CROSS-LINKING AND ELECTRON MICROSCOPY STUDIES OF THE STRUCTURE AND FUNCTIONING OF THE ESCHERICHIA COLI ATP SYNTHASE
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
The Journal of Experimental Biology 203, 29–33 (2000) 29
Printed in Great Britain © The Company of Biologists Limited 2000
JEB2341
CROSS-LINKING AND ELECTRON MICROSCOPY STUDIES OF THE STRUCTURE
AND FUNCTIONING OF THE ESCHERICHIA COLI ATP SYNTHASE
RODERICK A. CAPALDI*, BIRTE SCHULENBERG, JAMES MURRAY AND ROBERT AGGELER
Institute of Molecular Biology, University of Oregon, Eugene, OR 97403-1229, USA
*e-mail: rcapaldi@oregon.uoregon.edu
Accepted 21 October; published on WWW 13 December 1999
Summary
ATP synthase, also called F1Fo-ATPase, catalyzes the The utility of these two approaches in particular, and the
synthesis of ATP during oxidative phosphorylation. The important insights they give into the structure and
enzyme is reversible and is able to use ATP to drive a mechanism of the ATP synthase, are reviewed.
proton gradient for transport purposes. Our work has
focused on the enzyme from Escherichia coli (ECF1Fo). We
have used a combination of methods to study this enzyme, Key words: F1Fo-type ATPase, ATP synthase, electron microscopy,
including electron microscopy and chemical cross-linking. cross-linking, rotation.
Introduction
An F1Fo-type ATP synthase is found in the inner artifact of the staining procedure until our cryoelectron
mitochondrial membrane, the inner membrane of bacteria and microscopy images confirmed its presence in buffered
the thylakoid membrane of chloroplasts, where it functions to solutions without heavy atoms added (Gogol et al., 1987;
convert the free energy of the proton-motive force into the Lücken et al., 1990). In these first cryoelectron microscopy
chemical energy source ATP (for reviews, see Boyer, 1993; pictures of ECF1Fo, F1 appeared to extend to Fo at one side in
Junge et al., 1997; Weber and Senior, 1997). This large enzyme some of the images, as though there were two linkages between
complex is composed of two major parts, a water-soluble F1 the two parts.
sector made up of three α subunits, three β subunits and one More recently, we have obtained electron micrographs
copy of each of the γ, δ and ε subunits, and a membrane- of ECF1Fo in a monodisperse, detergent-solubilized form
embedded Fo sector consisting of one a, two b and 12 c (Wilkens and Capaldi, 1998a,b). Under these conditions, the
subunits. The overall molecular mass of the complex is overlap of molecules seen in membranous material is absent,
520 kDa. There are three catalytic nucleotide-binding sites simplifying the analysis of images. These new data establish
located on the β subunits of the F1 and one proton channel conclusively that there are two stalks in ECF1Fo (Fig. 1)
formed by the a and c subunits in the membrane-embedded Fo and, in concert with recent data for chloroplast F1Fo (Böttcher
sector. et al., 1998) and mitochondrial F1Fo (J. E. Walker, personal
The available evidence suggests that the coupling of communication), indicate that this is a common feature of
catalytic sites to proton translocation in both the ATP synthesis F1Fo-type proton-pumping ATPases and possibly of V1Vo-type
and hydrolysis modes is conformational and that this enzyme ATPases as well (Boekema et al., 1997).
works as a rotary molecular motor. The key to its functioning
is in the nature of the interaction between the F1 and Fo sectors.
