Critical Review Molecular Structure and Rotary Dynamics of Enterococcus hirae V1-ATPase
Ryota Iino1,2 Yoshihiro Minagawa3 Hiroshi Ueno4 Mayu Hara3 Takeshi Murata5,6
1
Department of Bioorganization Research, Okazaki Institute for Integrative Bioscience, Institute for Molecular Science, National Institutes of Natural Sciences, Aichi, Japan 2 Department of Functional Molecular Science, School of Physical Sciences, Graduate University for Advanced Studies (SOKENDAI), Kanagawa, Japan 3 Department of Applied Chemistry, University of Tokyo, Tokyo, Japan 4 Department of Physics, Faculty of Science and Engineering, Chuo University, Tokyo, Japan 5 Department of Chemistry, Graduate School of Science, Chiba University, Chiba, Japan 6 JST, PRESTO, Chiba, Japan
Abstract V1-ATPase is a rotary molecular motor in which the mechanical rotation of the rotor DF subunits against the stator A3B3 ring is driven by the chemical free energy of ATP hydrolysis. Recently, using X-ray crystallography, we solved the highresolution molecular structure of Enterococcus hirae V1ATPase (EhV1) and revealed how the three catalytic sites in the stator A3B3 ring change their structure on nucleotide binding and interaction with the rotor DF subunits. Furthermore,
recently, we also demonstrated directly the rotary catalysis of EhV1 by using single-molecule high-speed imaging and analyzed the properties of the rotary motion in detail. In this critical review, we introduce the molecular structure and rotary dynamics of EhV1 and discuss a possible model of its chemoC 2014 IUBMB Life, 66(9):624– mechanical coupling scheme. V 630, 2014
Keywords: enzyme mechanisms; protein structure; structural biology
Introduction Vacuolar ATPase (V-ATPase) is an ion pump that actively transports ions across the cell membrane by using the chemical free energy of ATP hydrolysis. Intracellular V-ATPases acidify membrane vesicles and fulfill a critical role in various cellular activities, including receptor-mediated endocytosis, membrane trafficking, and protein processing and degradation (1). Furthermore, V-ATPases located at cell surface are
C 2014 International Union of Biochemistry and Molecular Biology V
Volume 66, Number 9, September 2014, Pages 624–630 Address correspondence to: Ryota Iino, Okazaki Institute for Integrative Bioscience, Institute for Molecular Science, National Institutes of Natural Sciences, Aichi 444–8787, Japan. Tel: 181–564-59–5230. E-mail: [email protected] Received 16 August 2014; Accepted 3 September 2014 DOI 10.1002/iub.1311 Published online 17 September 2014 in Wiley Online Library (wileyonlinelibrary.com)
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involved in bone resorption and tumor metastasis and serve as a drug target in the treatment of osteoporosis and cancer (2). The water-soluble V1 moiety of V-ATPase catalyzes the ATP hydrolysis reaction and is called V1-ATPase (V1). V1 performs “rotary catalysis” (3,4) much like another widely recognized rotary molecular motor, F1-ATPase (F1), which is a water-soluble moiety of FoF1-ATP synthase (5–7). To understand the energy conversion mechanism of V1 functioning as a molecular motor, not only atomic-level information on the molecular structure of V1 but also information on its dynamics at the single-molecule level is indispensable. By means of single-molecule imaging performed under an optical microscope, the rotary catalysis of V1 was first verified using the enzyme isolated from Thermus thermophilus (TtV1), which acts as an ATP synthase (8). However, high-resolution structural information on the entire TtV1 complex is not yet available (9,10). Recently, we solved the crystal structure of Enterococcus hirae V1 (EhV1) (11), providing the first high-resolution
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FIG 1
Crystal structures of EhV1. A: A3B3 complex without bound nucleotides (PDB ID: 3VR2). B: A3B3 complex bound with two AMPPNP molecules (PDB ID: 3VR3). C: A3B3DF complex without bound nucleotides (PDB ID: 3VR4). D: A3B3DF complex bound with two AMPPNP molecules (PDB ID: 3VR6). On the right side, only C-terminal domains of the A3B3 rings and the a-helical coiled-coil portion of the D subunit are shown to clarify the distinct conformations of individual A or B subunits (for details, see Fig. 2) and the different structures of the three catalytic sites (indicated by red arrowheads). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
molecular structure of the V1 moiety. Recently, we have also verified the rotary catalysis of EhV1 by using single-molecule high-speed imaging and have analyzed the properties of the rotary motion in detail (12). In this critical review, we
Iino et al
introduce the molecular structure and rotary dynamics of EhV1. Furthermore, using the complementary information of structure and dynamics, we discuss a possible model of the chemomechanical coupling scheme of EhV1.
