ATP Synthase: Two rotary molecular motors
working together
George Oster
Hongyun Wang
University of California, Berkeley
Encyclopedia of Molecular Biology
Thomas E. Creighton
ISBN: 0-471-15302-8
Encyclopedia of Molecular Biology, 4 Volume Set
Thomas E. Creighton
ISBN: 0-471-15302-8
ATP Synthase: Two rotary molecular motors
working together
George Oster
Hongyun Wang
University of California, Berkeley
Introduction
ATP synthase—also called Fo F1 ATPase, or simply F-ATPase—is the universal protein that
terminates oxidative phosphorylation by synthesizing ATP from ADP and phosphate. Nearly
identical proteins are found in eukaryotic mitochondria and bacteria, and they all operate on the
same principle. Electron driven ion pumps set up concentration and electrical gradients across a
membrane. ATP synthase utilizes the energy stored in this electrochemical gradient to drive
nucleotide synthesis. It does this in a surprising way: by converting the electromotive force into a
rotary torque that is used to promote substrate binding and to liberate ATP from the catalytic site
where it was formed. Remarkably, this process can be reversed in certain circumstances: ATP
hydrolysis can drive the engine backwards so that F-ATPase functions as a proton pump. Indeed,
the vacuolar V-ATPases—the most ubiquitous intracellular proton pumps—are structurally similar
to ATP synthase and appear to operate according to the same principles.
ATP synthase is composed of at least 8 subunit types, whose stochiometry is denoted with
subscripts: (a3 , b3 , g, d, e, a, b2 , c10-14), that combine into two distinct regions. The geometric
arrangement of the subunits is shown schematically in Figure 1. The F1 portion is soluble and
consists of a hexamer, denoted a3 b 3 . This hexamer is arranged in an annulus about a central shaft
consisting of the coiled-coil g subunit. Subunits d and e generally isolate with F1 as well. The Fo
portion consists of three transmembrane subunits: a, b2 and c10-14. 10-14 copies of the c-subunit,
depending on the species, form a disk into which the g and e subunits insert. The remainder of Fo
consists of the transmembrane subunits a, and b2 ; the latter is attached by the d subunit to the a3 b 3
hexamer so that it anchors the a subunit to F1 . Thus there are two ‘stalks’ connecting Fo to F1 : ge
and b2 d.
The key to understanding how ATP synthase carries out its catalytic and synthetic roles lies in this
geometric organization. The entire protein can be divided into two operational regions denoted
suggestively as the ‘rotor’ and ‘stator’ for reasons that derive from the rotary mechanism by which
the protein operates. The rotor consists of gec10-14; the stator consists of a3 b 3 db2 a. Indeed, it turns
out that ATP synthase is two rotary engines in one. The Fo motor converts transmembrane
electrochemical energy into torque on the g shaft, and F1 uses ATP hydrolysis to turn the g shaft in
the opposite direction. Since they are connected, one drives the other in reverse: when Fo dominates,
the rotor turns clockwise (looking from the membrane, i.e. upwards in Figure 1) so that F1
synthesizes ATP. When F1 dominates, Fo is driven counterclockwise, whereupon it can pump
protons against an electrochemical gradient. Deciphering how this remarkable dual energy
transduction works is one of the great triumphs of modern chemistry.
Figure 1.
ATP synthesis in F1 is driven by the rotation of the g-shaft
We begin with the F1 motor because we now know precisely what it looks like. This is due to John
Walker and his x-ray crystallography group at Cambridge, who worked out the structure of the
a 3 b 3 g assembly [1]. Figure 2A shows the view from the membrane (looking upwards in Figure 1)
of a3 b 3 g. There are 6 nucleotide binding sites on the a3 b 3 hexamer, all lying at the interfaces
between the a and b subunits. The three catalytic sites alternate with three non-catalytic sites. The
catalytic sites lie mostly in the b subunit, while the noncatalytic sites lie mostly in the a subunit.
The role of the noncatalytic sites is uncertain, but may help hold the hexamer together. Figure 2B
shows a side view of one a and one b and g. Walker‘s structure revealed essential asymmetries in
the molecule’s structure that were the key to understanding its mechanism.
