document

© 2000 Nature America Inc. • http://structbio.nature.com©

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The F1Fo-ATP synthase found in chloroplasts, eubacteria andmitochondria uses the transmembrane proton motive forcegenerated by photosynthesis or oxidative phosphorylation todrive the synthesis of ATP from ADP and phosphate (see ref. 1for a review). It is composed of two major domains, a globularF1 catalytic domain and a membrane bound Fo proton-translo-cating domain, linked by a central stalk. In mitochondrial F1-ATPase, the central stalk contains the γ, δ and ε subunits2,3. Itpenetrates into the F1 catalytic domain in which an assembly ofthree α subunits and three β subunits (the (αβ)3 domain) arearranged alternately around an asymmetrical antiparallel α-helical coiled coil in the γ subunit4. The α and β subunitshave closely related structures. They both bind nucleotides, buteach β subunit contains a catalytic nucleotide binding sitewhereas the nucleotides bound to the α subunits do not partic-ipate directly in catalysis.

The structure of the bovine F1-ATPase provides strong sup-port for the ‘binding change mechanism’ of catalysis in whichthe catalytic sites have different nucleotide affinities5. One (the‘open’ site) has very low affinity for substrates, a second (the‘loose’ site) can bind substrates reversibly and a third (the ‘tight’site) has a very high affinity such that ATP can form sponta-neously from ADP and inorganic phosphate. In F1-ATPase, thecentral stalk, which is asymmetric, rotates during ATP hydroly-sis6,7 with an anticipated frequency of 100–200 Hz. The rotationof the central stalk, which proceeds in 120° steps8, changes theaffinities of the catalytic sites, taking each through cycles of‘open’, ‘loose’ and ‘tight’ states. In ATP synthase, the rotatingcentral stalk is, therefore, the key coupling element in theenzyme involved in transferring energy from the Fo membranedomain, where rotation is generated from the transmembraneproton motive force, into the catalytic sites some 100 Å abovethe membrane surface. A peripheral stalk linking the surface ofF1 to Fo probably acts as a stator to counter the tendency of the(αβ)3 domain to follow the rotation of the central stalk2,9. Thedetails of the structure of the central stalk and of structuralchanges that occur in this region during catalysis are key ele-ments in unraveling the enzyme’s mechanism.

Much of our current knowledge of the details of the catalyticmechanism of ATP synthase has come from X-ray crystallo-

nature structural biology • volume 7 number 11 • november 2000 1055

graphic studies of the F1 domain from bovine mitochondria, anddirect demonstrations of the rotary mechanism in the bacterial(αβ)3γ subcomplex6,7 and in F1-ATPase10 were based on this

The structure of the central stalk in bovine F1-ATPase at 2.4 Å resolutionClyde Gibbons1, Martin G. Montgomery1, Andrew G. W. Leslie2 and John E. Walker1

The central stalk in ATP synthase, made of γ, δ and ε subunits in the mitochondrial enzyme, is the key rotaryelement in the enzyme’s catalytic mechanism. The γ subunit penetrates the catalytic (αβ)3 domain and protrudesbeneath it, interacting with a ring of c subunits in the membrane that drives rotation of the stalk during ATPsynthesis. In other crystals of F1-ATPase, the protrusion was disordered, but with crystals of F1-ATPase inhibitedwith dicyclohexylcarbodiimide, the complete structure was revealed. The δ and ε subunits interact with aRossmann fold in the γ subunit, forming a foot. In ATP synthase, this foot interacts with the c-ring and couplesthe transmembrane proton motive force to catalysis in the (αβ)3 domain.

1The Medical Research Council Dunn Human Nutrition Unit, Hills Road, Cambridge CB2 2XY, UK. 2The Medical Research Council Laboratory of Molecular Biology, HillsRoad, Cambridge CB2 2QH, UK.

Correspondence should be addressed to J.E.W. email: [email protected] or A.G.W.L. email: [email protected]

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Fig. 1 Space filling model of bovine F1-ATPase. a, Side view and b, view from the membrane. Alternating α subunits (red) and β subunits(yellow) and the attached central stalk containing the γ, δ and ε subunits(shown in blue, green and magenta, respectively). The insert indicates thepositions of the different α and β subunits viewed from above. In the orig-inal structure4, the βTP, βDP and βE subunits had AMP-PNP, ADP and nonucleotide bound, respectively. The α subunits are named according to thecatalytic interface to which they contribute. The scale bar represents 20 Å.


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structural information. However, until the work describedbelow, our knowledge of the structure of the central stalk hasremained incomplete. The protruding part of this stalk was dis-ordered in crystals of F1-ATPase, although the (αβ)3 domain andthe penetrating α-helical coiled coil part of the central stalk wereresolved to high resolution in the same crystals. Similar disorderwas present in crystals of bovine F1-ATPase inhibited withefrapeptin11, aurovertin12, 4-chloro-7-nitrobenzofurazan chlo-ride13 and Mg-ADP fluoroaluminate14.

Significant progress has been made by analyzing the structureof the isolated ε subunit of the enzyme from Escherichia coli15,16

(equivalent to the δ subunit in the mitochondrial enzyme17), butthis approach of solving the structures of individual subunits,even when allied with covalent crosslinking experiments in theintact enzyme, would not give the detailed information neces-sary for a precise understanding of the rotary mechanism of themultisubunit enzyme complex. Recently, a first glimpse of thearchitecture of the central stalk came from the structural analysisof the F1 domain linked by its central stalk to a ring of 10 c sub-units from the Fo domain of the enzyme from Saccharomycescerevisiae18. Although the fold of the δ subunit was located usingthe known structure of the homologous bacterial ε subunit, therelatively modest resolution of the electron density map (3.9 Å)did not allow a detailed model to be built of the protruding cen-tral stalk region.

