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Crystallization and Preliminary X-ray Studies of V1-ATPase of Thermus thermophilus HB8 Complexed with Mg-ADP
Journal of Structural Biology 134, 88 –92 (2001) doi:10.1006/jsbi.2001.4358, available online at http://www.idealibrary.com on
CRYSTALLIZATION NOTE
Crystallization and Preliminary X-ray Studies of V1-ATPase of Thermus thermophilus HB8 Complexed with Mg-ADP Noriyuki Ishii,*,1,2 Shinya Saijo,† Takao Sato,† Nobuo Tanaka,† and Kazuaki Harata*,1 *Biophysical Chemistry Laboratory, National Institute of Bioscience and Human Technology, 1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan; and †Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Kanagawa 226-8501, Japan Received January 29, 2001, and in revised form April 9, 2001; published online June 13, 2001
sociated with a fairly small integral membrane sector, F0, which functions as a proton channel. In prokaryotes, for example, F1 consists of ␣33␥1␦1⑀1 subunits, and F0 of a 1 b 2 c 12 , though there are some more additional subunits in eukaryotic counterparts. On the other hand, V-type, or V0V1-, ATPase resides in endomembrane systems such as endosomes, lysosomes, Golgi-related vesicles, chromaffin granules, and the vacuolar membranes of plants, yeast, and Neurospora (Nelson, 1992b). V-type ATPase belongs to a class of ATP-driven proton pumps responsible for acidification of intracellular compartments in the cells. Although it had been believed that all H⫹-translocating ATPases in prokaryotic cells are F0F1-ATPases, some kinds of eubacteria as well as most archea have been found to hold V0V1-ATPases whose role is ATP synthesis coupled with proton flux across membranes (Inatomi, 1986; Kakinuma and Igarashi, 1990; Konishi et al., 1987; Nanba and Mukohata, 1987; Yokoyama et al., 1994). V0V1-ATPases consist of two functional sectors, as does the F0F1-ATPase, in this case designated as a peripheral V1 moiety and an integral membrane V0 moiety. The V1 moiety has an ATP synthesis/hydrolysis activity and is composed of two large subunits, A and B, and other smaller subunits. From electron microscopic studies (Bowman et al., 1989; Reidlinger et al., 1994; Yokoyama et al., 1994, 2000), the gross features of V1 have been shown to consist of a roughly spherical assembly with a 90- to 100-Å diameter, containing three copies each of the A and B subunits, which alternate in a hexagonal arrangement around an aqueous cavity in which the ␥ and ␦ subunits are located. The amino acid sequence analysis has shown that the A subunit in V1-ATPases is homologous to the  subunit in F1-
Crystals have been grown of the V1-ATPase sector of the V-type ATP synthase complex (V0V1) from the thermophilic eubacterium Thermus thermophilus HB8. These crystals are grown by the vapor diffusion method in the presence of 5 mM Mg-ADP, from solutions containing 100 mM sodium acetate and 2 M sodium formate, pH 5.5. The crystals diffracted X rays beyond 3.4 Å in resolution on a synchrotron radiation source. The crystals belong to the trigonal space group P3, with unit cell dimensions of a ⴝ b ⴝ 89.0 Å, c ⴝ 179.2 Å, and ␥ ⴝ 120°. The unit cell presumably contains one molecule of V1-ATPase and the Vm value is calculated as 3.0 Å3/Da. © 2001 Academic Press
Key Words: V1-ATPase; Thermus thermophilus; Xray crystallography.
Vacuolar (V)-type ATPase and F0F1-ATPase are two major subclasses, related by evolution, of a superfamily of proton translocating ATPases that do not involve the formation of phosphorylated enzymes as an intermediate (Forgac, 1989; Nelson, 1992a). F0F1-ATPase is the well-known ATP synthase. This important enzyme is present in the inner membranes of mitochondria, thylakoid membranes of chloroplasts, and plasma membranes of prokaryotic cells (Boyer, 1993; Senior, 1988; Futai et al., 1988). F-type ATPase is composed of several kinds of subunits and exists as a huge (⬃500 kDa) complex in which a water-soluble catalytic sector, F1, is as1 Present address: Biological Information Research Center, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan. 2 To whom correspondence should be addressed. E-mail: [email protected]. Fax: ⫹81-298-61-6194.
1047-8477/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. Crystals of Thermus thermophilus V1-ATPase grown in the presence of Mg-ADP. The approximate dimensions are 36 ⫻ 36 ⫻ 143 m. The inset shows an enlarged crystal having a typical shape.