In the early 1980’s, Paul Boyer at UCLA, proposed the surprising theory that, in the catalytic sites
of F1 , ATP was in chemical equilibrium with its reactants, ADP and phosphate [2]. So the
formation of ATP in the catalytic sites was essentially without energetic cost. However, since each
ATP, when hydrolyzed under cellular conditions, liberates about 12 kcal/mol, this energetic price
must be paid at some point. Boyer proposed that F1 pays this price in the mechanical work
necessary to liberate the nucleotide from the catalytic site. Further, ATP release proceeds
sequentially and cyclically around the a3 b 3 hexamer because the synthetic reactions are
synchronized via the rotation of the g shaft and cooperative coupling between the three catalytic
2
sites. Boyer’s ‘binding change’ mechanism neatly fits the Walker structure. The way in which this
works is found in the shape of the a3 b 3 hexamer and the g shaft.
Figure 2.
The g subunit is asymmetric and bowed. It fits into a central annulus in the a3 b 3 which is itself
asymmetric (Figure 2B). At the top of the a3 b 3 hexamer is a hydrophobic ‘sleeve’ in which the g
shaft rotates. Further down, however, the annulus is offset from the center, so that as g rotates, it
sequentially pushes outwards on each catalytic site. Figure 3 shows the relation between the g
rotation and the conformational change of b and catalytic site. In the hydrolysis direction, bending
of the lower part of b toward the upper part drives the counterclockwise rotation of the g (looking
from the membrane, upwards in Figure 3): panels A Æ B Æ C in Figure 3. In the synthesis
direction, clockwise rotation of the g forces the opening of the b and the catalytic site, reducing the
ATP binding affinity: panels C Æ B Æ A in Figure 3.
The catalytic sites do not act independently; rather they are synchronized so that each site traverses
the synthetic cycle in a more or less fixed phase with respect to the others. This synchronization is
orchestrated in two ways. As the g shaft rotates, it not only stresses each catalytic site, but it also
interacts electrostatically with the b subunits at two locations [3]. These interactions may mediate
phosphate and nucleotide binding, the necessary precursors to synthesis. In addition, the catalytic
sites appear to be elastically coupled so that the occupancy of one site affects the other two sites.
The consequence of this coupling is that, when ATP concentrations are low enough so that only one
site is occupied, hydrolysis proceeds much more slowly than when more than one site is occupied.
Together with the F1 molecular structure, the binding change model strongly supported the idea that
catalysis involved rotation of the g subunit. However, dramatic visual confirmation was provided by
in vitro experiments in which the a3 b 3 g subunits were isolated and attached to a bead. A
florescently tagged actin filament was attached to the g shaft and, when ATP was supplied, the
filament could clearly be seen to rotate. In fact, a complete revolution took place in 3 steps, and
consumed a single ATP per step [4]. Recently, more detailed experiments indicated that each 120˚
rotation appears to take place in two steps: an initial 90˚ rotation that depends on the solution
concentration of ATP, followed by a 30˚ rotation that is ATP independent [5]. The initial rotation is
associated with ATP binding, and the second rotation with product release.
3
The viscous drag on the actin filament could be estimated, which allowed the torque developed by
the F1 motor to be computed and compared with the free energy available from ATP hydrolysis.
The startling result was that the motor generated an average torque of more than 40 piconewton
-12
nanometers (40¥10
-9
N ¥ 10 m), more than six times the maximum force developed by kinesin
or myosin. More impressively, the motor operated near 100% mechanical efficiency; this
precludes any sort of heat engine that would be limited by the Carnot efficiency [6]. Several
models have been proposed that address the issue of torque generation and efficiency (see Box 1)
[6-9].
The energy to drive rotation derives from the hydrolysis cycle of ATP at the catalytic site.
Moreover, the conformational change that drives the hydrolysis motor must be nearly the reverse of
the motion that frees ATP from the catalytic site during synthesis. Examination of Walker’s
structure reveals that the major conformational change is a hinge-bending motion of about 30˚ in the
b subunits. The lower part of each b below the nucleotide binding site rotates inward and upwards
during which it pushes on the bowed g subunit, turning it much like one cranks an automobile jack
(Figure 3) [8].
