672
The rotary mechanism of ATP synthase
Daniela Stock*, Clyde Gibbons*, Ignacio Arechaga*,
Andrew GW Leslie† and John E Walker*‡
Since the chemiosmotic theory was proposed by Peter Mitchell
in the 1960s, a major objective has been to elucidate the
mechanism of coupling of the transmembrane proton motive
force, created by respiration or photosynthesis, to the
synthesis of ATP from ADP and inorganic phosphate. Recently,
significant progress has been made towards establishing the
complete structure of ATP synthase and revealing its
mechanism. The X-ray structure of the F1 catalytic domain has
been completed and an electron density map of the F1–c10
subcomplex has provided a glimpse of the motor in the
membrane domain. Direct microscopic observation of rotation
has 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: [email protected]
Current Opinion in Structural Biology 2000, 10:672–679
0959-440X/00/$ — see front matter
Published by Elsevier Science Ltd.
Abbreviations
EM
electron microscopy
F1
factor 1
Fo
factor oligomycin
OSCP oligomycin sensitivity conferring protein
Pi
inorganic phosphate
pmf
proton motive force
Introduction
ATP synthase contains a rotary motor involved in biological energy conversion. Respiratory complexes in
mitochondria and eubacteria, and photosynthetic complexes
in chloroplasts and photosynthetic eubacteria use energy
derived from the oxidation of nutrients and from light,
respectively, to generate a transmembrane proton motive
force (pmf) [1–3]. ATP synthase uses the pmf to make
ATP from ADP and inorganic phosphate (Pi). As summarised in Figure 1a,b, the enzyme has two major
structural domains, known as F1 (factor 1) and Fo (factor
oligomycin). The globular F1 catalytic domain in the mitochondrial enzyme is an assembly of five subunits with the
stoichiometry α3β3γ1δ1ε1. Subunits γ, δ and ε form a central
stalk linking the (αβ)3 subcomplex of F1 to the membrane
domain, Fo. The (αβ)3 subcomplex and Fo are also linked
by a peripheral stalk, sometimes called the stator [2].
In the F1 domain, the three α subunits and the three β subunits are arranged alternately around a central α-helical
coiled coil in the γ subunit [4]. This arrangement suggested
that the enzyme works by a mechanism involving the cyclic
modulation of nucleotide affinity in catalytic β subunits, as
required by the binding-change mechanism [1], by rotation
of the asymmetrical γ subunit. During ATP synthesis, the
rotation would be generated in Fo and fuelled by the pmf.
During ATP hydrolysis in F1Fo (or in F1 alone), the energy
released by hydrolysis would drive rotation in the opposite
direction and reverse the direction of proton translocation.
Subsequently, the rotation of the γ subunit in an (αβ)3γ
complex was observed directly by microscopy and was
shown to depend on ATP hydrolysis [5].
Recent structural results have provided additional insight
into the nature of the central stalk [63••]. This feature links
the F1 and Fo domains, and forms part of the rotor in the
ATP synthase molecular motor. The way in which the central stalk is linked to a ring of c subunits in the Fo domain has
been suggested from a low-resolution electron density map
of a subcomplex of the yeast enzyme. As yet, no structural
information is available on other key subunits in the Fo
domain, but a number of models have been proposed for
torque generation. A much clearer picture of the molecular
mechanism of the motor in ATP synthase is slowly emerging.
The central stalk
Until recently, the protruding part of the central stalk was
disordered in crystals of bovine F1-ATPase [4], although
the (αβ)3 domain and the penetrating α-helical coiled-coil
part of the central stalk were resolved in the same crystals.
By modification of the cryoprotection conditions, the crystal lattice of bovine F1-ATPase (covalently inhibited with
dicyclohexylcarbodiimide) has been shrunk, thereby
ordering the protruding central stalk region and allowing
the 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 a
new α/β domain in the γ subunit, containing a Rossmann
fold, that does not bind nucleotides. It appears to be a buttress, stabilising the lower section of the coiled-coil shaft.