In the present work, we describe the structure at 2.4 Å resolu-tion of bovine F1-ATPase covalently inhibited by dicyclohexyl-carbodiimide (F1-DCCD). In cryo-cooled crystals of F1-DCCD,the central stalk is ordered, allowing its features to be revealed inatomic detail, thereby providing new insights into the couplingmechanism of ATP synthase.

1056 nature structural biology • volume 7 number 11 • november 2000

Structure determinationThe structure of F1-DCCD was solved by molecularreplacement using the frozen native structure14 as a startingmodel (see Methods and Table 1), and the covalentlyattached dicyclohexyl-N-acylurea (DCU) was modeledinto the density. Because the stalk region is better orderedthan in any previous crystals of F1-ATPase, it could be builtinto the electron density using the structure of the E. coli εsubunit16 as a starting model. The new structure contains atotal of 3,316 amino acids, including residues 19–510 of thethree α subunits, residues 9–474 of two β subunits and9–475 of the third β subunit; residues 1–61, 67–96 and101–272 of the γ subunit, residues 15–145 of the δ subunitand residues 1–47 of the ε subunit. The remaining unre-solved sections are the N-terminal regions of the α and β subunits (residues 1–18 and -4–8, respectively; by con-vention, residue numbering starts at -4 in the β subunits),the C-terminal regions of the β subunits (residues 475–478of two β subunits and 476–478 of the third), two loopregions of five and four residues in the γ subunit (residues62–66 and 97–100), residues 1–14 and the C-terminalresidue (146) of the δ subunit, and three residues (48–50)at the C-terminus of the ε subunit.

The unit cell dimensions of F1-DCCD (Table 1) differsignificantly from those reported for the frozen native crys-

tals14. The a, b and c axes are 13.6 Å, 0.2 Å and 3.7 Å shorter,respectively. It is likely that these changes in unit cell parametersarise from modifications in the cryoprotection procedure (seeMethods). The shrinkage of the unit cell results from closerpacking of the F1 complexes in the crystal lattice involving moreextensive interaction between the central stalk and the head of anadjacent F1 complex. This increased interaction seems to beresponsible (at least in part) for the ordered and slightly twistedcentral stalk. The extended N-terminus (Ala 19–Ala 24) of the αE

subunit (see Fig. 1 for subunit nomenclature) interacts with the γsubunit of a neighboring complex, and the same region in theαTP subunit (Ala 19–Ala 24) is involved in a more extensive inter-action with the interface region of the δ and ε subunits of thesame neighboring complex. It is also possible that interactionwith the inhibitor DCCD played a part in ordering the centralstalk by presenting a more homogeneous population of inhibitedenzyme complexes.

After initial refinement, extra density interpreted as DCU wasfound associated with βDPGlu 199 near the interface between theβDP and αDP subunits. No other regions of density that could cor-respond to additional modifications by DCCD were present inthe electron density map, consistent with the observed stoi-chiometry at comparable levels of inhibition19–21.

Molecular architecture of F1-DCCDThe overall conformations of the α and β subunits are similar tothose in earlier structures4,11–14. They superimpose with rootmean square (r.m.s.) deviations for Cα atoms of 0.36–0.76 Å.However, the occupancy of the nucleotide binding sites is differ-ent. Mg–ADP is bound to the αTP subunit and Mg–ATP to the αE

and αDP subunits, although the larger temperature factor of the

Fig. 2 The structure of the central stalk. The color code for subunitsis the same as in Fig. 1. The light blue regions have been describedin earlier structures and new regions of structure in the γ subunitare dark blue. a, Side-on stereo view of stalk subunits (same viewas in Fig. 1a). b, Stereo view of stalk subunits, rotated 90° withrespect to (a), viewed from the membrane.

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γ-phosphate compared to the α-phosphate and β-phosphatesuggests that a mixed population of ADP and ATP may be pre-sent in both of these subunits. Mg–ADP is bound in the cat-alytic βDP and βTP subunits and, as in previous structures, thenucleotide binding site in the βE subunit is empty. The regionsof the γ subunit present in previous structures4,11–14 (residues1–44, 77–90 and 209–272) superimpose with an r.m.s. devationfor Cα atoms of 0.82 Å. This larger deviation is primarily due todifferences in the poorly ordered α-helical termini of previousF1-ATPase structures, and does not reflect a significant confor-mational change in the γ subunit. However, there has been a shiftof 1–2 Å in the lower segment of the γ subunit relative to the(αβ)3 domain. This shift is probably due to the lattice contactsdescribed above in the F1-DCCD crystals, which lead to theordering of the central stalk in this crystal form. The shift inposition of the γsubunit produces shifts of similar magnitudes inthe C-terminal domains of the αTP, βE and αE subunits, but notin the other subunits.

The main new feature in the model is the protruding part ofthe central stalk, which is composed of approximately half of theγ subunit and the entire δ and ε subunits (Fig. 1a). The overalllength of the central stalk from the C-terminus of the γ subunit(Fig. 2a) to the foot of the protruding region is 114 Å, and itextends 47 Å from the (αβ)3 domain. At its widest part, the pro-trusion has an oval cross section (54 × 41 Å) (Figs 1b, 2b).