These interactions have been worked out largely by a The central stalk contains the γ and ε subunits
combination of electron microscopy and cross-linking studies. Key evidence that the γ subunit contributes to the central
stalk came from the high-resolution structure of mitochondrial
F1 α3β3γ subcomplex of Abrahams et al. (1994). This structure
ECF1 is linked to the Fo sector by two stalks showed the γ subunit passing through a cavity within the α3β3
The bipartite nature of the F1Fo complex had been observed hexagon and extending from the bottom of the structure as
in early electron micrographs using negative staining to expected of a stalk component. Our finding that the γ subunit
optimize the contrast between protein and solvent, and such can be cross-linked in high yield to the c subunit ring (Watts
pictures appeared to show a stalk linking the two parts et al., 1996) established that the γ subunit extends the full
(Fernandez-Moran, 1962; Soper et al., 1979; Telford et al., length of the stalk. Also present in the central stalk is the ε
1984). However, it was uncertain whether this stalk was an subunit. This polypeptide has a two-domain structure (Wilkens30 R. A. CAPALDI AND OTHERS
During functioning, three catalytic sites must be coupled
alternately to one proton channel. To explain how this coupling
might occur, both Cox and colleagues (e.g. Cox et al., 1984)
and Boyer (1993) independently proposed that, during ATP
synthesis, a central element in the enzyme rotated in one
direction between catalytic sites, driven by a proton gradient.
During ATP hydrolysis in the three catalytic sites, the central
element rotates in the opposite direction. Electron microscopy
provided the first indication that this central element of the
F1 sector was the γ subunit (Gogol et al., 1989, 1990).
Furthermore, we were able to show that the γ subunit was not
fixed in the cavity within the α3β3 hexagon, but could be found
at each of the three α–β pairs (Gogol et al., 1990). Additional
evidence that the γ subunit could rotate within the F1 sector
was obtained from cross-linking studies (Duncan et al., 1995)
and by polarized absorption relaxation after photobleaching
(PARAP) measurements (Sabbert et al., 1996). Conclusive
evidence of ATP-hydrolysis-driven rotation of the γ subunit
Fig. 1. Electron microscopy images of detergent-dispersed F1Fo-
came with the video microscopy of single particles by Yoshida,
ATPase of Escherichia coli (ECF1Fo). Parts A–C show three
Kinosita and their colleagues (Noji et al., 1997). In our earlier
different classes of images resolved. A and C are mirror images of
one another and are side views that clearly separate a central and cryoelectron microscopy studies, we had observed movements
peripheral stalk. Part B is a view more end-on, in which the Fo sector of the γ subunit relative to the ε subunit. We now know that
is more symmetrical. The scale bar in C is 5 nm. The figure is the ε subunit moves in concert with the γ subunit, as shown by
reproduced from Wilkens and Capaldi (1998a) with permission. cross-linking experiments in which ε can be covalently linked
to γ without alteration of function (Aggeler et al., 1992; Tang
and Capaldi, 1996; Schulenberg et al., 1997). More recently,
et al., 1995; Uhlin et al., 1997). There is a C-terminal pair of rotation of the ε subunit has been observed directly by video
α-helices that fits below F1 and interacts with either the α or microscopy (Kato-Yamada et al., 1998). In our earlier
β subunit, depending on nucleotide binding in catalytic sites experiments, the interaction of ε with γ had been perturbed by
(Aggeler et al., 1995; Aggeler and Capaldi, 1996; Wilkens and the monoclonal antibody used to tag it, resulting in this subunit
Capaldi, 1998c). The N-terminal domain is a 10-stranded β remaining fixed at one β subunit.
sandwich that binds on one face to the γ subunit (Tang and The rotation of the γ–ε central stalk, two subunits that react
Capaldi, 1996; Watts et al., 1996) and by the bottom of the with the c subunit ring, suggests a model in which γ–ε and the
sandwich to the c subunit ring (Zhang and Fillingame, 1995). c subunits form a rotor, which moves relative to the rest of the
complex, α3β3δb2a. In such a scheme, the second stalk of δb2
The peripheral stalk contains the δ and b subunits should act as a stator positioning the α3β3 subunits in a fixed
Obvious candidates for the second stalk in the bacterial disposition relative to the a subunit. Rotation of the c subunit
enzyme are the δ and b subunits. Our nuclear magnetic ring relative to the a subunit then provides the proton-
resonance (NMR) structure determination of the δ subunit shows translocation mechanism. The evidence that the γ–ε–c12
a largely globular α-helical protein (Wilkens et al., 1997) which subcomplex is moving relative to the rest of the complex is so
cross-linking studies have established is attached near the top of far based only on cross-linking studies. The cross-linking of γ
F1 by interactions with an α subunit (Lill et al., 1996; Ogilvie et to ε, or of ε to c, does not block ATP-driven proton
al., 1997). Along with N-terminal segments of the three α translocation in ECF1Fo, whereas the cross-linking of either γ
subunits, it probably generates the cap seen at the top of ECF1Fo or ε to α or β does (Aggeler and Capaldi, 1992, 1996; Aggeler
in recent electron micrographs (Wilkens and Capaldi, 1998a,b). et al., 1992, 1993, 1995).
