, Schwarz L, Graber P: Direct indication for the existenceof a double stalk in CF0F1. J Mol Biol 1998, 281:757-762.

17. Karrasch S, Walker JE: Novel features in the structure of bovine • ATP synthase. J Mol Biol 1999, 290:379-384.Single-particle analysis of electron micrographs of bovine F1Fo-ATPase.Evidence is revealed for a peripheral stalk and for formerly unseen features.

18. Revington M, McLachlin DT, Shaw GS, Dunn SD: The dimerization • domain of the b subunit of the Escherichia coli F1Fo-ATPase.

J Biol Chem 1999, 274:31094-31101.A biochemical demonstration of the dimerisation of the bacterial b subunit,showing that the b subunits form a single stator that interacts extensivelywith the α and β subunits in F1.

19. Dmitriev O, Jones PC, Jiang WP, Fillingame RH: Structure of the • membrane domain of subunit b of the Escherichia coli FoF1 ATP

synthase. J Biol Chem 1999, 274:15598-15604.NMR studies of the membrane sector of the E. coli F1Fo-ATP synthasesubunit b, showing its α-helical nature.

20. Collinson IR, van Raaij MJ, Runswick MJ, Fearnley IM, Skehel JM,Orriss GL, Miroux B, Walker JE: ATP synthase from bovine heartmitochondria — in vitro assembly of a stalk complex in thepresence of F1-ATPase and in its absence. J Mol Biol 1994,242:408-421.

21. Soubannier V, Rusconi F, Vaillier J, Arselin G, Chaignepain S,Graves PV, Schmitter JM, Zhang JL, Mueller D, Velours J: The secondstalk of the yeast ATP synthase complex: identification ofsubunits showing cross-links with known positions of subunit 4(subunit b). Biochemistry 1999, 38:15017-15024.

22. Velours J, Paumard P, Soubannier V, Spannagel C, Vaillier J, Arselin G,Graves PV: Organisation of the yeast ATP synthase Fo: a studybased on cysteine mutants, thiol modification and cross-linkingreagents. Biochim Biophys Acta 2000, 1458:443-456.

23. Sorgen PL, Caviston TL, Perry RC, Cain BD: Deletions in thesecond stalk of F1Fo-ATP synthase in Escherichia coli. J BiolChem 1998, 273:27873-27878.

The rotary mechanism of ATP synthase Stock et al. 677

24. Sorgen PL, Bubb MR, Cain BD: Lengthening the second stalk ofF1Fo-ATP synthase in Escherichia coli. J Biol Chem 1999,274:36261-36266.

25. Ogilvie I, Aggeler R, Capaldi RA: Cross-linking of the delta subunitto one of the three alpha subunits has no effect on functioning, asexpected if delta is a part of the stator that links the F1 and Foparts of the Escherichia coli ATP synthase. J Biol Chem 1997,272:16652-16656.

26. Wilkens S, Zhou J, Nakayama R, Dunn SD, Capaldi RA: Localization • of the delta subunit in the Escherichia coli F1Fo-ATP synthase by

immune electron microscopy: the delta subunit binds on top ofthe F1. J Mol Biol 2000, 295:387-391.

Confirmation of the position of the N-terminal domain of the δ subunit atthe top of F1.

27. Wilkens S, Dunn SD, Chandler J, Dahlquist FW, Capaldi RA: Solutionstructure of the N-terminal domain of the delta subunit of theE. coli ATP synthase. Nat Struct Biol 1997, 4:198-201.

28. Jiang WP, Fillingame RH: Interacting helical faces of subunits aand c in the F1Fo-ATP synthase of Escherichia coli defined bydisulfide cross-linking. Proc Natl Acad Sci USA 1998,95:6607-6612.

29. Vik SB, Long JC, Wada T, Zhang D: A model for the structure ofsubunit a of the Escherichia coli ATP synthase and its role inproton translocation. Biochim Biophys Acta 2000, 1458:457-466.

30. Valiyaveetil FI, Fillingame RH: Transmembrane topography ofsubunit a in the Escherichia coli F1Fo-ATP synthase. J Biol Chem1998, 273:16241-16247.

31. Wada T, Long JC, Zhang D, Vik SB: A novel labeling approachsupports the five-transmembrane model of subunit a of theEscherichia coli ATP synthase. J Biol Chem 1999,274:17353-17357.

32. Girvin ME, Rastogi VK, Abildgaard F, Markley JL, Fillingame RH:Solution structure of the transmembrane H+-transporting subunitc of the F1Fo-ATP synthase. Biochemistry 1998, 37:8817-8824.