Figure 3
4
Box 1: How is torque generated?
The above description of the F1 motor begs the basic question of how the chemical energy liberated
in the hydrolysis cycle is converted into a mechanical torque. One solution (the ‘binding zipper’
model) proposes that, after an ATP diffuses into the catalytic site, the torque is generated during the
transition from weak to tight binding of the ATP onto the catalytic site [10] [9]. Recent work
supports this model [5, 11]. This accords with the observation that the power stroke of GroEL
accompanies ATP binding [12]. Figure 4 illustrates the ‘binding zipper’ model. What remains
unsettled is the precise role of the hydrolysis step. It appears that both ATP binding at one catalytic
site and hydrolysis at a second catalytic site are required to complete a 120˚ power stroke [11].
Moreover, the power stroke may proceed in two steps: a 90˚ step associated with ATP binding, and
a 30˚ step associated with hydrolysis or product release [5], What is not clear is whether the
hydrolysis step actually contributes to torque generation, or is simply permissive for the completion
of the binding transition of ATP at another site. The binding zipper model posits that the
electrostatic repulsion of the hydrolysis products is used not for torque generation, but to loosen the
tight binding of ATP to the catalytic site so that they can be released to repeat the power cycle.
There may be an elastic ‘recoil’ that contributes to the power stroke as the b subunit relaxes back to
its open conformation [10] [9].
Figure 4
Fo converts protonmotive force into rotary torque
There is currently no direct observation of rotation in the Fo portion of ATP synthase [13].
However, current thinking is that the Fo assembly converts the energy contained in the
transmembrane protonmotive force into a rotary torque at the interface between the a and the c
subunits (Figure 1). This torque turns the rotor (the c subunits, g and e subunits) which couples to
the F1 synthetic machine.
5
The c assembly consists of 10-14 subunits, each consisting of two transmembrane a-helices [14]
[15] [16]. There is one essential acidic amino acid (Asp61 in the E. Coli ATP synthase) which
binds protons. Since there are variants of ATP synthase that can operate on sodium rather than
protons, the interaction between the c subunit and the translocated ion has the property of an
electrostatic carrier mechanism [17].
The a subunit consists of 6 transmembrane a-helices which contain at least one essential basic
residue (Arg210 in E. Coli) [16, 18]. The interaction of these rotor and stator charges is essential
for torque generation, and several proposals have been put forward for how this could work [19]
[20] [21] [22] [23, 24]. Whatever the mechanism, the Fo motor must generate a torque sufficient to
liberate 3 ATP’s from the three catalytic sites in F1 for each revolution.
Under anaerobic conditions, the ATP synthase of the bacteria E. Coli can reverse its operation,
hydrolyzing ATP and turning the c subunits backwards so that it functions as a proton pump. This
is not surprising, since the F-ATPases are structurally similar to the most common proton pumps,
the vacuolar, or V-ATPases [13]. These pumps may have been the evolutionary precursors of ATP
synthase [25]. A striking difference between the two is that the F-ATPases have 10-14 acidic rotor
charges, whereas the V-ATPases have ~6. It can be shown that this enables the V-ATPases to
function more efficiently as ion pumps, at the expense of relinquishing their capability to synthesize
ATP [26].
Summary
Both the F1 and Fo motors can operate in both directions. F1 is a hydrolysis-driven 3-cylinder
engine which can be driven in reverse to synthesize ATP from ADP and phosphate. Fo is an iondriven rotary engine which can be driven in reverse to function as an ion pump. The F-ATPases are
structurally similar to, and presumably evolutionarily related to, the V-ATPase ion pumps [25].
Most ion pumps are thought to function by an ‘alternating access’ mechanism whereby an ion is
first bound strongly on the dilute side, then energy is supplied to move the ion such that it
communicates with the concentrated side and to weaken its binding affinity [27]. However, in
contrast with other ion pumps, the F and V-ATPases accomplish this by a rotary mechanism that is
driven indirectly by nucleotide hydrolysis, rather than by direct phosphorylation [28]. The Fo
motor is thought to be related also to the bacterial flagellar motor. Both can operate on sodium,
although the flagellar motor has 8 or more ‘stators’ and develops far more torque than Fo [29] [30].