There is little agreement between the structure of the
bovine γ subunit in the Rossmann fold region of the central stalk and a model of the same region of the Escherichia
coli γ subunit, deduced from a 4.4 Å resolution electron
density map of bacterial F1-ATPase [6].
The bovine structure confirms the structural homology
between the mitochondrial δ and bacterial (and chloroplast) ε subunits. Similar to the bacterial ε subunit [7], the
bovine δ subunit has two domains, an N-terminal β sandwich with 10 strands (residues 15–98) and a C-terminal
α-helical hairpin (residues 105–145). The 50 amino acid
bovine ε subunit has no counterpart in bacteria or chloroplasts. It has a helix-loop-helix structure and appears to
The rotary mechanism of ATP synthase Stock et al.
673
Figure 1
Structure of ATP synthase. (a,b) Summary of
current knowledge of the structure of ATP
synthase from mitochondria and eubacteria.
(a) Mitochondrial ATP synthase. The model is
based on EM studies of single particles [17•]. It
incorporates the structure of bovine F1-ATPase
[4,62•] and information from the electron
density map of the F1–c10 complex from
S. cerevisiae [36••]. The composition,
stoichiometry and arrangement of the subunits
in the peripheral stalk (subunits OSCP, F6,
b and d) come from biochemical and
reconstitution studies [20,33]. The position of
subunit a relative to the c10 ring was deduced
from studies of the bacterial enzyme [28].
Minor subunits (e, f, g, A6L) in the Fo domain
are not shown. They have no known functions
in the enzyme’s mechanism. (b) Eubacterial
ATP synthase. The overall model is also based
on EM studies [14,15•]. The core structure of
the central F1–c ring was deduced by
homology with the mitochondrial enzyme.
However, the c ring may contain 12 c subunits,
not 10 [45]. The positions of the subunits in the
peripheral stalk (subunits b and δ) are
supported by biochemical and EM studies
[14,18•,26•]. The δ subunit (structure
determined 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 was
also determined independently [7,11]. The
general structure of ATP synthase from
chloroplasts is very similar to that of the
bacterial enzyme. The main differences are that
the c ring may contain 14 c protomers [46••]
and that the two identical b subunits in some
eubacterial enzymes are replaced by
homologous, but not identical, subunits b and
b′. A similar arrangement of b and b′ subunits is
also found in other eubacterial species. (c) The
complete structure of bovine F1-ATPase shown
in 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 an
α-helical coiled coil in the γ subunit (blue).
Regions of the γ subunit present in the original
F1 structure [4] are shown in sky blue, those
regions determined in the latest structure [63••]
are in dark blue. The central stalk consists of
the γ, δ and ε subunits (blue, green and
magenta, respectively).
(a)
(b)
OSCP
α
δ
α
α
α
α
γ
α
γ
β
β
δ
F6
ε
c10
d
a
β
β
b
ε
b2
c9–12
a
(c)
stabilise the foot of the central stalk, where the γ, δ and
ε subunits all interact extensively. It is probable that all
three subunits contact the Fo domain.
In E. coli F1-ATPase, interactions between and within subunits have been examined by the introduction of cysteine
residues at specific sites and formation of disulfide crosslinks by oxidation. Cross-links observed within the
bacterial ε subunit [8•] and ε–γ cross-links [9,10] are consistent 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 in
the bovine structure. One possible interpretation is that the
Current Opinion in Structural Biology
bacterial ε subunit detaches wholly or partially from the
foot during the catalytic cycle, so that it can interact with
the lower surface of the (αβ)3 domain. However, the functional significance of such a rearrangement is obscure. A
critical re-examination of the formation of the α–ε and
β–ε cross-links is warranted.