33. Collinson IR, Runswick MJ, Buchanan SK, Fearnley IM, Skehel JM,van Raaij MJ, Griffiths DE, Walker JE: Fo membrane domain of ATPsynthase from bovine heart mitochondria: purification, subunitcomposition, and reconstitution with F1-ATPase. Biochemistry1994, 33:7971-7978.

34. Arnold I, Bauer MF, Brunner M, Neupert W, Stuart RA: Yeastmitochondrial F1Fo-ATPase: the novel subunit e is identical toTim11. FEBS Lett 1997, 411:195-200.

35. Arnold I, Pfeiffer K, Neupert W, Stuart RA, Schagger H: ATP synthaseof yeast mitochondria. Isolation of subunit j and disruption of theATP18 gene. J Biol Chem 1999, 274:36-40.

36. Stock D, Leslie AGW, Walker JE: Molecular architecture of the •• rotary motor in ATP synthase. Science 1999, 286:1700-1705.The 3.9 Å resolution structure of the yeast F1–c10 complex has provided thefirst insight into the arrangement of the c ring and its interactions with stalksubunits γ, δ and ε. The close contact between these subunits and the c ringsupports the idea that the γ, δ and ε subunits and the c ring rotate as anensemble. The unexpected finding of 10 subunit c protomers in the ring hasprofound implications for the mechanism of coupling and for the number ofprotons translocated through Fo for each ATP molecule synthesised in F1.

37. Hermolin J, Dmitriev OY, Zhang Y, Fillingame RH: Defining the • domain of binding of F1 subunit epsilon with the polar loop of Fo

subunit c in the Escherichia coli ATP synthase. J Biol Chem 1999,274:17011-17016.

Further confirmation of the interaction between bacterial ε and c subunits,providing evidence that the c ring rotates together with the central stalk.

38. Watts SD, Capaldi RA: Interactions between the F1 and Fo partsin the Escherichia coli ATP synthase. Associations involvingthe loop region of c subunits. J Biol Chem 1997,272:15065-15068.

39. Schulenberg B, Aggeler R, Murray J, Capaldi RA: The gamma-• epsilon-c subunit interface in the ATP synthase of Escherichia

coli. Cross-linking of the epsilon subunit to the c subunit ringdoes not impair enzyme function, that of gamma to c subunitsleads to uncoupling. J Biol Chem 1999, 274:34233-34237.

The authors infer that the ε subunit and c ring rotate as an ensemble duringcatalysis and that conformational changes in the γ subunit might occur.

40. Hendrix RW: Bacteriophage DNA packaging: RNA gears in a DNAtransport machine. Cell 1998, 94:147-150.

41. Valpuesta JM, Fernandez JJ, Carazo JM, Carrascosa JL: The three-dimensional structure of a DNA translocating machine at 10 Åresolution. Structure 1999, 7:289-296.

42. Thomas DR, Morgan DG, DeRosier DJ: Rotational symmetry of thec ring and a mechanism for the flagellar rotary motor. Proc NatlAcad Sci USA 1999, 96:10134-10139.

43. Beuron F, Maurizi MR, Belnap DM, Kocsis E, Booy FP, Kessel M,Steven AC: At sixes and sevens: characterization of the symmetrymismatch of the ClpAP chaperone-assisted protease. J Struct Biol1998, 123:248-259.

44. Jones PC, Fillingame RH: Genetic fusions of subunit c in the Fosector of H+-transporting ATP synthase. Functional dimers andtrimers and determination of stoichiometry by cross-linkinganalysis. J Biol Chem 1998, 273:29701-29705.

45. Jones PC, Jiang WP, Fillingame RH: Arrangement of the multicopyH+-translocating subunit c in the membrane sector of theEscherichia coli F1Fo-ATP synthase. J Biol Chem 1998,273:17178-17185.

46. Seelert H, Poetsch A, Dencher NA, Engel A, Stahlberg H, Müller DJ: •• Proton-powered turbine of a plant motor. Nature 2000, 405:418-419.Atomic force microscopy images of the c-subunit ring from chloroplast ATPsynthase show 14 protomers in the ring. Therefore, the number of c subunitsin ATP synthases may differ from species to species.

47. Schemidt RA, Qu J, Williams JR, Brusilow WSA: Effects of carbonsource on expression of Fo genes and on the stoichiometry of thec subunit in the F1Fo ATPase of Escherichia coli. J Bacteriol 1998,180:3205-3208.

48. Cherepanov DA, Mulkidjanian AY, Junge W: Transient accumulation • of elastic energy in proton translocating ATP synthase. FEBS Lett

1999, 449:1-6.A theoretical model is proposed for the generation of torque, involving anelastic element.

49. Oster G, Wang H: Reverse engineering a protein: the • mechanochemistry of ATP synthase. Biochim Biophys Acta 2000,

1458:482-510.Simplified physical models were developed for both the F1 and Fo sec-tors. The solutions of the resulting equations reproduce many of theempirical measurements.

