ATP Synthase: Two rotary molecular motors working together

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2 ATP Synthase: Two rotary molecular motors working together Introduction George Oster Hongyun Wang University of California, Berkeley ATP synthase also called F o F 1 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: (a 3, b 3, g, d, e, a, b 2, c ), that combine into two distinct regions. The geometric arrangement of the subunits is shown schematically in Figure 1. The F 1 portion is soluble and consists of a hexamer, denoted a 3 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 F 1 as well. The F o portion consists of three transmembrane subunits: a, b 2 and c copies of the c-subunit, depending on the species, form a disk into which the g and e subunits insert. The remainder of F o consists of the transmembrane subunits a, and b 2 ; the latter is attached by the d subunit to the a 3 b 3 hexamer so that it anchors the a subunit to F 1. Thus there are two stalks connecting F o to F 1 : ge and b 2 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

3 suggestively as the rotor and stator for reasons that derive from the rotary mechanism by which the protein operates. The rotor consists of gec ; the stator consists of a 3 b 3 db 2 a. Indeed, it turns out that ATP synthase is two rotary engines in one. The F o motor converts transmembrane electrochemical energy into torque on the g shaft, and F 1 uses ATP hydrolysis to turn the g shaft in the opposite direction. Since they are connected, one drives the other in reverse: when F o dominates, the rotor turns clockwise (looking from the membrane, i.e. upwards in Figure 1) so that F 1 synthesizes ATP. When F 1 dominates, F o 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 F 1 is driven by the rotation of the g-shaft We begin with the F 1 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 a 3 b 3 g. There are 6 nucleotide binding sites on the a 3 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 F 1, 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 F 1 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 a 3 b 3 hexamer because the synthetic reactions are synchronized via the rotation of the g shaft and cooperative coupling between the three catalytic 2

4 sites. Boyer s binding change mechanism neatly fits the Walker structure. The way in which this works is found in the shape of the a 3 b 3 hexamer and the g shaft. Figure 2. The g subunit is asymmetric and bowed. It fits into a central annulus in the a 3 b 3 which is itself asymmetric (Figure 2B). At the top of the a 3 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 F 1 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 a 3 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

5 The viscous drag on the actin filament could be estimated, which allowed the torque developed by the F 1 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 nanometers ( N 10-9 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

6 Box 1: How is torque generated? The above description of the F 1 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 F o converts protonmotive force into rotary torque There is currently no direct observation of rotation in the F o portion of ATP synthase [13]. However, current thinking is that the F o 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 F 1 synthetic machine. 5

7 The c assembly consists of 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 F o motor must generate a torque sufficient to liberate 3 ATP s from the three catalytic sites in F 1 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 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 F 1 and F o motors can operate in both directions. F 1 is a hydrolysis-driven 3-cylinder engine which can be driven in reverse to synthesize ATP from ADP and phosphate. F o 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 F o 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 F o [29] [30]. 6

8 The mechanism driving the F 1 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 F 1 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 F 1 motor, implying that there are important entropic steps in other motors that are absent in the F 1 motor. 7

9 References 1. Abrahams, J., et al., Structure at 2.8Å resolution of F 1 -ATPase from bovine heart mitochondria. Nature, : p Boyer, P., The binding change mechanism for ATP synthase--some probabilities and possibilities. Biochim. Biophys. Acta, : p Al-Shawi, M., C. Ketchum, and R. Nakamoto, Energy coupling, turnover, and stability of the F 0 F 1 ATP synthase are dependent on the energy of interaction between g and b subunits. J. Biol. Chem., (4): p Yasuda, R., et al., F 1 -ATPase is a highly efficient molecular motor that rotates with discrete 120 steps. Cell, : p Yasuda, R., et al., Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase. Nature, : p Kinosita, K., et al., F 1 -ATPase: A rotary motor made of a single molecule. Cell, : p Oosawa, F. and S. Hayashi, The loose coupling mechanism in molecular machines of living cells. Adv Biophys, : p Wang, H. and G. Oster, Energy transduction in the F1 motor of ATP synthase. Nature, : p Oster, G. and H. Wang, Why is the mechanical efficiency of F1-ATPase so high? Journal of Bioenergetics and Biomembranes, (5): p Oster, G. and H. Wang, Reverse engineering a protein: The mechanochemistry of ATP synthase. Biochimica et Biophysica Acta (Bioenergetics), (1-2): p 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, : p Bukau, B. and A. Horwich, The Hsp70 and Hsp60 chaperone machines. Cell, (3): p Finbow, M. and M. Harrison, The vacuolar H + -ATPase: a universal proton pump of eukaryotes. Biochem. J., : p Stock, D., A. Leslie, and J. Walker, Molecular architecture of the rotary motor in ATP synthase. Science, : p Seelert, H., et al., Proton-powered turbine of a plant motor. Nature, : p 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., (Pt 2): p Dimroth, P., Primary Sodium Ion Translocating Enzymes. Biochim. Biophys. Acta, (1-2): p Fillingame, R.H., Membrane sectors of F- and V-type H+-transporting ATPases. Current Opinion in Structural Biology, (4): p Elston, T., H. Wang, and G. Oster, Energy transduction in ATP synthase. Nature, : p