The peripheral stalk
There is general agreement that the F1 and Fo domains are
also connected by a second, peripheral, stalk [2]. This has
been observed by single-particle analysis using electron
microscopy (EM) in negative stain of bacterial [14,15•],
674
Proteins
Figure 2
50 Å
83 Å
(a)
α
β
α
β
γ
58 Å
δ
C
(b)
55 Å
2 1
10
3
9
δ
4
8
5 6 7
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.
chloroplast [16] and mitochondrial [17•] F1Fo-ATPases. Its
function has not been demonstrated, but it may act as a stator to counter the tendency of the (αβ)3 domain to follow
the rotation of the central stalk [2]. In E. coli, it contains
the δ subunit and the extrinsic membrane domains of two
identical b subunits that form a parallel α-helical coiled
coil [18•] (see Figure 1b). The membrane domains of the
b subunits (one transmembrane α helix each) also interact
and form part of Fo [19•]. In some other bacterial species
and in chloroplasts, the two identical b subunits are
replaced by single copies of homologous subunits b and b′.
The bovine peripheral stalk contains one copy each of the
OSCP (oligomycin sensitivity conferring protein) subunit
(the equivalent of bacterial δ), the extrinsic domain of subunit b and the d and F6 subunits [2] (see Figure 1a). It has
been assembled in vitro and interacting regions have been
The rotary mechanism of ATP synthase Stock et al.
675
Figure 3
(c)
(b)
(a)
Actin filament
c ring
Fo
Actin filament
γ
F1
Actin filament
ε
ATP
ATP
ADP + Pi
ADP + Pi
ATP
ADP + Pi
Current Opinion in Structural Biology
Observations of rotation in ATP synthase. The direct observation of rotation
using fluorescently labelled actin filaments attached to (a) the γ subunit in
the (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 are
associated with a nickel-coated glass surface. Counterclockwise rotation
dependent on ATP hydrolysis was observed in a fluorescence microscope.
defined [20]. In Saccharomyces cerevisiae, cross-links have
been observed between the b subunit and subunits β,
OSCP and d (in agreement with the bovine findings), and
also to the membrane subunit a (and other minor Fo subunits) [21,22]. The peripheral stalk subunits are poorly
conserved (relative to F1 components, for example) and
subunits b can be shortened and lengthened without having
a major effect on the enzyme’s activity [23,24].
NMR spectroscopy in organic solvents, has two transmembrane α helices linked by a polar loop [32]. The
C-terminal α helix contains a carboxyl group (Asp61) that
is also essential for proton translocation. The conservation
and arrangement of the b subunits was discussed above.
The Fo domains of mitochondrial enzymes contain a number of small subunits that appear to have no direct role in
catalysis [33–35]. They are absent from bacterial and
chloroplast enzymes.
For many years, it has been known that the δ and OSCP
subunits 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. This
arrangement has been confirmed by cross-linking experiments [25] and EM [26•]. The structure of the N-terminal
domain of the E. coli δ subunit has been established by
NMR studies [27].
The Fo domain
In E. coli, the Fo domain is composed of three subunits
with the stoichiometry a1b2c9–12 (Figure 1b). The a and
c subunits are in contact and protons are thought to be
translocated through the interface between them [28,29].
Both subunits are conserved in all F-ATPases. The E. coli
a subunit is hydrophobic and is probably folded into five
transmembrane α helices [30,31]. It contains basic and
acidic residues (Arg210, His245, Glu196, Glu219) that are
essential for proton translocation. The c subunit is also
hydrophobic. The protomer structure, determined by
The first view of the structure of the Fo domain came from
an electron density map of F1-ATPase associated with a
ring of 10 c subunits from S. cerevisiae [36••] (see Figure 2).
This F1–c10 complex was formed from ATP synthase during the crystallisation process, when other subunits
dissociated. The electron density map contains a number
of important features. First, the 10 c protomers appear to
have secondary structure similar to the c protomer structure determined by NMR. The map also shows that the
C-terminal α helices form an outer ring, with the N-terminal α helices in a second inner ring. Second, the map shows
that the extensive footprint of the central stalk sits asymmetrically on the polar loop regions of six c subunits. This
arrangement is consistent with the rotation of the central
stalk and the c ring as an ensemble, as are covalent crosslinks between the E. coli ε and c subunits that do not affect
the enzyme’s activity [37•,38,39•]. Third, 10 c subunits are
found in the ring and not 12, as was widely anticipated.