An entirely new α/β domain has been identified in the γ sub-unit (Figs 2a, 4a). It consists of a five-stranded β-sheet (1–5)(Fig. 4a) and six α-helices (a–f). Strands 1–3 and α-helices band c form a Rossmann fold, which is linked by helix d to a β-hairpin formed by strands 4 and 5. Helices a and f are extend-ed by 9 and 12 residues, respectively, relative to previous modelsof the bovine enzyme. They are associated in an antiparallelcoiled coil shaft that passes through the middle of the (αβ)3 sub-complex. Pro 40 induces a kink in helix a, and there is a similarbut less pronounced bend at the corresponding position (Leu 217) in helix f. Two short loop regions of the γ subunit havenot been included in the model. In the loop from residues 62–66(between helix a and strand 1), there is weak electron density forthe main chain, but not for side chains. The loop from residues97–100, between helix b and strand 2, is disordered and has noassociated electron density. The position of the α/β domain sug-gests that it may provide structural stability to the lower sectionof the coiled coil shaft. Although the α/β fold is similar to anucleotide binding fold, there is no evidence that nucleotidesbind to this region of the γ subunit. The loops that are involvedin nucleotide binding in other proteins are glycine-rich, whereasthe corresponding loops in the γ subunit (between strand 1 andhelix b, and strands 4 and 5) contain large side chains thatwould prevent binding of nucleotides. A search of the ProteinData Bank (PDB) using DALI22 did not reveal any structurallyhomologous proteins. In chloroplast γ subunits, the loopbetween strand 5 (residues 170–179) and helix e (residues187–192) contains an insert of ∼ 35 amino acids not present inmitochondria or nonphotosynthetic eubacteria23. This loopincludes two Cys residues that are reduced in the active enzymeduring photosynthesis and oxidized to a disulfide in the inactiveenzyme in the dark24.


An extension of the a and f helices by 12 and 20 residues,respectively (relative to the bovine model), was found in a 4.4 Åresolution electron density map of E. coli F1-ATPase25. Four rodshaped features were interpreted as additional α-helices (denot-ed B and D–F) within the γ subunit. When the N-terminal andC-terminal helices of the γ subunit of the E. coli polyalaninemodel (PDB accession code 1D8S) were superimposed on the F1-DCCD structure, there was little or no agreement with theseadditional proposed α-helical regions. Bacterial helix B runsparallel to β-strand 5 in F1-DCCD and may correspond to it. Nopart of the bovine model was found in the vicinity of bacterialhelix D, and helices E and F overlap approximately with nonheli-cal regions of the bovine δ and ε subunits. E. coli helix C, corre-sponding to the bovine radial helix b, is in the same orientation,but it is displaced laterally by about 4 Å. This difference may bedue either to differences in the catalytic states of the two struc-tures, or to low sequence homology in this region.

The sequence identity (60%) between the bovine δ subunitand bacterial ε subunit suggested that they are functionallyequivalent17,26, and the similarity in fold confirms this view —127 residues superimpose with an r.m.s. deviation of 1.64 Å forCα atoms. As in the bacterial ε subunit, the bovine δ subunit hastwo domains (Fig. 4b), an N-terminal β-sandwich (residues15–98) and a C-terminal α-helical hairpin (residues 105–145).The β-sandwich consists of 10 β-strands with a hydrophobicinterior. A 310-helix (residues 99–104) is in the loop connectingthe β-sandwich to the two C-terminal helices. The second ofthese helices is slightly shorter than the first (38 and 43 Å, respec-tively) and is two α-helical turns shorter than the equivalent fea-ture in the E. coli ε subunit. The structural alignment of thebovine δ subunit and E. coli ε subunit does not support a predic-tion that the bovine δ subunit would have a longer loop betweenthe two C-terminal helices, and an even shorter C-terminalhelix16. No electron density was observed for residues 1–14 of thebovine δ subunit. This region is susceptible to proteolytic degra-dation26. However, from N-terminal sequence analysis, there wasno evidence of its proteolytic removal in F1-DCCD crystals, andtherefore this region is disordered.

The position of the δ subunit relative to the (αβ)3 subcomplexis quite different in the structures of F1-DCCD and the yeastF1–c10 complex18 (PDB entry 1QO1). Following superposition ofthe (αβ)3 domain, a rotation of 37° is required to superimposethe δ subunits. A more detailed model of the yeast structurereveals that the interface between the γ and δ subunits is essen-tially the same in both structures (D. Stock, pers. comm.). Theshift in the position of the δ subunit results from a change in thetwist of the coiled coil region of the γ subunit, but the details ofthis conformational change must await completion of the refine-ment of the yeast enzyme model.

The bovine ε subunit has no counterpart in either bacterial orchloroplast ATP synthases, although homologs are present in

Fig. 3 Stereo view of the electron density for strands 3, 2, 1, 4 and 5(from left to right) of the γ subunit. The 2Fo - Fc map was calculatedbefore including any new features of the γ subunit in the model. Thecontour level is 90% of the r.m.s. density.


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mitochondria of other eukaryotic species. It is 50 amino acidslong and is folded into a helix-loop-helix structure (Fig. 4c). Theonly Trp residue in the entire bovine mitochondrial F1-ATPase26

is found at position 4. It is buried in a hydrophobic pocketformed by Tyr 3, Tyr 11 and Tyr 14. The C-terminal region(residues 38–47) forms an extension of the β-sheet of the γ sub-unit (Fig. 2).

Intersubunit interactions in the central stalkThe γ, δ and ε subunits interact extensively (Table 2), particular-ly in the region at the foot of the central stalk. Here the N-termi-nal region of the ε subunit (residues 1–28) is in a shallow cleftbetween the two domains of the δ subunit (Fig. 2). This cleft isformed principally by residues from the C-terminal α-helix,from the interdomain linker, and from interstrand loops alongone edge of the β-sandwich. The C-terminal region of the ε sub-unit (residues 37–50) packs against helix d and strand 3 of the γsubunit. The interface between the γ and δ subunits involvesresidues in the γ subunit, both from the kinked part of helix a(residues 40–51) and from helix f (residues 201–214), which lieacross one face of the β-sandwich formed by strands 1, 2, 5, 8 and9 of the δ subunit (Fig. 2b).