6
The mechanism driving the F1 hydrolysis motor may carry hints for other nucleotide hydrolysis
fueled motors, such as kinesin, myosin and dynein. However, there are important structural
differences that may make the comparison difficult [6]. For example, the above mentioned motors
all ‘walk’ along a polymer track to which they bind tightly during a portion of their
mechanochemical cycle. The power stroke of the F1 motor is driven by the b subunit which pushes
on the g shaft, but does not appear to bind tightly to it; that is, it does not ‘walk’ around the g shaft.
Moreover, no other motor operates with nearly the efficiency as the F1 motor, implying that there
are important entropic steps in other motors that are absent in the F1 motor.
7
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Boyer, P., The binding change mechanism for ATP synthase--some
probabilities and possibilities. Biochim. Biophys. Acta, 1993. 1140: p. 215-250.
Al-Shawi, M., C. Ketchum, and R. Nakamoto, Energy coupling, turnover, and
stability of the F0F1 ATP synthase are dependent on the energy of interaction
between g and b subunits. J. Biol. Chem., 1997. 272(4): p. 2300-2306.
Yasuda, R., et al., F1-ATPase is a highly efficient molecular motor that rotates
with discrete 120° steps. Cell, 1998. 93: p. 1117-1124.
Yasuda, R., et al., Resolution of distinct rotational substeps by submillisecond
kinetic analysis of F1-ATPase. Nature, 2001. 410: p. 898-904.
Kinosita, K., et al., F1-ATPase: A rotary motor made of a single molecule. Cell,
1998. 93: p. 21-24.
Oosawa, F. and S. Hayashi, The loose coupling mechanism in molecular
machines of living cells. Adv Biophys, 1986. 22: p. 151-183.
Wang, H. and G. Oster, Energy transduction in the F1 motor of ATP synthase.
Nature, 1998. 396: p. 279-282.
Oster, G. and H. Wang, Why is the mechanical efficiency of F1-ATPase so high?
Journal of Bioenergetics and Biomembranes, 2000. 32(5): p. 459-69.
Oster, G. and H. Wang, Reverse engineering a protein: The mechanochemistry
of ATP synthase. Biochimica et Biophysica Acta (Bioenergetics), 2000. 1458(12): p. 482-510.
Menz, R., J. Walker, and A. Leslie, Structure of Bovine Mitochondrial F1-ATPase
with Nucleotide Bound to All Three Catalytic Sites: Implications for the
Mechanism of Rotary Catalysis. Cell, 2001. 106: p. 331-341.
Bukau, B. and A. Horwich, The Hsp70 and Hsp60 chaperone machines. Cell,
1998. 92(3): p. 351-366.
Finbow, M. and M. Harrison, The vacuolar H+-ATPase: a universal proton pump
of eukaryotes. Biochem. J., 1997. 324: p. 697-712.
Stock, D., A. Leslie, and J. Walker, Molecular architecture of the rotary motor in
ATP synthase. Science, 1999. 286: p. 1700-1705.
Seelert, H., et al., Proton-powered turbine of a plant motor. Nature, 2000. 405: p.
418-419.
Fillingame, R.H., Coupling H+ transport and ATP synthesis in F1F0-ATP
synthases: glimpses of interacting parts in a dynamic molecular machine. J.
Exp. Biol., 1997. 200(Pt 2): p. 217-224.
Dimroth, P., Primary Sodium Ion Translocating Enzymes. Biochim. Biophys.
Acta, 1997. 1318(1-2): p. 11-51.
Fillingame, R.H., Membrane sectors of F- and V-type H+-transporting ATPases.
Current Opinion in Structural Biology, 1996. 6(4): p. 491-8.
Elston, T., H. Wang, and G. Oster, Energy transduction in ATP synthase. Nature,
1998. 391: p. 510-514.
8
20.