Therefore, there is a symmetry mismatch between the
676
Proteins
Figure 4
(a)
H+
(b)
(c)
Na+
a subunit
c subunits
a subunit
140°
c subunits
c
a
H+
Na+
Current Opinion in Structural Biology
Models of the generation of rotation by movement of ions through the
Fo domain of ATP synthase. (a) A two-channel model proposed by
Junge [2,58]. Two half channels across the interface between the
a subunit and the c ring are linked by rotation of the c ring. (b) A
single-channel model [60•] for the Na+-motive ATP synthase in
P. modestum. Sodium ions enter via a channel in the interface between
the a subunit and the c ring, and bind to c protomers near to the
cytoplasmic surface where they are released. (c) A model based on
pH-induced structural changes observed by NMR of the c protomer in
organic solvents [61••]. Deprotonation of Asp61 and release of the
proton triggers a 140° rotation of the c protomer C-terminal (outer)
helix and concomitant movement of the c ring. The observed direction
of rotation in Figure 3 is counterclockwise, as viewed from the
membrane towards F1, and driven by ATP hydrolysis. In Figure 4, the
direction of rotation during ATP synthesis is counterclockwise, as
viewed from F1 towards the membrane.
F1 and Fo domains, which may help to facilitate rotation by
avoiding the deeper energy minima that would accompany
matching symmetries. Symmetry mismatch has been discussed in relation to other macromolecular assemblies that
contain rotating elements [40–43].
Direct observation of rotation
The number of c subunits in the c ring
Based on metabolic labelling and mechanistic models of
the generation of rotation, the notion has grown up that
E. coli Fo contains 12 c subunits arranged in a ring and, by
implication, that mitochondrial and chloroplast Fo domains
also contain 12 c subunits similarly arranged. Cross-linking
experiments and genetic fusions [44,45] have been interpreted as supporting this view. This notion has been
challenged by the F1–c10 structure (above) [36••] and by
the observation of 14-fold symmetry in rings of c subunits
from spinach chloroplasts [46••]. At the present time, the
possibility that subunits were lost from the S. cerevisiae
c ring during crystallisation cannot be excluded, unlikely
as this proposal seems. However, there are now clear indications that the c-ring symmetry may differ among species.
The c-ring symmetry may also vary within a single species
according to physiological conditions [47]. If the concept of
symmetry mismatch is an important general feature of
ATP synthases, it would argue against c-ring stoichiometries divisible by three. It also implies that the number of
protons that transverse the membrane for each ATP synthesised is nonintegral, possibly between three and four in
mitochondria. As the generation of each ATP requires a
120° rotation of the central stalk, an elastic element, possibly in the γ subunit, may be needed to store energy and
release it in quanta, as required by a stepping motor
mechanism [48•,49•] (see below).
By attachment of fluorescent actin filaments to either the
γ or ε subunit, rotation of the central stalk driven by ATP
hydrolysis has been observed by microscopy of tethered
α3β3γ [5,50•] complexes and of F1 itself [51,52•,53•] (see
Figure 3a,b). The main characteristics of this rotation are
that it is highly efficient in energy usage, that it proceeds
in 120° steps [54] and that the rotation is counterclockwise
as viewed from the tip of the central stalk protrusion.
Attempts have also been made to observe the rotation in
F1Fo-ATPase preparations by attaching actin filaments to
the c ring on the surface distal from F1 [55••,56••]
(Figure 3c). Although technical objections have been
voiced concerning these experiments [57•], they can be
reasonably interpreted as showing that the F1–c ring rotates
as an ensemble in response to ATP hydrolysis in F1.