Comparison of the aligned sequences using the programCLUSTALW27 of the γ and δ (bacterial ε) subunits from mito-chondrial, chloroplast, and bacterial sources revealed no signifi-cant conservation of residues forming the subunit interfaces. In

the mitochondrial ε subunit, five residues are strictly conserved.Trp 4 is located at the interface with γ and δ subunits, Arg 5 isinvolved in hydrogen bonding to the γsubunit, Tyr 11 forms partof the hydrophobic pocket for Trp 4 and is also involved inhydrogen bonding to the δ subunit, and Arg 23 and Lys 27 formhydrogen bonds to the δ subunit.

The extensive areas of the intersubunit interfaces (Table 2)involving the bovine ε subunit emphasizes its important role instabilizing the foot of the central stalk. The absence of an equiva-lent subunit in the bacterial (and chloroplast) enzymes mayexplain why the bacterial equivalent of the δ subunit is readilydissociated from the F1-ATPase complex while this has not beenobserved for the mitochondrial enzyme.

In E. coli F1-ATPase and F1Fo-ATPase, interactions within andbetween subunits have been examined by covalent crosslinking(Table 3). Typical Cα–Cα distances for disulfide linked Cysresidues lie in the range 4.4–6.8 Å (ref. 28). The distances forintrasubunit crosslinks between pairs of residues in the bovine δsubunit lie close to this range, consistent with the close structur-al homology to the bacterial ε subunit. The distances betweenresidues in the bovine δ and γ subunits, although somewhatlonger, suggests that they pack in a similar way to the bacterial εand γ subunits. In contrast, the corresponding distances involv-ing the bovine α and β subunits with the δ subunit lie farbeyond the range that would be consistent with their being incontact. A possible explanation is that the bacterial ε subunitdetaches and binds to a second site near to the (αβ)3 domain29.Alternatively, rotation of the central stalk is accompanied eitherby subunit rearrangements in this region, or by a deeper pene-tration of the γ subunit into the central cavity of the (αβ)3 com-plex during catalysis, bringing the bacterial ε subunit near to theC-terminal regions of the α and β subunits, and concomitantlythe (αβ)3 domain in close proximity to Fo and the membranesurface. The latter seems unlikely, as it would require large con-formational changes in the (αβ)3 complex in order to accom-modate the relatively bulky α/β domain of the γ subunit withinthe central cavity.

Mechanistic implicationsThe rotating central stalk in ATP synthase is an essential compo-nent in indirectly coupling the proton motive force across themembrane to ATP formation in the catalytic sites in the (αβ)3

domain. The role of the α-helical coiled coil region in the γ sub-

Fig. 4 The structures of the individual stalk subunits. a, The γ subunit with part of the α-helical coiled coil region(residues 1–19 and 235–272) removed for clarity. The color scheme is the same as in Fig. 2. The β-strands 1 to 5and α-helices a to f are indicated. The red lines from residues 62–66 and 97–100 represent regions without inter-pretable electron density. Helices c and e are 310-helices. The position of Arg 75, possibly involved in the ‘catch’mechanism (see Fig. 5), is indicated. b, The bovine δ subunit. c, The bovine ε subunit; (a) and (b) have been rotat-ed relative to Fig. 2a by 110° and 30°, respectively.

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Fig. 5 Interaction of γArg 75 with residues in the α and β subunits toform part of the catalytic ‘catch’. Distances are in Å.



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unit, in modulating the nucleotide affinities of catalytic sites in βsubunits by rotation of the central stalk, has been describedbefore. From the complete structure of the central stalkdescribed here, two new features have emerged.

First, in the region where the γsubunit emerges from the (αβ)3

domain, a conserved Arg residue at position 75 interacts withβDPGlu 395 and αEAsp 409 in the so-called DELSEED regions(Fig. 5). Residue γArg 75 is in a loop linking strand 1 (residues68–71) to helix b (residues 77–96), the ‘radial α-helix’ observedin earlier F1-crystal structures (Fig. 4a). The γsubunit rotation inF1-ATPase is known to proceed in 120° steps8, suggesting theexistence of three stable conformational intermediates in therotary catalytic cycle. Polar interactions such as those involvingγArg 75 (Fig. 5) and the previously described ‘catch’ residues(γArg 254, γGln 255)4 may contribute to the stabilization of theseintermediates. This is consistent with mutagenesis of the (αβ)3γcomplex from the thermophilic Bacillus PS3; replacing theresidue equivalent to βGlu 395 with Ala resulted in a five-folddecrease in ATPase activity30.

Second, the new structure demonstrates the extensive area ofthe foot of the central stalk, which involves parts of all three ofits constituent subunits (Fig. 1b). Regions of the γ and δ sub-units in these parts are highly polar and contain several residuesthat extend away from the (αβ)3 subcomplex towards the mem-brane domain. In the γ subunit, three carboxyl groups (γAsp 194, γAsp 195 and γAsp 197) in the exposed lower face ofthe foot extend downwards, suggesting that, in ATP synthase,they may interact with basic residues in loop regions of the c-ring. The details of these interactions with the c-ring mustawait a high resolution structure of the F1–c-ring complex or ofthe intact ATP synthase itself.