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FIG 2
Conformations of the A and B subunits in EhV1. A: A and B subunits in A3B3 complex without bound nucleotides (PDB ID: 3VR2). B: A and B subunits in A3B3 complex bound with two AMPPNP molecules (PDB ID: 3VR3). C: A and B subunits in A3B3DF complex without bound nucleotides (PDB ID: 3VR4). Conformations of the A (left) and B (right) subunits superimposed at the N-terminal b-barrel domain are shown. O and O0 : open conformation. C: closed conformation. CR: more closed “closer” conformation. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
the A and B subunits (indicated by red arrowheads in Fig. 1), although most of the amino acid residues responsible for the catalysis are from the A subunit. The overall architecture of EhV1 resembles that of the subcomplex of bovine and yeast mitochondrial F1 (13,14), implying a common evolutionary origin. However, the crystal structures of EhV1 display certain distinct features that are not observed in the structure of F1. First, even in the absence of the rotor DF subunits and bound nucleotides, the conformations of each A and B subunit in the A3B3 ring are not identical (Fig. 2A). Each A and B subunit shows open (O or O0 ) or closed (C) conformations. These distinct conformations result in the different structures of the three catalytic sites and the asymmetric A3B3 ring (Fig. 1A). The three catalytic sites are termed “empty,” “bound,” and “bindable” sites, respectively. This asymmetric structure of the rotor-less stator ring is distinct from that of F1, which displays a three times symmetric structure and three identical catalytic sites in the absence of bound nucleotides in the crystal structure (15) and in observations made using high-speed atomic force microscopy (16–18). When the nonhydrolyzable ATP analog AMPPNP is added, it binds to the “bound” and “bindable” sites of the A3B3 complex, which results in a conformational change of the A (O0 to C) and B (O to O0 ) subunits (Fig. 2B) and a transformation of the “bindable” site into the “bound” site (Fig. 1B). A second crucial feature is the effect of the rotor DF subunits on the structure of the three catalytic sites. Insertion of the DF complex into the A3B3 ring induces conformational changes in the A and B subunits even in the absence of bound nucleotides, and consequently, the more closed “closer” (CR) conformations of the A and B subunits appear (Fig. 2C). This results in not only the formation of the “bound” site from the “bindable” site but also the formation of the “tight” site from the “bound” site (Figs. 1A and 1C). The “tight” site presumably corresponds to the catalytic site in the pre-ATP-hydrolysis stage. Conversely, the binding of AMPPNP to the “bound” and “tight” sites of the A3B3DF complex does not cause further conformational changes of the A and B subunits, and nucleotide-free and nucleotide-bound A3B3DF complexes show almost identical structures (Figs. 1C and 1D). Almost identical nucleotide-free and nucleotide-bound structures were also reported previously in the case yeast F1 (19). These results imply that the interactions between the stator and rotor are as critical as nucleotide binding in determining the structure of the catalytic sites of rotary molecular motors.
Molecular Structure of EhV1
Rotary Dynamics of EhV1
The crystal structures of the A3B3 and A3B3DF complexes of EhV1 are shown in Fig. 1 (11). The A and B subunits form an alternately arranged stator A3B3 ring (Figs. 1A and 1B), and the D and F subunits form the rotor complex that penetrates into the central hole of the A3B3 ring (Figs. 1C and 1D). The nucleotide-binding catalytic sites are located at the interface of
Recently, we established the single-molecule rotation assay of EhV1 (12); this experimental system is depicted schematically in Fig. 3A. In this system, the stator A3B3 ring of EhV1 was fixed on the glass surface by using the His6-tag present at the N-terminus of the A subunits. Next, a streptavidin-coated 40nm gold colloid was attached to biotinylated cysteine residues
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FIG 3
Single-molecule rotation assay of EhV1. A: Schematic drawing of the single-molecule rotation assay of EhV1. B: Examples of clear and unclear states in the rotation of EhV1. Left: Time-revolution plots. Center: XY plots. Right: Distribution of rotary angles. The two clear states are indicated by 1 and 3 and the one unclear state is indicated by 2. C: Distribution of the dwell times of the clear (top) and unclear (bottom) states. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
in the rotor DF subunits. The rotary motion of EhV1 was observed by using total internal reflection dark-field microscopy (20) at an imaging rate of 500–10,000 frames per second. In the presence of ATP, EhV1 showed unidirectional successive rotation in the counterclockwise direction, and the rotary catalysis of EhV1 was successfully verified. However, EhV1 exhibited two distinct reversible states of rotation, namely, clear and unclear (Figs. 3B and 3C). This result suggests that the interactions between the rotor and stator subunits in isolated EhV1 are less stable than those in TtV1 and F1. However, the unclear state occurs only in the isolated EhV1 and is not detected in the entire V-ATPase complex
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(Ueno et al., unpublished). In the entire V-ATPase, two peripheral stalks stabilize the interactions between the rotor and stator subunits. Thus, we concluded that the tight chemomechanical coupling of EhV1 is achieved at least in the clear state and analyzed the rotary dynamics in the clear state in detail. In the clear rotation state, EhV1 showed only three pausing positions separated by 120 at all ATP concentrations ranging from below to above the Michaelis constant, at which distinct elementary reaction steps of the ATP hydrolysis, such as ATP binding, phosphate bond cleavage, or product release, become the rate-limiting steps of the rotation (Fig. 4A). In contrast to the rotation of thermophilic Bacillus PS3 F1 (21) and
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FIG 4
Rotation of EhV1 at various concentrations of ATP or ATPcS. Examples of the clear rotation at various ATP or ATPcS concentrations. A: Left: 40 mM ATP, considerably higher than the Michaelis constant (Km, 154 mM). Center: 100 mM ATP, near the Km. Right: 10 mM ATP, considerably lower than the Km. B: Left: 3 mM ATPcS, considerably higher than the Km (9 mM). Center: 10 mM ATP, near the Km. Right: 3 mM ATP, lower than the Km. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Escherichia coli F1 (22), the rotation of EhV1 was not resolved into any substeps, and this was also the case with TtV1 (23). These results strongly suggest that the 120 stepping rotation without substeps is a common property of V1 and that the chemomechanical coupling scheme of V1 differs from that of F1 (7,24). The 120 stepping rotation of EhV1 without substeps was also confirmed using a slowly hydrolyzed ATP analog, ATPcS (Fig. 4B). Interestingly, in the slow rotation featuring considerably longer pauses that is driven by ATPcS, EhV1 showed only the clear states; no unclear states were observed. This result indicates that the probability of the transition between clear
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and unclear states depends on the duration of the pause and implies that the conformational relaxation of the stator and/or rotor subunits is part of the underlying cause of the transition between clear and unclear states.