Cross-linking studies indicate that the C-terminal part of the Cross-linking of δ or b to α also has no effect on the
b subunits interacts with the C-terminal region of the δ subunit functioning of the ATP synthase (Ogilvie et al., 1997; Rodgers
and with the α subunit (Rodgers et al., 1997; Rodgers and and Capaldi, 1998), as expected if these two subunits are the
Capaldi, 1998) and that these interactions are close to the top stator element. A summary of our cross-linking experiments to
of the F1 sector. The two b subunits extend as paired α helices date in ECF1Fo is shown in Fig. 2. Additional evidence for the
through the length of F1Fo, with interactions inside the lipid rotation of γ and ε during ATP hydrolysis in ECF1Fo (Aggeler
bilayer (Dmitriev et al., 1999). et al., 1997; Zhou et al., 1997) and of γ during ATP synthesis
(Bulygin et al., 1998) has been provided by cross-linking
experiments, and rotation of the c subunit ring relative to subunit
Two stalks, a central rotor and a peripheral stator a is implied by recent cross-linking studies of Fillingame and
F1 is unusual in being a trimeric enzyme (three α–β pairs). coworkers (Jones et al., 1998). However, convincing evidenceStructure and function of E. coli ATP synthase 31
β α β 60
50
Number of particles
40
α α
δ 30
20
10
γ
b2 0
4.5 5 5.5 6 6.5 7
Particle length (mm)
ε
Fig. 3. Size distribution of F1Fo-ATPase of Escherichia coli
(ECF1Fo) single particles when examined in side view. Cross-
hatched bars are molecules in Mg2+ADP. Open bars are for enzyme
c10-12 a in Mg2+AMP.PNP.
torque generated in relation to energy conservation during
coupling within the enzyme complex. However, as pointed out
Fig. 2. A summary of the cross-links generated so far between by Cherepanov et al. (1999), there is likely to be an elasticity
subunits of F1Fo-ATPase of Escherichia coli (ECF1Fo). Red bow-ties of the transmission between Fo and F1 when four small steps
show cross links that inhibit both ATP hydrolysis and ATP synthesis. of rotation of c relative to a are coupled to one 120 ° rotation
The blue bow-tie differentiates the cross link between γ and c from of γ–ε. We have been looking for conformational changes in
others because, in this case, ATP synthesis is affected but not ATP ECF1Fo that could correspond to this storage and release of
hydrolysis. Yellow bow-ties show cross links that have no effect on elastic energy. We have recently found that there are significant
functioning of the enzyme. nucleotide-dependent alterations in the relationship between F1
and Fo that should be part of the functioning of the enzyme.