Mechanism of inhibition of F1-ATPase by DCCDOne molecule of DCCD reacted per F1-ATPase complex. Theadditional density corresponding to DCU is associated withβDPGlu 199 (Fig. 6a) towards the end of helix C. The modifiedGlu is found at the interface between the βDP and αDP subunits(Fig. 6b) and the side chain χ1 angle has changed from -70° inthe native enzyme to -154° in the inhibited form. The DCUmoiety is bound in a hydrophobic cleft, but one face is almostentirely solvent exposed. Residues, in the βDP subunit, con-tributing to the binding site include Val 164 and Met 167 from

helix B (immediately following the nucleotide binding loop (P-loop)) and Val 420 and Phe 424 from the C-terminaldomain. In the structural transition from the closed conforma-tion of the βDP subunit to the open conformation of the βE sub-unit, there is a large movement of helix B and the C-terminaldomain relative to helix C. The mechanism of inhibition byDCCD is probably due to steric hindrance of this conformation-al change by the bulky DCU group.

Conformational changes at the active sitesThe presence of DCU modified Glu 199 in the βDP subunit hasno significant effect on the conformation of the residues at theβDP subunit catalytic site. However, in the βTP subunit catalyticsite, there is a dramatic change in the side chain conformationof αTPArg 373 (equivalent to αArg 376 in the E. coli enzyme).


Fig. 6 The DCCD binding pocket in bovine F1-ATPase. a, Stereo view of the electron density corresponding to the DCU moiety attached to βDPGlu 199.The side chains forming the pocket are all in the βDP subunit. b, The interface between the βDP and αDP subunits viewed from the exterior of the (αβ)3

subcomplex. Nucleotides (both ADP) are in black ball-and-stick representation. The DCU moiety is shown as an orange space filling model.

Table 1 Data collection and refinement statistics

Space group P212121

Unit cell dimensions (Å) a = 267.2, b = 107.2, c = 135.9Resolution (Å) 20.0–2.4Number of reflections 140,093Rejected measurements (%) 0.6Rmerge

1 0.088 (0.331)I / σ1 8.1 (2.1)Completeness1 (%) 92.2 (68.6)Multiplicity1 2.4 (1.8)Number of protein atoms2 25,363Number of nucleotide atoms 148Number of DCU atoms2 16Number of water molecules2 909R-factor3 (%) 22.5Free R-factor (%) 28.1R.m.s deviations

Bonds (Å) 0.008Angles (°) 1.1

1The statistics for the highest resolution bin (20–2.4 Å) are given inparentheses.2Hydrogen atoms were excluded.3R = Σ|Fo - Fc| / Σ|Fo|, where Fo and Fc are the observed and calculated struc-ture factor amplitudes, respectively, for 133,686 reflections used in therefinement.

a b


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This residue has been shown to be essential for full activity31,32

and in previous structures the guanidinium group is posi-tioned to interact with the β-phosphate and/or γ-phosphategroups of the nucleotide bound to the catalytic β subunit. Inthe F1-DCCD structure, the guanidinium group of αTPArg 373has moved 10 Å and now interacts with the 2′ hydroxyl of theribose. It seems unlikely that this conformational change is dueto the DCCD inhibition, as αTPArg 373 is 70 Å from the DCUgroup. Superimposing the F1-DCCD and frozen native struc-tures shows that there are no significant conformationalchanges in the βTP, αDP and βDP subunits that could be associat-ed with DCCD binding. A more likely explanation is the smallmovement (1–2 Å) of the αTP subunit relative to the βTP sub-unit induced by the twisting of the γ subunit, which in turnarises from lattice contacts as discussed above. It is not clearwhy this leads to repositioning of the Arg side chain, but thisresult suggests that the preferred conformation of αTPArg 373is critically dependent on the interaction between the α sub-unit and the γ subunit, and a similar conformational changemay well occur during the catalytic rotation of the central stalkof the enzyme.

MethodsInhibition of F1-ATPase by DCCD. Bovine F1-ATPase was purified as described33 except thatthe Sephacryl S-300 column was replaced by aHiLoad 26/60 Superdex 200pg column(Pharmacia Biotech, St. Albans, Herts., UK) and5 mM 2-mercaptoethanol in the buffer wasreplaced with 5 mM dithiothreitol. The puri-fied enzyme was stored as an ammonium sul-fate precipitate, which was collected bycentrifugation (31,000 g) at 4 °C, redissolved inminimal DCCD buffer (250 mM sucrose, 50 mMMES, pH 6.0, 4 mM Na-ATP and 2 mM EDTA)and desalted on a Sephadex G-25 column(Pharmacia). The enzyme was reacted with asolution of DCCD in absolute ethanol (20 mM).The final concentration of DCCD was 100 µM.After reaction for 1 h at room temperature,the ATP concentration was readjusted to4 mM. The activity of F1-ATPase was measuredat 30 min intervals with an ATP regeneratingsystem coupled to NADH oxidation. Maximuminhibition (96%) of the enzyme activity wasattained after reaction for 2 h. DCCD inhibitsbovine F1-ATPase by covalent modification ofthe γ-carboxylate of Glu 199 in one of the cat-alytic β subunits, producing the DCU deriva-tive34.

Crystallization of F1-DCCD. Freshly inhibitedF1-ATPase (10 mg ml-1) with its boundnucleotides was crystallized in 50 µl microdial-ysis buttons with SpectraPor dialysis mem-branes (5000 molecular weight cutoff). An


equal volume of inside buffer (100 mM Tris-HCl, pH 7.2, 200 mMsodium chloride, 4 mM magnesium chloride, 0.04% (w/v) sodiumazide, 0.004% (w/v) phenylmethylsulphonyl fluoride, 14% (w/v)polyethylene glycol (PEG) 6000, 660 µM ADP and 100 µM DCCD) wasadded to the protein solution (final concentration 5 mg ml-1) anddialyzed against outside buffer (50 mM Tris-HCl, pH 7.5, 400 mMsodium chloride, 10 mM magnesium chloride, 0.02% (w/v) sodiumazide, 1 mM EDTA, 0.004% (w/v) phenylmethylsulphonyl fluoride,9% (w/v) PEG 6000, 660 µM ADP and 100 µM DCCD). After threedays, the outside buffer was replaced and the PEG 6000 concentra-tion was increased to between 10.25% and 11.75% (in 0.25% steps).After 3 weeks, the crystals were fully grown, with typical dimen-sions of 250 µm × 100 µm × 100 µm.