A Possible Chemomechanical Coupling Scheme of EhV1 A possible model of the chemomechanical coupling scheme of EhV1 is shown in Fig. 5A, together with the corresponding crystal structures. In this model, we assume that the crystal
Structure and Dynamics of Enterococcus hirae V1
FIG 5
A possible model of the chemomechanical coupling scheme of EhV1. A: The chemomechanical coupling scheme for 120 rotation, described together with the corresponding crystal structures. The three circles indicate the three catalytic sites, and the green arrows indicate rotor DF subunits. ATP* indicates tightly bound ATP in the prehydrolysis state. This model assumes that the two crystal structures of A3B3 complexes resemble the two intermediate states (states 2 and 3) in the rotation of EV1. Note that in state 2, because of the strong interactions between the DF subunits and the “tight” site in the A3B3DF complex, not “empty” but another intermediate state is likely to exist. B: Chemomechanical coupling scheme for 360 rotation. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
structure of nucleotide-bound A3B3DF (Fig. 1D) corresponds to the most stable structure during the pause of the rotation. The tightly bound ATP (ATP*) is hydrolyzed into ADP and inorganic phosphate (Pi) at the “tight” site (state 1 in Fig. 5A). If the effects of the DF subunits are ignored, ATP hydrolysis at the “tight” site results in the conformational change of EhV1 into a nucleotide-free A3B3-like structure (Fig. 1A), and the “empty” and “tight” sites appear to transform into “bindable” and “empty” sites, respectively (state 2). However, strong interac-
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tions between the DF subunits and the “tight” site exist in the A3B3DF complex (11). These strong interactions will prevent the conformational change of the “tight” site into the “empty” site, and another intermediate state is likely to exist. Next, this conformational change almost concomitantly induces ATP binding to the “bindable” site, simultaneous releases of ADP and Pi, and 120 rotation of the rotor DF subunits. On ATP binding and 120 rotation, the “bindable” site changes into the “bound” site, and a nucleotide-bound A3B3-like structure (Fig.
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1B) appears transiently (state 3). Finally, EhV1 transforms into the more stable nucleotide-bound A3B3DF structure, 120 ahead (state 4, identical to state 1). Our model predicts that after ATP binds to the “empty” site at 0 , it is hydrolyzed into ADP and Pi at 240 (Fig. 5B). Furthermore, ADP and Pi are also released at the same angle, 240 . Currently, we are not certain whether three elementary reaction steps occurring at same angle (i.e., at 240 ) are appropriate for the cooperative and coordinated rotary catalysis of EhV1, because in F1, ATP hydrolysis into ADP and Pi, ADP release, and Pi release occur at distinct angles (7,24). We will refine our model further by performing the singlemolecule rotation assay on a hybrid EhV1 that contains a single mutated catalytic site featuring altered ATP binding and hydrolysis abilities, as has been performed in the case of F1 (25,26).
Perspective In addition to structural and single-molecule analyses, computational molecular simulations will be extremely helpful in the study of EhV1. Coarse-grained and all-atom molecular dynamics simulations can reveal transient and unstable conformational intermediates and the pathways of conformational changes (27–29). Ab initio quantum mechanical/molecular mechanical calculations can provide information on the detailed molecular mechanism of the ATP hydrolysis reaction that occurs at the catalytic sites (30–32). Our next approach will be to use a combination of structural, single-molecule, and computational analyses to fully understand the operation mechanism of EhV1.
References [1] Forgac, M. (2007) Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology. Nat. Rev. Mol. Cell Biol. 8, 917–929. [2] Bowman, E. J., and Bowman, B. J. (2005) V-ATPases as drug targets. J. Bioenerg. Biomembr. 37, 431–435. [3] Boyer, P. D. (1993) The binding change mechanism for ATP synthase—some probabilities and possibilities. Biochim. Biophys. Acta 1140, 215–250. [4] Stewart, A. G., Laming, E. M., Sobti, M., and Stock, D. (2014) Rotary ATPases—dynamic molecular machines. Curr. Opin. Struct. Biol. 25, 40–48. [5] Walker, J. E. (2013) The ATP synthase: the understood, the uncertain and the unknown. Biochem. Soc. Trans. 41, 1–16. [6] Yoshida, M., Muneyuki, E., and Hisabori, T. (2001) ATP synthase—a marvellous rotary engine of the cell. Nat. Rev. Mol. Cell Biol. 2, 669–677. [7] Iino, R., and Noji, H. (2013) Operation mechanism of F(o) F(1)-adenosine triphosphate synthase revealed by its structure and dynamics. IUBMB Life 65, 238–246. [8] Imamura, H., Nakano, M., Noji, H., Muneyuki, E., Ohkuma, S., et al. (2003) Evidence for rotation of V1-ATPase. Proc. Natl. Acad. Sci. USA 100, 2312–2315. [9] Maher, M. J., Akimoto, S., Iwata, M., Nagata, K., Hori, Y., et al. (2009) Crystal structure of A3B3 complex of V-ATPase from Thermus thermophilus. EMBO J. 28, 3771–3779. [10] Nagamatsu, Y., Takeda, K., Kuranaga, T., Numoto, N., and Miki, K. (2013) Origin of asymmetry at the intersubunit interfaces of V1-ATPase from Thermus thermophilus. J. Mol. Biol. 425, 2699–2708.