However, because the detergents used to isolate the complex destabilise interactions of the c ring with the a subunit,
these experiments should not be taken as definitive proof
of the rotation of the F1–c ring in an intact F1Fo complex
that is capable of synthesising, as well as hydrolysing, ATP.
Definitive proof may require rotation to be observed under
conditions in which ATP is being synthesised.
Generation of torque
A hypothetical model of how rotation might be generated
was developed by Junge et al. [58], based upon models of
bacterial flagellar rotation (see [3,49•,59] for a detailed
description and further discussion of this model)
(Figure 4a). A related model has been described to explain
the generation of rotation by the Na+-motive F1Fo-ATPase
from the bacterium Propionigenium modestum [60•]
The rotary mechanism of ATP synthase Stock et al.
(Figure 4b). In this model, the carboxyl sidechains of the
essential residue Glu65 in subunit c are negatively charged
when they enter the interface between the c ring and subunit a. The positive charge of Arg227 in subunit a attracts
the negative charge of the essential carboxylate in subunit
c and also prevents ion leakage. Once this carboxylate has
been neutralised by a Na+ ion from the periplasm, it will
move by thermal vibrations, bringing the next negatively
charged carboxylate into the channel. Electrostatic forces
strongly bias the rotation, making it effectively unidirectional. As in the Junge model, the central stalk is attached
to the c ring, which drives its rotation directly.
A radically different model for the generation of rotation of
the central stalk has been advanced on the basis of NMR
studies in organic solvents of the c protomer from E. coli, in
which reduction in pH and protonation of Asp61 cause the
C-terminal α helix to rotate by 140° about its helix axis. It
is proposed that this rotation either drives the rotation of
the c ring (Figure 4c) or, alternatively, generates rotation of
the central stalk without the c ring itself turning [61••].
Conclusions
The rather extensive current knowledge of how ATP synthase works is based largely upon accurate and novel
structures of subcomplexes of the enzyme [4,36••,62•,63••];
striking progress had been made using this approach in the
past six years. However, current models for explaining the
generation of rotation in Fo are tentative and require further experimental validation. It is unlikely that the
mechanism of rotation in ATP synthase will be understood
fully until accurate molecular models of the entire enzyme
complex in different conformational states have been
established. Determination of these structures requires
either the crystallisation of the intact ATP synthase complex or the establishment of an accurate low-resolution
model by EM of single complexes, which can then be used
as a framework for building a molecular model from
structures of subcomplexes and individual subunits.
References and recommended reading
Papers 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 the
FoF1 ATP synthase. Annu Rev Biophys Biomol Struct 1999,
28:205-234.
4.
Abrahams JP, Leslie AGW, Lutter R, Walker JE: Structure at 2.8 Å
resolution of F1-ATPase from bovine heart mitochondria. Nature
1994, 370:621-628.
677
crystallography. Proc Natl Acad Sci USA 1999,
96:13697-13702.
7.
Uhlin U, Cox GB, Guss JM: Crystal structure of the epsilon subunit
of 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 Escherichia
coli ATP synthase — tyrosine 205 of the gamma-subunit is in the
interface between the F1 and Fo parts and can interact with both
the epsilon and c oligomer. J Biol Chem 1996, 271:28341-28347.
10. Tang CL, Capaldi RA: Characterization of the interface between
gamma and epsilon subunits of Escherichia coli F1-ATPase. J Biol
Chem 1996, 271:3018-3024.
11. Wilkens S, Capaldi RA: Solution structure of the epsilon subunit of
the F1-ATPase from Escherichia coli and interactions of this
subunit with beta subunits in the complex. J Biol Chem 1998,
273:26645-26651.
12. Aggeler R, Haughton MA, Capaldi RA: Disulfide bond formation
between the COOH-terminal domain of the beta subunits and the
gamma and epsilon subunits of the Escherichia coli F1-ATPase.