Cryoprotection and data collection. Using a protocol suggestedby R.I. Menz, the PEG 6000 concentration in the outside buffer sur-rounding the crystals was increased to 14% and the glycerol concen-tration was increased stepwise to 5%, 10% and 20% (2 h at eachconcentration). Then crystals were harvested with a cryoloop (0.2 µM, Hampton Research, Laguna Niguel, California, USA),plunged into liquid nitrogen and stored at 100 K. Single crystalswere placed in a gaseous nitrogen stream at 100 K on station 9.6 atCLRC-SRS (Daresbury, UK). Diffraction data were collected on anADSC Quantum Four CCD detector to 2.4 Å resolution using X-raysat a wavelength of 0.87 Å. The crystals belong to the orthorhombicspace group P212121 and the unit cell dimensions are a = 267.2 Å, b =107.2 Å, c = 135.9 Å. The diffraction data were integrated withMOSFLM35 and processed further with programs from the suitesCCP436 and CNS37.

Structure solution and refinement. The structure of F1-DCCDwas solved by molecular replacement with AmoRe38 using thefrozen native structure14 (PDB accession code 1E1Q) as a startingmodel. In all subsequent refinement steps, 5% of the data was setaside for calculation of the free R-factor. Upon refinement with REF-MAC36,39 using all data between 20 Å and 2.4 Å resolution, the freeR-factor and R-factor fell from 34.4% to 31.7% and 35.3% to 26.6%,respectively. At this point it became possible to model new features

Table 2 Intersubunit interfaces in the central stalk

Interface γ–δ γ–ε δ–εArea in interface (Å2) 971 957 1122Interface surface areaof each subunit (%)1 6 (γ); 12 (δ) 6 (γ); 21 (ε) 14 (δ); 24 (ε)Hydrophobicity2 (%) 68.5 60.8 58.6

1Relative to total surface area.2Surface area involving carbon atoms is considered to be hydrophobic.

Table 3 Covalent crosslinks introduced into E. coli F1-ATPase and distances betweenequivalent residues in bovine F1-DCCD

Subunits Bacterial F11 Bovine F1 References

Crosslink Distance2 (Å) Equivalent residues Distance2 (Å)ε–ε M49–A126 7.2 δ L62–δ A140 8.5 46

F61–V130 6.4 δ K74–δ A144 8.9A94–L128 6.5 δ A108–δ V142 7.1A101–L121 5.5 δ A115–δ I135 5.7

ε–γ ε S10–γ Y228 – δ S22–γ Y214 11.5 47,48ε T43–γ Y205 – δ Q56–γ Y193 10.5ε H38–γ Y205 – δ H51–γ Y193 10.7

β–ε β E381–ε S108 – β E395–δ A122 47.3 (βDP) 4952.4 (βE)54.8 (βTP)

β E381-ε M138 – β E395-δ L1453 37.4 (βE) 50,5150.8 (βDP)55.7 (βTP)

α–ε α S411-ε S108 – α S408-δ A122 43.4 (αE) 29,5260.6 (αTP)49.5 (αDP)

1The residues listed in the bacterial enzyme were replaced by Cys residues and a disulfidecrosslink was observed to form, as indicated, under oxidizing conditions.2The corresponding Cα–Cα distance. Values for the bacterial ε subunit are from the crystal struc-ture of the isolated ε subunit16. For bovine F1, the Cα–Cα distances are from the F1-DCCD struc-ture. For α and β subunits, the distances for different protomers are indicated. The Cα–Cαdistance for residues in a disulfide bond is typically 4.4–6.8 Å (ref. 28).3There is no residue in the bovine δ subunit equivalent to bacterial εMet 138, which lies in ashort C-terminal extension. Therefore, the distance is measured to bovine δLeu 145, the finalstructured residue and the penultimate residue in the chain.


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in the central stalk, initially as polyalanine chains. Residues 15–145of the δ subunit were built into the density as polyalanine using thestructure of the equivalent E. coli ε subunit (PDB accession code1AQT)16. After modeling the N-terminal helix of the ε subunit, the γsubunit was completed, including the Rossmann fold (Fig. 3), andthen the C-terminal extended strand of the ε subunit was built. Thedensity for the loops and side chains of the stalk region wasimproved greatly by using the CNS suite of programs, probablybecause of the algorithm used to calculate the bulk solvent correc-tion. Nucleotides, glycerol and sulfate were removed from themodel during refinement with CNS. Side chains for the polyalanineregions of the γ, δ and ε subunits were added during multiplerounds of refinement and manual model building with the programO40. The coordinates for DCU were obtained from the CambridgeSmall Molecule Database (entry CYHXUR02; ref. 41). A dictionaryentry for the DCU modified Glu was created and, in a final stage inthe refinement, it was built into the βDP subunit with O. Followingfurther refinement (REFMAC), the stereochemistry of the final

1. Boyer, P.D. The ATP synthase: a splendid molecular machine. Annu. Rev. Biochem.66, 717–749 (1997).

2. Walker, J.E. ATP synthesis by rotary catalysis (Nobel Lecture). AngewandteChemie-International Edition 37, 2309–2319 (1998).