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[11] Arai, S., Saijo, S., Suzuki, K., Mizutani, K., Kakinuma, Y., et al. (2013) Rotation mechanism of Enterococcus hirae V1-ATPase based on asymmetric crystal structures. Nature 493, 703–707. [12] Minagawa, Y., Ueno, H., Hara, M., Ishizuka-Katsura, Y., Ohsawa, N., et al. (2013) Basic properties of rotary dynamics of the molecular motor Enterococcus hirae V1-ATPase. J. Biol. Chem. 288, 32700–32707. [13] Abrahams, J. P., Leslie, A. G., Lutter, R., and Walker, J. E. (1994) Structure at 2.8 Å resolution of F1-ATPase from bovine heart mitochondria. Nature 370, 621–628. [14] Kabaleeswaran, V., Puri, N., Walker, J. E., Leslie, A. G., and Mueller, D. M. (2006) Novel features of the rotary catalytic mechanism revealed in the structure of yeast F1 ATPase. EMBO J. 25, 5433–5442. [15] Shirakihara, Y., Leslie, A. G., Abrahams, J. P., Walker, J. E., Ueda, T., et al. (1997) The crystal structure of the nucleotide-free a3b3 subcomplex of F1ATPase from the thermophilic Bacillus PS3 is a symmetric trimer. Structure 5, 825–836. [16] Uchihashi, T., Iino, R., Ando, T., and Noji, H. (2011) High-speed atomic force microscopy reveals rotary catalysis of rotorless F-ATPase. Science 333, 755– 758. [17] Iino, R., and Noji, H. (2012) Rotary catalysis of the stator ring of F(1)-ATPase. Biochim. Biophys. Acta 1817, 1732–1739. [18] Iino, R., and Noji, H. (2013) Intersubunit coordination and cooperativity in ring-shaped NTPases. Curr. Opin. Struct. Biol. 23, 229–234. [19] Kabaleeswaran, V., Shen, H., Symersky, J., Walker, J. E., Leslie, A. G., et al. (2009) Asymmetric structure of the yeast F1 ATPase in the absence of bound nucleotides. J. Biol. Chem. 284, 10546–10551. [20] Ueno, H., Nishikawa, S., Iino, R., Tabata, K. V., Sakakihara, S., et al. (2010) Simple dark-field microscopy with nanometer spatial precision and microsecond temporal resolution. Biophys. J. 98, 2014–2023. [21] Yasuda, R., Noji, H., Yoshida, M., Kinosita, K., Jr., and Itoh, H. (2001) Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1ATPase. Nature 410, 898–904. [22] Bilyard, T., Nakanishi-Matsui, M., Steel, B. C., Pilizota, T., Nord, A. L., et al. (2013) High-resolution single-molecule characterization of the enzymatic states in Escherichia coli F1-ATPase. Philos. Trans. R. Soc. Lond. Ser. B: Biol. Sci. 368, 20120023. [23] Imamura, H., Takeda, M., Funamoto, S., Shimabukuro, K., Yoshida, M., et al. (2005) Rotation scheme of V1-motor is different from that of F1-motor. Proc. Natl. Acad. Sci. USA 102, 17929–17933. [24] Watanabe, R., Iino, R., and Noji, H. (2010) Phosphate release in F1-ATPase catalytic cycle follows ADP release. Nat. Chem. Biol. 6, 814–820. [25] Ariga, T., Muneyuki, E., and Yoshida, M. (2007) F1-ATPase rotates by an asymmetric, sequential mechanism using all three catalytic subunits. Nat. Struct. Mol. Biol. 14, 841–846. [26] Okuno, D., Fujisawa, R., Iino, R., Hirono-Hara, Y., Imamura, H., et al. (2008) Correlation between the conformational states of F1-ATPase as determined from its crystal structure and single-molecule rotation. Proc. Natl. Acad. Sci. USA 105, 20722–20727. [27] Koga, N., and Takada, S. (2006) Folding-based molecular simulations reveal mechanisms of the rotary motor F1-ATPase. Proc. Natl. Acad. Sci. USA 103, 5367–5372. [28] Pu, J., and Karplus, M. (2008) How subunit coupling produces the c-subunit rotary motion in F1-ATPase. Proc. Natl. Acad. Sci. USA 105, 1192–1197. [29] Ito, Y., and Ikeguchi, M. (2014) Molecular dynamics simulations of F1ATPase. Adv. Exp. Med. Biol. 805, 411–440. [30] Dittrich, M., Hayashi, S., and Schulten, K. (2003) On the mechanism of ATP hydrolysis in F1-ATPase. Biophys. J. 85, 2253–2266. [31] Dittrich, M., Hayashi, S., and Schulten, K. (2004) ATP hydrolysis in the bTP and bDP catalytic sites of F1-ATPase. Biophys. J. 87, 2954–2967. [32] Hayashi, S., Ueno, H., Shaikh, A. R., Umemura, M., Kamiya, M., et al. (2012) Molecular mechanism of ATP hydrolysis in F1-ATPase revealed by molecular simulations and single-molecule observations. J. Am. Chem. Soc. 134, 8447–8454.