50. Hisabori T, Kondoh A, Yoshida M: The gamma subunit in chloroplast • F1-ATPase can rotate in a unidirectional and counter-clockwise

manner. FEBS Lett 1999, 463:35-38.The authors demonstrate that rotation of the γ subunit driven by ATPhydrolysis, as first observed in the bacterial enzyme, also occurs in thechloroplast enzyme.

51. Kato-Yamada Y, Noji H, Yasuda R, Kinosita K, Yoshida M: Directobservation of the rotation of epsilon subunit in F1-ATPase. J BiolChem 1998, 273:19375-19377.

52. Noji H, Hasler K, Junge W, Kinosita K, Yoshida M, Engelbrecht S: • Rotation of Escherichia coli F1-ATPase. Biochem Biophys Res

Comm 1999, 260:597-599.The authors provide evidence of rotation in intact F1-ATPase.

53. Omote H, Sambonmatsu N, Sambongi Y, Iwamato-Kihara A, Yanagida T, • Wada Y, Futai M: The gamma-subunit rotation and torque

generation in F1-ATPase from wild-type or uncoupled mutantEscherichia coli. Proc Natl Acad Sci USA 1999, 96:7780-7784.

Further demonstration of rotation of the γ subunit in the E. coli F1 domain.Significantly, a mutation of the γ subunit, known to cause uncoupling, had noeffect on torque generation.

54. Yasuda R, Noji H, Kinosita K, Yoshida M: F1-ATPase is a highlyefficient molecular motor that rotates with discrete 120o steps.Cell 1998, 93:1117-1124.

55. Sambongi Y, Iko Y, Tanabe M, Omote H, Iwamoto-Kihara A, Ueda I, •• Yanagida T, Wada Y, Futai M: Mechanical rotation of the c subunit

oligomer in ATP synthase (F0F1): direct observation. Science1999, 286:1722-1724.

This paper presents the first direct evidence for rotation of the c ring in anATP synthase complex. Objections have been raised concerning the inter-pretation of these experiments [57•]. The major remaining concern iswhether the ATP synthase is intact.

56. Panke O, Gumbiowski K, Junge W, Engelbrecht S: F-ATPase: •• specific observation of the rotating c subunit oligomer of EFoEF1.

FEBS Lett 2000, 472:34-38.This paper describes one specific experimental approach to the direct obser-vation of the rotation of the c ring. The specificity of the attachment of the

678 Proteins

actin filament to subunit c was ensured by the introduction of a ‘strep-tag’sequence in the C-terminal region of subunit c.

57. Tsunoda SP, Aggeler R, Noji H, Kinosita K, Yoshida M, Capaldi RA: • Observations of rotation within the FoFl-ATP synthase: deciding

between rotation of the Foc-subunit ring and artifact. FEBS Lett2000, 470:244-248.

A critique of experiments directly demonstrating rotation in ATP synthase.

58. Junge W, Lill H, Engelbrecht E: ATP synthase: an electrochemicaltransducer with rotary mechanics. Trends Biol Sci 1997,22:420-423.

59. Elston T, Wang HY, Oster G: Energy transduction in ATP synthase.Nature 1998, 391:510-513.

60. Dimroth P, Wang H, Grabe M, Oster G: Energy transduction in the • sodium F-ATPase of Propionigenium modestum. Proc Natl Acad

Sci USA 1999, 96:4924-4929.A novel mechanochemical model for the generation of rotation in sodium-dependent ATP synthase involving a single channel, rather than two halfchannels, as proposed for the proton-dependent ATP synthase.

61. Rastogi VK, Girvin ME: Structural changes linked to proton •• translocation by subunit c of the ATP synthase. Nature 1999,

402:263-268.The authors describe the structural changes undergone by the c protomerthat accompany deprotonation of essential residue Asp61, as determinedby NMR in organic solvents. A novel model is proposed for the rotation ofsubunit c and for its interactions with subunit a.

62. Braig K, Menz IR, Montgomery MG, Leslie AGW, Walker JE: • Structure of bovine F1-ATPase inhibited by Mg2+ADP and

aluminium fluoride. Structure 2000, 8:567-573.A description of a transition state in the catalytic cycle of F1-ATPase.

Now publishedThe work referred to in the text as (C Gibbons, MG Montgomery,AGW Leslie, JE Walker, unpublished data) is now published:

63. Gibbons C, Montgomery MG, Leslie AGW, Walker JE: The structure of •• the central stalk at 2.4 Å resolution. Nat Struct Biol 2000, 7:1055-1061.A description of the structure of the central stalk of F1Fo-ATPase determinedin the context of an intact F1-ATPase assembly.

The rotary mechanism of ATP synthase Stock et al. 679