3. Orriss, G.L. et al. The delta- and epsilon subunits of bovine F1-ATPase interact toform a heterodimeric subcomplex. Biochem. J. 314, 695–700 (1996).

4. Abrahams, J.P., Leslie, A.G.W., Lutter, R. & Walker, J.E. Structure at 2.8 Åresolution of F1-ATPase from bovine heart mitochondria. Nature 370, 621–628(1994).

5. Boyer, P.D. The binding change mechanism for ATP synthase: some probabilitiesand possibilities. Biochim. Biophys. Acta 1140, 215–250 (1993).

6. Noji, H., Yasuda, R., Yoshida, M. & Kinosita, K. Direct observation of the rotationof F1-ATPase. Nature 386, 299–302 (1997).

7. Noji, H. et al. Rotation of Escherichia coli F1-ATPase. Biochem. Biophys. Res.Comm. 260, 597–599 (1999).

8. Yasuda, R., Noji, H., Kinosita, K. & Yoshida, M. F1-ATPase is a highly efficientmolecular motor that rotates with discrete 120° steps. Cell 93, 1117–1124 (1998).

9. Karrasch, S. & Walker, J.E. Novel features in the structure of bovine ATP synthase.J. Mol. Biol. 290, 379–384 (1999).

10. Kato-Yamada, Y., Noji, H., Yasuda, R., Kinosita, K. & Yoshida, M. Directobservation of the rotation of ε subunit in F1-ATPase. J. Biol. Chem. 273,19375–19377 (1998).

11. Abrahams, J.P. et al. The structure of bovine F1-ATPase complexed with thepeptide antibiotic efrapeptin. Proc. Natl. Acad. Sci. USA 93, 9420–9424 (1996).

12. van Raaij, M., Abrahams, J.P., Leslie, A.G.W. & Walker, J.E. The structure of bovineF1-ATPase complexed with the antibiotic inhibitor aurovertin B. Proc. Natl. Acad.Sci. USA 93, 6913–6917 (1996).

13. Orriss, G.L., Leslie, A.G.W., Braig, K. & Walker, J.E. Bovine F1-ATPase covalentlyinhibited with 4-chloro-7-nitrobenzofurazan: the structure provides furthersupport for a rotary catalytic mechanism. Structure 6, 831–837 (1998).

14. Braig, K., Menz, R.I., M.G., M., Leslie, A.G.W. & Walker, J.E. Structure of bovinemitochondrial F1-ATPase inhibited by Mg2+ ADP and aluminium fluoride.Structure 8, 567–573 (2000).

15. Wilkens, S., Dahlquist, F.W., McIntosh, L.P., Donaldson, L.W. & Capaldi, R.A.Structural features of the ε subunit of the Escherichia coli ATP synthasedetermined by NMR spectroscopy. Nature Struct. Biol. 2, 961–967 (1995).

16. Uhlin, U., Cox, G.B. & Guss, J.M. Crystal structure of the epsilon subunit of theproton-translocating ATP synthase from Escherichia coli. Structure 5, 1219–1230(1997).

17. Walker, J.E., Runswick, M.J. & Saraste, M. Subunit equivalence in Escherichia coliand bovine heart mitochondrial F1F0-ATPases. FEBS Lett. 146, 393–396 (1982).

18. Stock, D., Leslie, A.G.W. & Walker, J.E. Molecular architecture of the rotary motorin ATP synthase. Science 286, 1700–1705 (1999).

19. Matsuno-Yagi, A. & Hatefi, Y. Inhibitory chemical modifications of F1-ATPase:effects on the kinetics of adenosine 5′-triphosphate synthesis and hydrolysis inreconstituted systems. Biochemistry 23, 3508–3514 (1984).

20. Satre, M., Lunardi, J., Pougeois, R. & Vignais, P.V. Inactivation of Escherichia coliBF1-ATPase by dicyclohexylcarbodiimide. Chemical modification of the β subunit.Biochemistry 18, 3134–3141 (1979).

21. Tommasino, M. & Capaldi, R.A. Effect of dicyclohexylcarbodiimide on unisite andmultisite catalytic activities of the adenosine triphosphatase of Escherichia coli.Biochemistry 24, 3972–3976 (1985).

22. Holm, L. & Sander, C. Touring protein fold space with Dali/FSSP. Nucleic Acids Res.26, 316–319 (1998).

23. Miki, J., Maeda, M., Mukohata, Y. & Futai, M. The γ subunit of ATP synthase fromspinach-chloroplasts: primary structure deduced from the cloned cDNAsequence. FEBS Lett. 232, 221–226 (1988).

24. Nalin, C.M. & McCarty, R.E. Role of a disulfide bond in the γ subunit in activationof the ATPase of chloroplast coupling Factor-1. J. Biol. Chem. 259, 7275–7280(1984).

25. Hausrath, A.C., Gruber, G., Matthews, B.W. & Capaldi, R.A. Structural features ofthe gamma subunit of the Escherichia coli F1-ATPase revealed by a 4.4-Åresolution map obtained by X-ray crystallography. Proc. Natl. Acad. Sci. USA 96,13697–13702 (1999).

26. Walker, J.E. et al. Primary structure and subunit stoichiometry of F1-ATPase frombovine mitochondria. J. Mol. Biol. 184, 677–701 (1985).

27. Thompson, J.D., Higgins, D.G. & Gibson, T.J. CLUSTAL W: improving the sensitivityof progressive multiple sequence alignment through sequence weighting,

position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22,4673–4680 (1994).