Structure and Dynamics of Enterococcus hirae V1
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Ryota Iino1,2 Yoshihiro Minagawa3 Hiroshi Ueno4 Mayu Hara3 Takeshi Murata5,6
1
Department of Bioorganization Research, Okazaki Institute for Integrative Bioscience, Institute for Molecular Science, National Institutes of Natural Sciences, Aichi, Japan 2 Department of Functional Molecular Science, School of Physical Sciences, Graduate University for Advanced Studies (SOKENDAI), Kanagawa, Japan 3 Department of Applied Chemistry, University of Tokyo, Tokyo, Japan 4 Department of Physics, Faculty of Science and Engineering, Chuo University, Tokyo, Japan 5 Department of Chemistry, Graduate School of Science, Chiba University, Chiba, Japan 6 JST, PRESTO, Chiba, Japan
Abstract V1-ATPase is a rotary molecular motor in which the mechanical rotation of the rotor DF subunits against the stator A3B3 ring is driven by the chemical free energy of ATP hydrolysis. Recently, using X-ray crystallography, we solved the highresolution molecular structure of Enterococcus hirae V1ATPase (EhV1) and revealed how the three catalytic sites in the stator A3B3 ring change their structure on nucleotide binding and interaction with the rotor DF subunits. Furthermore,
recently, we also demonstrated directly the rotary catalysis of EhV1 by using single-molecule high-speed imaging and analyzed the properties of the rotary motion in detail. In this critical review, we introduce the molecular structure and rotary dynamics of EhV1 and discuss a possible model of its chemoC 2014 IUBMB Life, 66(9):624– mechanical coupling scheme. V 630, 2014
Keywords: enzyme mechanisms; protein structure; structural biology
Introduction Vacuolar ATPase (V-ATPase) is an ion pump that actively transports ions across the cell membrane by using the chemical free energy of ATP hydrolysis. Intracellular V-ATPases acidify membrane vesicles and fulfill a critical role in various cellular activities, including receptor-mediated endocytosis, membrane trafficking, and protein processing and degradation (1). Furthermore, V-ATPases located at cell surface are
C 2014 International Union of Biochemistry and Molecular Biology V
Volume 66, Number 9, September 2014, Pages 624–630 Address correspondence to: Ryota Iino, Okazaki Institute for Integrative Bioscience, Institute for Molecular Science, National Institutes of Natural Sciences, Aichi 444–8787, Japan. Tel: 181–564-59–5230. E-mail: [email protected] Received 16 August 2014; Accepted 3 September 2014 DOI 10.1002/iub.1311 Published online 17 September 2014 in Wiley Online Library (wileyonlinelibrary.com)
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involved in bone resorption and tumor metastasis and serve as a drug target in the treatment of osteoporosis and cancer (2). The water-soluble V1 moiety of V-ATPase catalyzes the ATP hydrolysis reaction and is called V1-ATPase (V1). V1 performs “rotary catalysis” (3,4) much like another widely recognized rotary molecular motor, F1-ATPase (F1), which is a water-soluble moiety of FoF1-ATP synthase (5–7). To understand the energy conversion mechanism of V1 functioning as a molecular motor, not only atomic-level information on the molecular structure of V1 but also information on its dynamics at the single-molecule level is indispensable. By means of single-molecule imaging performed under an optical microscope, the rotary catalysis of V1 was first verified using the enzyme isolated from Thermus thermophilus (TtV1), which acts as an ATP synthase (8). However, high-resolution structural information on the entire TtV1 complex is not yet available (9,10). Recently, we solved the crystal structure of Enterococcus hirae V1 (EhV1) (11), providing the first high-resolution
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FIG 1
Crystal structures of EhV1. A: A3B3 complex without bound nucleotides (PDB ID: 3VR2). B: A3B3 complex bound with two AMPPNP molecules (PDB ID: 3VR3). C: A3B3DF complex without bound nucleotides (PDB ID: 3VR4). D: A3B3DF complex bound with two AMPPNP molecules (PDB ID: 3VR6). On the right side, only C-terminal domains of the A3B3 rings and the a-helical coiled-coil portion of the D subunit are shown to clarify the distinct conformations of individual A or B subunits (for details, see Fig. 2) and the different structures of the three catalytic sites (indicated by red arrowheads). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
molecular structure of the V1 moiety. Recently, we have also verified the rotary catalysis of EhV1 by using single-molecule high-speed imaging and have analyzed the properties of the rotary motion in detail (12). In this critical review, we
Iino et al
introduce the molecular structure and rotary dynamics of EhV1. Furthermore, using the complementary information of structure and dynamics, we discuss a possible model of the chemomechanical coupling scheme of EhV1.