Dimroth, P., et al., Energy transduction in the sodium F-ATPase of
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Junge, W., H. Lill, and S. Engelbrecht, ATP Synthase: An Electro-Chemical
Transducer with Rotatory Mechanics. Trends Biochem. Sci., 1997. 22(11): p.
420-423.
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Vik, S.B. and B.J. Antonio, A mechanism of proton translocation by F1F0 ATP
synthases suggested by double mutants of the a subunit. J. Biol. Chem., 1994.
269: p. 30364-30369.
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Fillingame, R.H., W. Jiang, and O.Y. Dmitriev, Coupling H+ transport to rotary
catalysis in F-type ATP synthases: structure and organization of the
transmembrane rotary motor. J. Exp. Biol., 2000. 203(1): p. 9-17.
24.
Rastogi, V. and M. Girvin, Structural changes linked to proton translocation by
subunit c of the ATP synthase. Nature, 1999. 402: p. 263-268.
25.
Cross, R. and L. Taiz, Gene duplication as a means for altering H+/ ATP ratios
during the evolution of F0F1 ATPases and synthases. FEBS Lett., 1990. 259(2):
p. 227-229.
26.
Grabe, M., H. Wang, and G. Oster, The mechanochemistry of the V-ATPase
proton pumps. Biophys. J., 2000. 78(6): p. 2798-281.
27.
Alberts, B., et al., Molecular Biology of the Cell. 3d ed. 1994, New York: Garland.
28.
Khan, S., Rotary chemiosmotic machines. Biochimica et Biophysica Acta, 1997.
1322(2-3): p. 86-105.
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Berg, H., Torque generation by the flagella rotary motor. Biophys. J., 1995. 68(4
Suppl): p. 163s-166s.
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Muramoto, K., et al., High-speed rotation and speed stability of the sodiumdriven flagellar motor in Vibrio alginolyticus. J. Mol. Biol., 1995. 251: p. 50-58.
31.
Pedersen, P.L., Y.H. Ko, and S. Hong, ATP Synthases in the Year 2000:
Evolving Views about the
Structures of These Remarkable Enzyme Complexes. Journal of Bioenergetics and
Biomembranes, 2000. 32(4): p. 325-32.
32.
Uhlin, U., G.B. Cox, and J.M. Guss, Crystal structure of the epsilon subunit of
the proton-translocating ATP synthase from Escherichia coli. Structure, 1997.
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33.
Dmitriev, O., et al., Structure of the membrane domain of subunit b of the
Escherichia coli F0F1 ATP synthase. Journal of Biological Chemistry, 1999.
274(22): p. 15598-604.
34.
Bianchet, M., et al., The 2.8-Å structure of rat liver F1-ATPase: Configuration of a
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Supplementary Reading
1.
J. Weber, A. Senior, Biochim. Biophys. Acta 1319, 19-58 (1997).
2.
M. Yoshida, E. Muneyuki, T. Hisabori, Nature Reviews, 1-21 (2001).
9
Figure Captions
Figure 1. Structural models proposed for ATP synthases constructed by Pedersen, Ko and Hong
[31], based on the crystal structure for a3 b 3 gc1 0 (1qo1) [14] and other crystal structures for
various subunits (1aqt, 1b9u, 1bmf, 1mab, 1c17) [32] [33] [1] [34] [24]. The c assembly consists of
10-14 c subunits [14] [15]. The g subunit attaches to the c subunits. The a subunit is attached to
the a3 b 3 hexamer via the b2 and d subunits. Torque is generated by the protonmotive force at the
interface of the a and c subunits. This leads to the functional subdivision into two counter-rotating
assemblies, usually denoted as the ‘rotor’ and ‘stator’. The rotor consists of subunits gec10-14 and
the stator consists of subunits a3 b 3 db2 a.
Figure 2. (A) The view from the membrane (looking upwards in Figure 1) of a3 b 3 g. (B) A side
view of only one a, one b and the g. The a subunit is in yellow, the two coils of the g subunit are
blue and purple. The b subunit is in two colors: the stationary upper barrel segment is in green, and
the lower hinge segment is red. Three catalytic sites are indicated by arrows. In the Walker
structure, the three catalytic sites have different occupancies: one is empty (bE); another one is
occupied by an ADP (bD) and the third one is occupied by an AMPPNP (bT).