28. Richardson, J.S. The Anatomy and taxonomy of protein structures. Adv. ProteinChem. 34, 167–339 (1981).

29. Aggeler, R. & Capaldi, R.A. Nucleotide-dependent movement of the epsilonsubunit between alpha and beta subunits in the Escherichia coli F1Fo-type ATPase.J. Biol. Chem. 271, 13888–13891 (1996).

30. Hara, K.Y. et al. The role of the DELSEED motif of the beta subunit in rotation ofF1-ATPase. J. Biol. Chem. 275, 14260–14263 (2000).

31. Le, N.P. et al. Escherichia coli ATP synthase alpha subunit Arg-376: the catalyticsite arginine does not participate in the hydrolysis/synthesis reaction but isrequired for promotion to the steady state. Biochemistry 39, 2778–2783 (2000).

32. Senior, A.E., Nadanaciva, S. & Weber, J. Rate acceleration of ATP hydrolysis byF1Fo-ATP synthase. J. Exp. Biol. 203, 35–40 (2000).

33. Lutter, R. et al. Crystallisation of F1-ATPase from bovine heart mitochondria. J.Mol. Biol. 229, 787–790 (1993).

34. Esch, F.S., Bohlen, P., Otsuka, A.S., Yoshida, M. & Allison, W.S. Inactivation ofbovine mitochondrial F1-ATPase with dicyclohexyl-carbodiimide [14C] leads to themodification of a specific glutamic-acid residue in the beta subunit. J. Biol. Chem.256, 9084–9089 (1981).

35. Leslie, A.G.W. Joint CCP4 and EACMB Newsletter Protein Crystallography, Vol. 26(Daresbury Laboratory, Warrington; 1992).

36. Collaborative Computational Project, Number 4. The CCP4 Suite: programs forprotein crystallography. Acta Crystallogr. D 50, 760–763 (1994).

37. Brunger, A.T. et al. Crystallography & NMR system: a new software suite formacromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998).

38. Navaza, J. AMoRe: an automated package for molecular replacement. ActaCrystallogr. A 50, 157–163 (1994).

39. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecularstructures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255(1997).

40. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard. Improved methods for bindingprotein models in electron density maps and the location of errors in thesemodels. Acta Crystallogr. A 47, 110–119 (1991).

41. Toniolo, C. et al. Crystal structure of DCCD. Int. J. Pep. Prot. Res. 215, 77 (1988).42. Laskowski, R.A., Macarthur, M.W., Moss, D.S. & Thornton, J.M. PROCHECK: a

program to check the stereochemical quality of protein structures. J. Appl.Crystallogr. 26, 283–291 (1993).

43. Jones, S. & Thornton, J.M. Prediction of protein-protein interaction sites usingpatch analysis. J. Mol. Biol. 272, 133–143 (1997).

44. Esnouf, R.M. An extensively modified version of MolScript that includes greatlyenhanced coloring capabilities. J. Mol. Graph. Modell. 15, 132–134 (1997).

45. Merritt, E.A. & Bacon, D.J. Raster3D: photorealistic molecular graphics. MethodsEnzymol. 277, 505–524 (1997).

46. Schulenberg, B. & Capaldi, R.A. The epsilon subunit of the F1Fo complex ofEscherichia coli: cross-linking studies show the same structure in situ as whenisolated. J. Biol. Chem. 274, 28351–28355 (1999).

47. Aggeler, R., Chicas-Cruz, K., Cai, S.X., Keana, J.F.W. & Capaldi, R.A. Introduction ofreactive cysteine residues in the ε subunit of Escherichia coli F1-ATPase,modification of these sites with tetraflorophenyl azide maleimides, andexamination of changes in the binding of the epsilon subunit when differentnucleotides are in catalytic sites. Biochemistry 31, 2956–2961 (1992).

48. Watts, S.D., Tang, C.L. & Capaldi, R.A. The stalk region of the Escherichia coli ATPsynthase: tyrosine 205 of the γ subunit is in the interface between the F1 and Fo

parts and can interact with both the epsilon and c oligomer. J. Biol. Chem. 271,28341–28347 (1996).

49. Aggeler, R., Haughton, M.A. & Capaldi, R.A. Disulfide bond formation betweenthe COOH-terminal domain of the β subunits and the γ subunits and ε subunits ofthe Escherichia coli F1-ATPase: structural implications and functionalconsequences. J. Biol. Chem. 270, 9185–9191 (1995).

50. Tang, C.L. & Capaldi, R.A. Characterization of the interface between gamma andepsilon subunits of Escherichia coli F1-ATPase. J. Biol. Chem. 271, 3018–3024 (1996).

51. Wilkens, S. & Capaldi, R.A. Solution structure of the epsilon subunit of the F1-ATPase from Escherichia coli and interactions of this subunit with beta subunitsin the complex. J. Biol. Chem. 273, 26645–26651 (1998).

52. Aggeler, R., Weinreich, F. & Capaldi, R.A. Arrangement of the ε subunit in theEscherichia coli ATP synthase from the reactivity of cysteine residues introducedat different positions in this subunit. Biochim. Biophys. Acta 1230, 62–68 (1995).

model was verified using the program PROCHECK42. Intersubunitinteractions were analyzed using the program Areaimol from theCCP4 suite, and the Protein-Protein Interaction (PPI) Server soft-ware43. The data processing and refinement statistics are presentedin Table 1. Figures were produced using the programs Bobscript44

and Raster3D45.

Coordinates. The structure has been submitted to the Protein DataBank (accession code 1E79).

AcknowledgmentsWe thank the staff at the SRS, Daresbury, UK, for their support during datacollection, and D. Stock and J. Li for their help during CNS refinement. C.G. issupported by a Medical Research Council PhD studentship.

Received 14 June, 2000; accepted 14 August, 2000.


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