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FIG 2
Conformations of the A and B subunits in EhV1. A: A and B subunits in A3B3 complex without bound nucleotides (PDB ID: 3VR2). B: A and B subunits in A3B3 complex bound with two AMPPNP molecules (PDB ID: 3VR3). C: A and B subunits in A3B3DF complex without bound nucleotides (PDB ID: 3VR4). Conformations of the A (left) and B (right) subunits superimposed at the N-terminal b-barrel domain are shown. O and O0 : open conformation. C: closed conformation. CR: more closed “closer” conformation. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
the A and B subunits (indicated by red arrowheads in Fig. 1), although most of the amino acid residues responsible for the catalysis are from the A subunit. The overall architecture of EhV1 resembles that of the subcomplex of bovine and yeast mitochondrial F1 (13,14), implying a common evolutionary origin. However, the crystal structures of EhV1 display certain distinct features that are not observed in the structure of F1. First, even in the absence of the rotor DF subunits and bound nucleotides, the conformations of each A and B subunit in the A3B3 ring are not identical (Fig. 2A). Each A and B subunit shows open (O or O0 ) or closed (C) conformations. These distinct conformations result in the different structures of the three catalytic sites and the asymmetric A3B3 ring (Fig. 1A). The three catalytic sites are termed “empty,” “bound,” and “bindable” sites, respectively. This asymmetric structure of the rotor-less stator ring is distinct from that of F1, which displays a three times symmetric structure and three identical catalytic sites in the absence of bound nucleotides in the crystal structure (15) and in observations made using high-speed atomic force microscopy (16–18). When the nonhydrolyzable ATP analog AMPPNP is added, it binds to the “bound” and “bindable” sites of the A3B3 complex, which results in a conformational change of the A (O0 to C) and B (O to O0 ) subunits (Fig. 2B) and a transformation of the “bindable” site into the “bound” site (Fig. 1B). A second crucial feature is the effect of the rotor DF subunits on the structure of the three catalytic sites. Insertion of the DF complex into the A3B3 ring induces conformational changes in the A and B subunits even in the absence of bound nucleotides, and consequently, the more closed “closer” (CR) conformations of the A and B subunits appear (Fig. 2C). This results in not only the formation of the “bound” site from the “bindable” site but also the formation of the “tight” site from the “bound” site (Figs. 1A and 1C). The “tight” site presumably corresponds to the catalytic site in the pre-ATP-hydrolysis stage. Conversely, the binding of AMPPNP to the “bound” and “tight” sites of the A3B3DF complex does not cause further conformational changes of the A and B subunits, and nucleotide-free and nucleotide-bound A3B3DF complexes show almost identical structures (Figs. 1C and 1D). Almost identical nucleotide-free and nucleotide-bound structures were also reported previously in the case yeast F1 (19). These results imply that the interactions between the stator and rotor are as critical as nucleotide binding in determining the structure of the catalytic sites of rotary molecular motors.
Molecular Structure of EhV1
Rotary Dynamics of EhV1
The crystal structures of the A3B3 and A3B3DF complexes of EhV1 are shown in Fig. 1 (11). The A and B subunits form an alternately arranged stator A3B3 ring (Figs. 1A and 1B), and the D and F subunits form the rotor complex that penetrates into the central hole of the A3B3 ring (Figs. 1C and 1D). The nucleotide-binding catalytic sites are located at the interface of
Recently, we established the single-molecule rotation assay of EhV1 (12); this experimental system is depicted schematically in Fig. 3A. In this system, the stator A3B3 ring of EhV1 was fixed on the glass surface by using the His6-tag present at the N-terminus of the A subunits. Next, a streptavidin-coated 40nm gold colloid was attached to biotinylated cysteine residues
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FIG 3
Single-molecule rotation assay of EhV1. A: Schematic drawing of the single-molecule rotation assay of EhV1. B: Examples of clear and unclear states in the rotation of EhV1. Left: Time-revolution plots. Center: XY plots. Right: Distribution of rotary angles. The two clear states are indicated by 1 and 3 and the one unclear state is indicated by 2. C: Distribution of the dwell times of the clear (top) and unclear (bottom) states. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
in the rotor DF subunits. The rotary motion of EhV1 was observed by using total internal reflection dark-field microscopy (20) at an imaging rate of 500–10,000 frames per second. In the presence of ATP, EhV1 showed unidirectional successive rotation in the counterclockwise direction, and the rotary catalysis of EhV1 was successfully verified. However, EhV1 exhibited two distinct reversible states of rotation, namely, clear and unclear (Figs. 3B and 3C). This result suggests that the interactions between the rotor and stator subunits in isolated EhV1 are less stable than those in TtV1 and F1. However, the unclear state occurs only in the isolated EhV1 and is not detected in the entire V-ATPase complex
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(Ueno et al., unpublished). In the entire V-ATPase, two peripheral stalks stabilize the interactions between the rotor and stator subunits. Thus, we concluded that the tight chemomechanical coupling of EhV1 is achieved at least in the clear state and analyzed the rotary dynamics in the clear state in detail. In the clear rotation state, EhV1 showed only three pausing positions separated by 120 at all ATP concentrations ranging from below to above the Michaelis constant, at which distinct elementary reaction steps of the ATP hydrolysis, such as ATP binding, phosphate bond cleavage, or product release, become the rate-limiting steps of the rotation (Fig. 4A). In contrast to the rotation of thermophilic Bacillus PS3 F1 (21) and
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FIG 4
Rotation of EhV1 at various concentrations of ATP or ATPcS. Examples of the clear rotation at various ATP or ATPcS concentrations. A: Left: 40 mM ATP, considerably higher than the Michaelis constant (Km, 154 mM). Center: 100 mM ATP, near the Km. Right: 10 mM ATP, considerably lower than the Km. B: Left: 3 mM ATPcS, considerably higher than the Km (9 mM). Center: 10 mM ATP, near the Km. Right: 3 mM ATP, lower than the Km. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Escherichia coli F1 (22), the rotation of EhV1 was not resolved into any substeps, and this was also the case with TtV1 (23). These results strongly suggest that the 120 stepping rotation without substeps is a common property of V1 and that the chemomechanical coupling scheme of V1 differs from that of F1 (7,24). The 120 stepping rotation of EhV1 without substeps was also confirmed using a slowly hydrolyzed ATP analog, ATPcS (Fig. 4B). Interestingly, in the slow rotation featuring considerably longer pauses that is driven by ATPcS, EhV1 showed only the clear states; no unclear states were observed. This result indicates that the probability of the transition between clear
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and unclear states depends on the duration of the pause and implies that the conformational relaxation of the stator and/or rotor subunits is part of the underlying cause of the transition between clear and unclear states.