of c subunit rotation from visualizing it in real time, as in the Thus, single particles of F1Fo show the now characteristic
video microscopy experiments, has not yet been provided. pattern of F1 separated by two stalks when Mg2+ADP is bound,
but the picture is altered with ATP in catalytic sites (in the
presence of Mg2+AMP.PNP). There is a contraction of the
Elasticity of the F1Fo complex during rotation complex in the ATP state such that the F1 and Fo sectors are
Most studies of the rotary motor model have focused on the closer together and the two stalks are only partly discerned. As
Mg2+ADP Mg2+ATP
1
2
δ
β α β δ
β α β
b2 b2
ε
γ
3 γ ε
Fig. 4. A diagram of the motions of the F1Fo-type
ATP synthase during functioning. Mutations can c12 a H+ c12 a
affect coupling in three ways: (1) by affecting
rotation, (2) by affecting the up–down movements,
and (3) by affecting the proton channel directly.32 R. A. CAPALDI AND OTHERS
shown in Fig. 3, the average particle length of side views is Aggeler, R., Ogilvie, I. and Capaldi, R. A. (1997). Rotation of a γ–ε
10 % shorter in the presence of ATP than in the presence of subunit domain in the E. coli F1Fo ATP synthase complex. The γ–ε
ADP, a contraction of 1.5–2.0 nm. Such a conformational subunits are essentially randomly distributed relative to the α3β3δ
change would require alterations in both stalks. If the domain in the intact complex. J. Biol. Chem. 272, 19621–19624.
conformational changes described above represent energy Boekema, E. J., Ubbink-Kok, T., Lolkema, J. S., Brisson, A. and
Konings, W. N. (1997). Visualization of the peripheral stalk in V-
storage by elastic strain, the uncoupling of catalytic site and
type ATPase: evidence for a stator structure essential to rotational
proton-translocation events seen in many different mutants of catalysis. Proc. Natl. Acad. Sci. USA 94, 14291–14293.
ECF1Fo may be due to perturbation of rotation or of the Böttcher, B., Schwarz, L. and Graber, P. (1998). Direct indication
up–down movements as well as to direct disruption of the for the existence of a double stalk in C FoF1. J. Mol. Biol. 281,
proton channel (Fig. 4). 757–762.
Boyer, P. D. (1993). The binding change mechanism for ATP
synthase – some probabilities and possibilities. Biochim. Biophys.
Epilogue
Acta 1140, 215–250.
As reviewed here, electron microscopy and cross-linking Bulygin, V. V., Duncan, T. M. and Cross, R. L. (1998). Rotation
studies of the E. coli ATP synthase have contributed of the ε subunit during catalysis by E. coli FoF1 ATPase synthase.
significantly to our understanding of the structure and J. Biol. Chem. 273, 31765–31769.
functioning of F1Fo-type ATPases. The dynamic nature of the Cherepanov, D. A., Mulkidjanian, A. Y. and Junge, W. (1999).
functioning of the enzyme means that a full understanding of Transient accumulation of elastic energy in proton translocating
its mechanism will require high-resolution structural data for ATP synthase. FEBS Lett. 449, 1–6.
the intact complex trapped in different conformations. Cross- Cox, G. B., Jans, D. A., Fimmel, A. L., Gibson, F. and Hatch, L.
linking to fix subunits in position and, thereby, trap specific (1984). The mechanism of ATP synthase: conformational changes
by rotation of the b subunit. Biochim. Biophys. Acta 768, 201–208.
conformations can continue to provide new insights into the
Dmitriev, O., Jones, P. C., Jiang, W. and Fillingame, R. H. (1999).
workings of the ATP synthase, particularly if ECF1 and Structure of the membrane domain of subunit b of the E. coli FoF1
ECF1Fo can be crystallized in a variety of trapped states. Our ATP synthase. J. Biol. Chem. 274, 15598–15604.
efforts to obtain such crystals continue. Duncan, T. M., Bulygin, V. V., Zhou, Y., Hutcheon, M. L. and
Cross, R. L. (1995). Rotation of subunits during catalysis of
Work in this laboratory is supported by National Institutes Escherichia coli F1-ATPase. Proc. Natl. Acad. Sci. USA 92,
of Health grants HL 58671 and HL 24526. We thank our 10964–10968.
many colleagues who have contributed to the studies Fernandez-Moran, H. (1962). Cell membrane ultrastructure. Low
described here. temperature electron microscopy and X-ray diffraction of
lipoprotein components in lamellar systems. Circulation 26,
1039–1065.
References Gogol, E. P., Aggeler, R., Sagermann, M. and Capaldi, R. A.