10 20. Dimroth, P., et al., Energy transduction in the sodium F-ATPase of Propionigenium modestum. Proc. Natl. Acad. Sci. USA, (9): p Junge, W., H. Lill, and S. Engelbrecht, ATP Synthase: An Electro-Chemical Transducer with Rotatory Mechanics. Trends Biochem. Sci., (11): p 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., : p 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., (1): p Rastogi, V. and M. Girvin, Structural changes linked to proton translocation by subunit c of the ATP synthase. Nature, : p Cross, R. and L. Taiz, Gene duplication as a means for altering H + / ATP ratios during the evolution of F 0 F 1 ATPases and synthases. FEBS Lett., (2): p Grabe, M., H. Wang, and G. Oster, The mechanochemistry of the V-ATPase proton pumps. Biophys. J., (6): p 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, (2-3): p Berg, H., Torque generation by the flagella rotary motor. Biophys. J., (4 Suppl): p. 163s-166s. 30. Muramoto, K., et al., High-speed rotation and speed stability of the sodiumdriven flagellar motor in Vibrio alginolyticus. J. Mol. Biol., : p 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, (4): p 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, (9): p Dmitriev, O., et al., Structure of the membrane domain of subunit b of the Escherichia coli F0F1 ATP synthase. Journal of Biological Chemistry, (22): p Bianchet, M., et al., The 2.8-Å structure of rat liver F 1 -ATPase: Configuration of a critical intermediate in ATP synthesis/hydrolysis. Proc. Natl. Acad. Sci. USA, : p Supplementary Reading 1. J. Weber, A. Senior, Biochim. Biophys. Acta 1319, (1997). 2. M. Yoshida, E. Muneyuki, T. Hisabori, Nature Reviews, 1-21 (2001). 9

11 Figure Captions Figure 1. Structural models proposed for ATP synthases constructed by Pedersen, Ko and Hong [31], based on the crystal structure for a 3 b 3 gc 10 (1qo1) [14] and other crystal structures for various subunits (1aqt, 1b9u, 1bmf, 1mab, 1c17) [32] [33] [1] [34] [24]. The c assembly consists of c subunits [14] [15]. The g subunit attaches to the c subunits. The a subunit is attached to the a 3 b 3 hexamer via the b 2 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 gec and the stator consists of subunits a 3 b 3 db 2 a. Figure 2. (A) The view from the membrane (looking upwards in Figure 1) of a 3 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 (b E ); another one is occupied by an ADP (b D ) and the third one is occupied by an AMPPNP (b T ). 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 F o 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

12 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 P i. The transition ATP ADP + Pi weakens the ATP binding and distributes it over ADP and P i 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 P i ) 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 F o 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

13 δ F 1 α 3 β 3 b 2 ε F o a c Membrane

14 α βe α βt βd α Fig. 2A

15 α β T

16 α β Helix C Helix B

17 Helix C Helix B

18 Helix C Helix B

19 ATP β 1 β 2 β 3 Multistep binding transition drives rotation ATP β 1 β β 2 3 ATP ATP docking ADP+P i Hydrolysis Keq ~ 1 β 1 β 2 β 3 ADP ADP release ADP β 1 β β 3 2 P i Phosphate release β 1 β β 2 3