Structural implications and functional consequences. J Biol Chem
1995, 270:9185-9191.
13. Aggeler R, Weinreich F, Capaldi RA: Arrangement of the epsilon
subunit in the Escherichia coli ATP synthase from the reactivity of
cysteine 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 into
focus. 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. coli
ATP synthase.
16. Bottcher B, Schwarz L, Graber P: Direct indication for the existence
of 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 extensively
with 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 synthase
subunit 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 heart
mitochondria — in vitro assembly of a stalk complex in the
presence 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 second
stalk of the yeast ATP synthase complex: identification of
subunits showing cross-links with known positions of subunit 4
(subunit b). Biochemistry 1999, 38:15017-15024.
5.
Noji H, Yasuda R, Yoshida M, Kinosita K: Direct observation of the
rotation of F1-ATPase. Nature 1997, 386:299-302.
22. Velours J, Paumard P, Soubannier V, Spannagel C, Vaillier J, Arselin G,
Graves PV: Organisation of the yeast ATP synthase Fo: a study
based on cysteine mutants, thiol modification and cross-linking
reagents. Biochim Biophys Acta 2000, 1458:443-456.
6.
Hausrath AC, Gruber G, Matthews BW, Capaldi RA: Structural
features of the gamma subunit of the Escherichia coli F1ATPase revealed by a 4.4 Å resolution map obtained by X-ray
23. Sorgen PL, Caviston TL, Perry RC, Cain BD: Deletions in the
second stalk of F1Fo-ATP synthase in Escherichia coli. J Biol
Chem 1998, 273:27873-27878.
678
Proteins
24. Sorgen PL, Bubb MR, Cain BD: Lengthening the second stalk of
F1Fo-ATP synthase in Escherichia coli. J Biol Chem 1999,
274:36261-36266.
41. Valpuesta JM, Fernandez JJ, Carazo JM, Carrascosa JL: The threedimensional structure of a DNA translocating machine at 10 Å
resolution. Structure 1999, 7:289-296.
25. Ogilvie I, Aggeler R, Capaldi RA: Cross-linking of the delta subunit
to one of the three alpha subunits has no effect on functioning, as
expected if delta is a part of the stator that links the F1 and Fo
parts of the Escherichia coli ATP synthase. J Biol Chem 1997,
272:16652-16656.
42. Thomas DR, Morgan DG, DeRosier DJ: Rotational symmetry of the
c ring and a mechanism for the flagellar rotary motor. Proc Natl
Acad Sci USA 1999, 96:10134-10139.
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 of
the F1. J Mol Biol 2000, 295:387-391.
Confirmation of the position of the N-terminal domain of the δ subunit at
the top of F1.
27.
Wilkens S, Dunn SD, Chandler J, Dahlquist FW, Capaldi RA: Solution
structure of the N-terminal domain of the delta subunit of the
E. coli ATP synthase. Nat Struct Biol 1997, 4:198-201.
28. Jiang WP, Fillingame RH: Interacting helical faces of subunits a
and c in the F1Fo-ATP synthase of Escherichia coli defined by
disulfide 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 of
subunit a of the Escherichia coli ATP synthase and its role in
proton translocation. Biochim Biophys Acta 2000, 1458:457-466.
30. Valiyaveetil FI, Fillingame RH: Transmembrane topography of
subunit a in the Escherichia coli F1Fo-ATP synthase. J Biol Chem
1998, 273:16241-16247.
31. Wada T, Long JC, Zhang D, Vik SB: A novel labeling approach
supports the five-transmembrane model of subunit a of the
Escherichia 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 subunit
c 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 ATP
synthase from bovine heart mitochondria: purification, subunit
composition, and reconstitution with F1-ATPase. Biochemistry
1994, 33:7971-7978.
34. Arnold I, Bauer MF, Brunner M, Neupert W, Stuart RA: Yeast
mitochondrial F1Fo-ATPase: the novel subunit e is identical to
Tim11. FEBS Lett 1997, 411:195-200.