Figure 3. Side views of one a, one b and the g. Three panels show the relation between the rotation
of the g and the bending of the b. (A) The b is open. (B) The b is bent halfway upwards and the g
is turned counterclockwise (looking upwards). (C) The b is bent more and the g is further turned in
the counterclockwise direction. The color scheme used here is the same as that in Figure 2. Abut
the catalytic site, there are two a helices: Helix B and Helix C. Helix B is associated with the lower
part of the b; Helix C is associated with the upper part. In the hydrolysis direction, the ATP
binding transition drives Helix B towards Helix C, which drives the bending of b. The bending
motion of b pushes on the eccentric g coiled-coil causing it to rotate within the barrel bearing
(panels A Æ B Æ C). In the synthesis direction, the torque from Fo drives the clockwise (looking
upwards) rotation of the g. The rotation of the eccentric g pushes on the lower part of b and forces
Helix B to move away from Helix C, which reduces the ATP binding affinity (panels C Æ B Æ
A). Movies of the rotational sequence can be downloaded from the authors’ web site.
Figure 4. The binding zipper model. The hydrolysis cycle proceeds clockwise around the diagram,
synthesis is counterclockwise. In the hydrolysis direction, an ATP in solution first diffuses to the
10
catalytic site and is weakly bound (ATP docking). The rate of this step is affected by the ATP
concentration in solution. The weakly bound ATP may dissociate from the catalytic site, returning
to the solution. Occasionally, it proceeds from weak binding to tight binding (the binding
transition). During the binding transition, the bonds between the ATP and the catalytic site form
sequentially, and the binding affinity increases gradually. In this way the conformational change of
the catalytic site is coupled to the binding affinity. Thus, during the multi-step ATP binding
transition, the binding free energy is used efficiently to generate a nearly constant force to rotate the
g shaft. ATP concentration in solution does not affect the binding transition, but only how often
ATP attempts docking to the catalytic site. After the binding transition, the ATP is in chemical
equilibrium with ADP and Pi . The transition ATP ‡ ADP + Pi weakens the ATP binding and
distributes it over ADP and Pi so that the hydrolysis products can be released and the cycle
repeated. Release may require the assistance of nucleotide binding to an adjacent catalytic site. The
rotation of g concurrent with ADP and Pi release is driven by the ATP binding transition at the
other two catalytic sites (not shown in the figure). In the binding zipper model, the rotation of the g
shaft is continuous and the ATP binding affinity is continuously coupled to the g rotation [10] [9].
The synthesis direction is counterclockwise around the diagram. A catalytic site first binds ADP
and phosphate (not necessarily in that order). While trapped in the catalytic site, reactants (ADP and
Pi ) and product (ATP) are in chemical equilibrium with equilibrium constant ~ 1. ATP is formed at
the catalytic site from ADP and Pi with little energy cost. However, the ATP is tightly bound at the
catalytic site. The mechanical torque from Fo on g is used to reduce the ATP binding affinity. In
this step, the mechanical torque is converted to the binding free energy of ATP. Most of the 12
kcal/mol price of synthesis is paid in this step. When the binding affinity is low enough, the ATP is
dislodged from the catalytic site by thermal fluctuations. Molecular dynamics simulations confirm
this sequence of events.
11
δ
α3β3
F1
γ
b2
ε
c10-14
Fo
a
Membrane
Fig. 1
α
βE
α
βT
βD
α
Fig. 2A
α
γ
βT
Fig. 2B
α
β
γ
Helix C
Helix B
Fig. 3A
Helix C
Helix B
Fig. 3B
Helix C
Helix B
Fig. 3C
γ
β1
•
β3
β1
•
•
ATP
β1
γ
β2 β
3
β2
Multistep binding transition
drives rotation
ATP docking
β2
γ
ATP
γ
ADP+Pi
ADP
γ
ADP
release
ADP
β1
β2
γ
β3
Pi
γ
Phosphate
release
β3
Hydrolysis
Keq ~ 1
ATP
β1
β2
γ
β3
Fig. 4