A Possible Chemomechanical Coupling Scheme of EhV1 A possible model of the chemomechanical coupling scheme of EhV1 is shown in Fig. 5A, together with the corresponding crystal structures. In this model, we assume that the crystal
Structure and Dynamics of Enterococcus hirae V1
FIG 5
A possible model of the chemomechanical coupling scheme of EhV1. A: The chemomechanical coupling scheme for 120 rotation, described together with the corresponding crystal structures. The three circles indicate the three catalytic sites, and the green arrows indicate rotor DF subunits. ATP* indicates tightly bound ATP in the prehydrolysis state. This model assumes that the two crystal structures of A3B3 complexes resemble the two intermediate states (states 2 and 3) in the rotation of EV1. Note that in state 2, because of the strong interactions between the DF subunits and the “tight” site in the A3B3DF complex, not “empty” but another intermediate state is likely to exist. B: Chemomechanical coupling scheme for 360 rotation. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
structure of nucleotide-bound A3B3DF (Fig. 1D) corresponds to the most stable structure during the pause of the rotation. The tightly bound ATP (ATP*) is hydrolyzed into ADP and inorganic phosphate (Pi) at the “tight” site (state 1 in Fig. 5A). If the effects of the DF subunits are ignored, ATP hydrolysis at the “tight” site results in the conformational change of EhV1 into a nucleotide-free A3B3-like structure (Fig. 1A), and the “empty” and “tight” sites appear to transform into “bindable” and “empty” sites, respectively (state 2). However, strong interac-
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tions between the DF subunits and the “tight” site exist in the A3B3DF complex (11). These strong interactions will prevent the conformational change of the “tight” site into the “empty” site, and another intermediate state is likely to exist. Next, this conformational change almost concomitantly induces ATP binding to the “bindable” site, simultaneous releases of ADP and Pi, and 120 rotation of the rotor DF subunits. On ATP binding and 120 rotation, the “bindable” site changes into the “bound” site, and a nucleotide-bound A3B3-like structure (Fig.
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1B) appears transiently (state 3). Finally, EhV1 transforms into the more stable nucleotide-bound A3B3DF structure, 120 ahead (state 4, identical to state 1). Our model predicts that after ATP binds to the “empty” site at 0 , it is hydrolyzed into ADP and Pi at 240 (Fig. 5B). Furthermore, ADP and Pi are also released at the same angle, 240 . Currently, we are not certain whether three elementary reaction steps occurring at same angle (i.e., at 240 ) are appropriate for the cooperative and coordinated rotary catalysis of EhV1, because in F1, ATP hydrolysis into ADP and Pi, ADP release, and Pi release occur at distinct angles (7,24). We will refine our model further by performing the singlemolecule rotation assay on a hybrid EhV1 that contains a single mutated catalytic site featuring altered ATP binding and hydrolysis abilities, as has been performed in the case of F1 (25,26).
Perspective In addition to structural and single-molecule analyses, computational molecular simulations will be extremely helpful in the study of EhV1. Coarse-grained and all-atom molecular dynamics simulations can reveal transient and unstable conformational intermediates and the pathways of conformational changes (27–29). Ab initio quantum mechanical/molecular mechanical calculations can provide information on the detailed molecular mechanism of the ATP hydrolysis reaction that occurs at the catalytic sites (30–32). Our next approach will be to use a combination of structural, single-molecule, and computational analyses to fully understand the operation mechanism of EhV1.
References [1] Forgac, M. (2007) Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology. Nat. Rev. Mol. Cell Biol. 8, 917–929. [2] Bowman, E. J., and Bowman, B. J. (2005) V-ATPases as drug targets. J. Bioenerg. Biomembr. 37, 431–435. [3] Boyer, P. D. (1993) The binding change mechanism for ATP synthase—some probabilities and possibilities. Biochim. Biophys. Acta 1140, 215–250. [4] Stewart, A. G., Laming, E. M., Sobti, M., and Stock, D. (2014) Rotary ATPases—dynamic molecular machines. Curr. Opin. Struct. Biol. 25, 40–48. [5] Walker, J. E. (2013) The ATP synthase: the understood, the uncertain and the unknown. Biochem. Soc. Trans. 41, 1–16. [6] Yoshida, M., Muneyuki, E., and Hisabori, T. (2001) ATP synthase—a marvellous rotary engine of the cell. Nat. Rev. Mol. Cell Biol. 2, 669–677. [7] Iino, R., and Noji, H. (2013) Operation mechanism of F(o) F(1)-adenosine triphosphate synthase revealed by its structure and dynamics. IUBMB Life 65, 238–246. [8] Imamura, H., Nakano, M., Noji, H., Muneyuki, E., Ohkuma, S., et al. (2003) Evidence for rotation of V1-ATPase. Proc. Natl. Acad. Sci. USA 100, 2312–2315. [9] Maher, M. J., Akimoto, S., Iwata, M., Nagata, K., Hori, Y., et al. (2009) Crystal structure of A3B3 complex of V-ATPase from Thermus thermophilus. EMBO J. 28, 3771–3779. [10] Nagamatsu, Y., Takeda, K., Kuranaga, T., Numoto, N., and Miki, K. (2013) Origin of asymmetry at the intersubunit interfaces of V1-ATPase from Thermus thermophilus. J. Mol. Biol. 425, 2699–2708.