Abrahams, J. P., Leslie, A. G. W., Lutter, R. and Walker, J. E. (1989). Cryoelectron microscopy of Escherichia coli F1
(1994). Structure at 2.8 Å resolution of F1-ATPase from bovine adenosinetriphosphatase decorated with monoclonal antibodies to
heart mitochondria. Nature 370, 621–628. individual subunits of the complex. Biochemistry 28, 4717–4724.
Aggeler, R., Cai, S. X., Keana, J. F. W., Koike, T. and Capaldi, Gogol, E. P., Johnson, E., Aggeler, R. and Capaldi, R. A. (1990).
R. A. (1993). The γ subunit of the E. coli F1 ATPase can be cross Ligand-dependent structural variations in Escherichia coli F1
linked near the glycine-rich loop region of a β subunit when ATPase revealed by cryoelectron microscopy. Proc. Natl. Acad.
ADP+Mg2+ occupies catalytic sites but not when ATP+Mg2+ is Sci. USA 87, 9585–9589.
bound. J. Biol. Chem. 268, 20831–20837. Gogol, E. P., Lücken, U. and Capaldi, R. A. (1987). The stalk
Aggeler, R. and Capaldi, R. A. (1992). Cross linking of the γ subunit connecting the F1 and Fo domains of ATP synthase visualized by
of the E. coli ATPase (ECF1) via cysteines introduced by site- electron microscopy of unstained specimens. FEBS Lett. 219,
directed mutagenesis. J. Biol. Chem. 267, 21355–21359. 274–278.
Aggeler, R. and Capaldi, R. A. (1996). Nucleotide dependent Jones, P. C., Jiang, W. and Fillingame, R. H. (1998). Arrangement
movement of the ε subunit between α and β subunits in the of the multicopy H+ translocating subunit c in the membrane sector
Escherichia coli F1Fo type ATPase. J. Biol. Chem. 271, of the E. coli F1Fo ATP synthase. J. Biol. Chem. 273, 17178–17185.
13888–13891. Junge, W., Lill, H. and Engelbrecht, S. (1997). ATP synthase: an
Aggeler, R., Chicas-Cruz, K., Cai, S-X., Keana, J. F. W. and electrochemical transducer with rotary mechanics. Trends
Capaldi, R. A. (1992). Introduction of reactive cysteine residues Biochem. Sci. 22, 420–423.
in the ε subunit of Escherichia coli F1 ATPase. Modification of Kato-Yamada, Y., Noji, H., Yasuda, R., Kinosita, K. and Yoshida,
these sites with tetrafluorophenlyazide-maleimides and M. (1998). Direct observation of the rotation of ε subunit in F1
examination of changes in the binding of the ε subunit when ATPase. J. Biol. Chem. 273, 19375–19377.
different nucleotides are in catalytic sites. Biochemistry 31, Lill, H., Hensel, F., Junge, W. and Engelbrecht, S. (1996). Cross
2956–2961. linking of engineered δ to (αβ)3 in chloroplast F1 ATPase. J. Biol.
Aggeler, R., Haughton, M. A. and Capaldi, R. A. (1995). Disulfide Chem. 271, 32737–32742.