35. Arnold I, Pfeiffer K, Neupert W, Stuart RA, Schagger H: ATP synthase
of yeast mitochondria. Isolation of subunit j and disruption of the
ATP18 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 the
first insight into the arrangement of the c ring and its interactions with stalk
subunits γ, δ and ε. The close contact between these subunits and the c ring
supports the idea that the γ, δ and ε subunits and the c ring rotate as an
ensemble. The unexpected finding of 10 subunit c protomers in the ring has
profound implications for the mechanism of coupling and for the number of
protons 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 F 1 and Fo parts
in the Escherichia coli ATP synthase. Associations involving
the 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 ring
does not impair enzyme function, that of gamma to c subunits
leads to uncoupling. J Biol Chem 1999, 274:34233-34237.
The authors infer that the ε subunit and c ring rotate as an ensemble during
catalysis and that conformational changes in the γ subunit might occur.
40. Hendrix RW: Bacteriophage DNA packaging: RNA gears in a DNA
transport machine. Cell 1998, 94:147-150.
43. Beuron F, Maurizi MR, Belnap DM, Kocsis E, Booy FP, Kessel M,
Steven AC: At sixes and sevens: characterization of the symmetry
mismatch of the ClpAP chaperone-assisted protease. J Struct Biol
1998, 123:248-259.
44. Jones PC, Fillingame RH: Genetic fusions of subunit c in the Fo
sector of H+-transporting ATP synthase. Functional dimers and
trimers and determination of stoichiometry by cross-linking
analysis. J Biol Chem 1998, 273:29701-29705.
45. Jones PC, Jiang WP, Fillingame RH: Arrangement of the multicopy
H+-translocating subunit c in the membrane sector of the
Escherichia 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 ATP
synthase show 14 protomers in the ring. Therefore, the number of c subunits
in ATP synthases may differ from species to species.
47.
Schemidt RA, Qu J, Williams JR, Brusilow WSA: Effects of carbon
source on expression of Fo genes and on the stoichiometry of the
c 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 an
elastic 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 F 1 and Fo sectors. The solutions of the resulting equations reproduce many of the
empirical 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 ATP
hydrolysis, as first observed in the bacterial enzyme, also occurs in the
chloroplast enzyme.
51. Kato-Yamada Y, Noji H, Yasuda R, Kinosita K, Yoshida M: Direct
observation of the rotation of epsilon subunit in F1-ATPase. J Biol
Chem 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 mutant
Escherichia 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 no
effect on torque generation.
54. Yasuda R, Noji H, Kinosita K, Yoshida M: F1-ATPase is a highly
efficient 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. Science
1999, 286:1722-1724.
This paper presents the first direct evidence for rotation of the c ring in an
ATP synthase complex. Objections have been raised concerning the interpretation of these experiments [57•]. The major remaining concern is
whether 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 observation of the rotation of the c ring. The specificity of the attachment of the
The rotary mechanism of ATP synthase Stock et al.
679
57.
•
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 protomer
that accompany deprotonation of essential residue Asp61, as determined
by NMR in organic solvents. A novel model is proposed for the rotation of
subunit c and for its interactions with subunit a.
58. Junge W, Lill H, Engelbrecht E: ATP synthase: an electrochemical
transducer with rotary mechanics. Trends Biol Sci 1997,
22:420-423.
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.
59. Elston T, Wang HY, Oster G: Energy transduction in ATP synthase.
Nature 1998, 391:510-513.
Now published
actin filament to subunit c was ensured by the introduction of a ‘strep-tag’
sequence in the C-terminal region of subunit c.
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 Lett
2000, 470:244-248.
A critique of experiments directly demonstrating rotation in ATP synthase.
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 sodiumdependent ATP synthase involving a single channel, rather than two half
channels, as proposed for the proton-dependent ATP synthase.
The 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 determined
in the context of an intact F1-ATPase assembly.