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[11] Arai, S., Saijo, S., Suzuki, K., Mizutani, K., Kakinuma, Y., et al. (2013) Rotation mechanism of Enterococcus hirae V1-ATPase based on asymmetric crystal structures. Nature 493, 703–707. [12] Minagawa, Y., Ueno, H., Hara, M., Ishizuka-Katsura, Y., Ohsawa, N., et al. (2013) Basic properties of rotary dynamics of the molecular motor Enterococcus hirae V1-ATPase. J. Biol. Chem. 288, 32700–32707. [13] Abrahams, J. P., Leslie, A. G., Lutter, R., and Walker, J. E. (1994) Structure at 2.8 Å resolution of F1-ATPase from bovine heart mitochondria. Nature 370, 621–628. [14] Kabaleeswaran, V., Puri, N., Walker, J. E., Leslie, A. G., and Mueller, D. M. (2006) Novel features of the rotary catalytic mechanism revealed in the structure of yeast F1 ATPase. EMBO J. 25, 5433–5442. [15] Shirakihara, Y., Leslie, A. G., Abrahams, J. P., Walker, J. E., Ueda, T., et al. (1997) The crystal structure of the nucleotide-free a3b3 subcomplex of F1ATPase from the thermophilic Bacillus PS3 is a symmetric trimer. Structure 5, 825–836. [16] Uchihashi, T., Iino, R., Ando, T., and Noji, H. (2011) High-speed atomic force microscopy reveals rotary catalysis of rotorless F-ATPase. Science 333, 755– 758. [17] Iino, R., and Noji, H. (2012) Rotary catalysis of the stator ring of F(1)-ATPase. Biochim. Biophys. Acta 1817, 1732–1739. [18] Iino, R., and Noji, H. (2013) Intersubunit coordination and cooperativity in ring-shaped NTPases. Curr. Opin. Struct. Biol. 23, 229–234. [19] Kabaleeswaran, V., Shen, H., Symersky, J., Walker, J. E., Leslie, A. G., et al. (2009) Asymmetric structure of the yeast F1 ATPase in the absence of bound nucleotides. J. Biol. Chem. 284, 10546–10551. [20] Ueno, H., Nishikawa, S., Iino, R., Tabata, K. V., Sakakihara, S., et al. (2010) Simple dark-field microscopy with nanometer spatial precision and microsecond temporal resolution. Biophys. J. 98, 2014–2023. [21] Yasuda, R., Noji, H., Yoshida, M., Kinosita, K., Jr., and Itoh, H. (2001) Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1ATPase. Nature 410, 898–904. [22] Bilyard, T., Nakanishi-Matsui, M., Steel, B. C., Pilizota, T., Nord, A. L., et al. (2013) High-resolution single-molecule characterization of the enzymatic states in Escherichia coli F1-ATPase. Philos. Trans. R. Soc. Lond. Ser. B: Biol. Sci. 368, 20120023. [23] Imamura, H., Takeda, M., Funamoto, S., Shimabukuro, K., Yoshida, M., et al. (2005) Rotation scheme of V1-motor is different from that of F1-motor. Proc. Natl. Acad. Sci. USA 102, 17929–17933. [24] Watanabe, R., Iino, R., and Noji, H. (2010) Phosphate release in F1-ATPase catalytic cycle follows ADP release. Nat. Chem. Biol. 6, 814–820. [25] Ariga, T., Muneyuki, E., and Yoshida, M. (2007) F1-ATPase rotates by an asymmetric, sequential mechanism using all three catalytic subunits. Nat. Struct. Mol. Biol. 14, 841–846. [26] Okuno, D., Fujisawa, R., Iino, R., Hirono-Hara, Y., Imamura, H., et al. (2008) Correlation between the conformational states of F1-ATPase as determined from its crystal structure and single-molecule rotation. Proc. Natl. Acad. Sci. USA 105, 20722–20727. [27] Koga, N., and Takada, S. (2006) Folding-based molecular simulations reveal mechanisms of the rotary motor F1-ATPase. Proc. Natl. Acad. Sci. USA 103, 5367–5372. [28] Pu, J., and Karplus, M. (2008) How subunit coupling produces the c-subunit rotary motion in F1-ATPase. Proc. Natl. Acad. Sci. USA 105, 1192–1197. [29] Ito, Y., and Ikeguchi, M. (2014) Molecular dynamics simulations of F1ATPase. Adv. Exp. Med. Biol. 805, 411–440. [30] Dittrich, M., Hayashi, S., and Schulten, K. (2003) On the mechanism of ATP hydrolysis in F1-ATPase. Biophys. J. 85, 2253–2266. [31] Dittrich, M., Hayashi, S., and Schulten, K. (2004) ATP hydrolysis in the bTP and bDP catalytic sites of F1-ATPase. Biophys. J. 87, 2954–2967. [32] Hayashi, S., Ueno, H., Shaikh, A. R., Umemura, M., Kamiya, M., et al. (2012) Molecular mechanism of ATP hydrolysis in F1-ATPase revealed by molecular simulations and single-molecule observations. J. Am. Chem. Soc. 134, 8447–8454.
Structure and Dynamics of Enterococcus hirae V1
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