bond formation between the COOH terminal domain of the β Lücken, U., Gogol, E. P. and Capaldi, R. A. (1990). Structure of
subunits and the γ and ε subunits of the E. coli F1 ATPase. J. Biol. the ATP synthase complex (ECF1Fo) of Escherichia coli from
Chem. 270, 9185–9191. cryoelectron microscopy. Biochemistry 29, 5339–5343.Structure and function of E. coli ATP synthase 33 Noji, H., Yasuda, R., Yoshida, M. and Kinosita, J., Jr (1997). Uhlin, U., Cox, G. B. and Goss, J. M. (1997). Crystal structure of Direct observation of the rotation of F1 ATPase. Nature 386, the ε subunit of the proton translocating ATP synthase from E. coli. 299–302. Structure 5, 1219–1230. Ogilvie, I., Aggeler, R. and Capaldi, R. A. (1997). Cross linking Watts, S. D., Tang, C. and Capaldi, R. A. (1996). The stalk region of the δ to one of the three α subunits has no effect on of E. coli ATP synthase: Tyr 205 of the γ subunit is in the interface functioning, as expected if δ is a part of the stator that links F 1 between F1 and Fo parts and can interact with both the ε and the c and Fo parts of the E. coli ATP synthase. J. Biol. Chem. 272, oligomer. J. Biol. Chem. 271, 28341–28347. 16652–16656. Weber, J. and Senior, A. E. (1997). Catalytic mechanism of F1 Rodgers, A. J. W. and Capaldi, R. A. (1998). The second stalk ATPase. Biochim. Biophys. Acta 1319, 19–58. composed of the b and δ subunits connects Fo to F1 via the α Wilkens, S. and Capaldi, R. A. (1998a). Two stalks link the F1 to subunit in E. coli ATP synthase. J. Biol. Chem. 273, the Fo part in the E. coli ATP synthase. A rotor and a stator? Nature 29406–29410. 393, 29. Rodgers, A. J. W., Wilkens, S., Aggeler, R., Morris, M. B., Wilkens, S. and Capaldi, R. A. (1998b). Electron microscopic Howitt, S. M. and Capaldi, R. A. (1997). The subunit δ-subunit evidence of two stalks linking the F1 and Fo parts of the Escherichia b domain of the E. coli F1Fo ATPase: the b subunits interact with coli ATP synthase. Biochim. Biophys. Acta 1365, 93–97. F1 as a dimer and through the δ subunit. J. Biol. Chem. 272, Wilkens, S. and Capaldi, R. A. (1998c). Solution structure of the ε 31058–31064. subunit of the F1 ATPase from E. coli and interactions of this Sabbert, D., Engelbrecht, S. and Junge, W. (1996). Intersubunit subunit with β subunits in the complex. J. Biol. Chem. 273, rotation in active F-ATPase. Nature 381, 623–625. 26645–26651. Schulenberg, B., Wellmer, F., Lill, H., Junge, W. and Engelbrecht, Wilkens, S., Dahlquist, F. W., Mcintosh, L. P., Donaldson, L. W. S. (1997). Cross-linking of chloroplast FoF1-ATPase subunit ε to γ and Capaldi, R. A. (1995). Structural features of the ε subunit of without effect on activity. ε and γ are parts of the rotor. Eur. J. the Escherichia coli ATP synthase (ECF1Fo) from NMR- Biochem. 249, 131–141. spectroscopy. Nature Struct. Biol. 2, 961–967. Soper, J. W., Decker, G. L. and Pederson, P. L. (1979). Wilkens, S., Dunn, S. D., Chandler, J., Dahlquist, F. W. and Mitochondrial ATPase complex. A dispersed, cytochrome Capaldi, R. A. (1997). Solution structure of the N terminal domain deficient, oligomycin-sensitive preparation from rat liver (residues 1–134) of the δ subunit of the E. coli ATP synthase mitochondria containing molecules with a tripartite structural (ECF1Fo). Nature Struct. Biol. 4, 198–201. arrangement. J. Biol. Chem. 254, 11170–11176. Zhang, Y. and Fillingame, R. H. (1995). Subunits coupling H+ Tang, C. and Capaldi, R. A. (1996). Characterization of the interface transport and ATP synthesis in the Escherichia coli ATP synthase: between γ and ε subunits of Escherichia coli F1-ATPase. J. Biol. Cys–Cys cross linking of F1 subunit ε to the polar loop of Fo subunit Chem. 271, 3018–3024. c. J. Biol. Chem.270, 24609–24614. Telford, J. N., Langworthy, T. A. and Racker, E. (1984). Three Zhou, Y., Duncan, T. M. and Cross, R. L. (1997). Subunit rotation proton pumps, morphology and movements. J. Bioenerg. in E. coli FoF1 ATP synthase during oxidative phosphorylation. Biomembr. 16, 335–351. Proc. Natl. Acad. Sci. USA 94, 10583–10587.
You can also read