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ATPases, and the B subunit in V1-ATPases is homologous to the ␣ subunits in F1-ATPases. Thus one can expect that the active sites for ATP synthesis/ hydrolysis for V1-ATPases should be located on the A subunit. The V0 moiety has a proton pump activity. Remarkable achievements have been reported recently in the structural studies of F0F1-ATPases (Abrahams et al., 1994; Bianchet et al., 1998; Shirakihara et al., 1997; Stock et al., 1999) as well as in kinetic mechanisms, including experimental demonstration of the rotation of the centrally located ␥ subunit in the ␣33 hexagon of the F1 moiety, coupled to ATP hydrolysis (Noji et al., 1997; Yasuda et al., 1998). The structure and mechanism reported for F0F1-ATPases are thought to be basically similar to those for V0V1-ATPases. However, information obtained about the enzymatic mechanism of ATP synthesis by F0F1-ATPases cannot tell us what is different about the mechanism used by V0V1ATPases to generate a proton gradient. A structure determination at high resolution is indispensable in order to understand these differences. Comparison
FIG. 2. Oscillation photograph of a crystal of Thermus thermophilus V1-ATPase taken with the use of the synchrotron radiation source. The oscillation range during a 10-min exposure was 5° and the distance from the crystal to the CCD detector was 300 mm. The resolution at the edge of the detector is 3.3 Å.
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of structures between F0F1-ATPases and V0V1ATPases would be expected to explain the known differences in enzymatic properties, such as the low specific ATPase activity, high K m values, and resistance to azide inhibition of V0V1-ATPases. A structural comparison at high resolution would also be expected to elucidate the common features involved in biological energy by which the ATP synthesis/ hydrolysis reaction is coupled to proton translocation across a membrane. The proton translocating ATPase found in the plasma membrane of an aerobic thermophilic eubacterium, Thermus thermophilus, belongs to the class of V0V1-ATPases (Yokoyama et al., 1990). The V1 moiety from T. thermophilus HB8 consists of four subunits with a stoichiometry of A 3 B 3 ␥ 1 ␦ 1 . The molecular masses of the individual subunits are approximately 66 ( A), 55 (B), 30 (␥), and 11 kDa (␦) (Yokoyama et al., 1998). The crystallization of the noncatalytic B subunit has been reported (Nogi et al., 1999). We here report the crystallization of V1-ATPase from T. thermophilus HB8. The protein was purified, as reported previously, without further modification (Yokoyama et al., 1990, 1994). Prior to the crystallization, the concentration of V1-ATPase was adjusted as required, and the protein solution was then incubated in the presence of 5 mM Mg-ADP at 328 K for 10 min in the buffer containing 20 mM Tris–HCl, pH 7.0. Crystallization conditions were screened by use of the sparse-matrix approach (Jancarik and Kim, 1991). The hanging drop vapor diffusion method was employed throughout the crystallization study. Crystals of V1-ATPase were initially observed for several different conditions, all containing sodium acetate. The final crystallization procedure was as follows: 2 l of a solution containing 16.2 mg/ml Thermus V1-ATPase in 20 mM Tris– HCl, pH 7.2, and the same volume of a reservoir solution were equilibrated against 500 l of reservoir at 298 K for 2–3 weeks. The reservoir solution contained 100 mM sodium acetate, 2 M sodium formate, and 5 mM Mg-ADP, adjusting pH to 5.5. Almost no difference was seen in the appearance and growth manner of the crystals at incubation temperatures between 288 and 298 K. Crystals of Thermus V1-ATPase with approximate dimensions of 36 ⫻ 36 ⫻ 143 m were frequently obtained as shown in Fig. 1. Larger crystals were rare, probably due to the tendency of V1-ATPase to unfold at the interfaces, forming a film of unstructured polypeptides that inhibited further equilibration. The crystals were analyzed with polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate after being gathered by centrifugation. No difference was seen between the mobilities of each subunit from the
TABLE I Crystallographic Data for the Initial Data Collection Source Beamline Mode Detector Wavelength (Å) Maximum resolution (Å) Distance (mm) Oscillation angle (degrees) Frame Exposure time (s) Dezinger Mosaicity (degrees) Number of crystals Crystal dimensions Crystal system Cell dimensions (Å) a ⫽ b ⫽ c ⫽ Space group Resolution range (Å) Measured reflection Unique reflection Multiplicity 具I/ 典 Rmergea Completeness (%)
PF BL-6A 3 GeV Multi-bunch ADSC Quantum 4R CCD detector 1.000 3.4 300 5 18 600 Used 0.220–0.440 1 0.036 mm ⫻ 0.036 mm ⫻ 0.143 mm Trigonal 89.0 179.2 P3 20–6.5 8563 2970 2.8 4.5 0.142 96.5
Note. The unit cell is a ⫽ b ⫽ 89.0 Å, c ⫽ 179.2 Å, ␣ ⫽  ⫽ 90°, ␥ ⫽ 120°, and the space group is P3. a Rmerge ⫽ ¥兩I i ⫺ 具I典兩/¥I i, where I i is the measured intensity of an individual reflection, and 具I典 is the mean intensity of symmetry-related measurements of this reflection.
crystals and from the intact V1-ATPase solution (data not shown). The crystals obtained were mounted in cryo-loops followed by rapid dipping through a buffer containing the same components as the reservoir solution plus 30% glycerol as a cryo-protectant. Intensity data were collected at 105 K using synchrotron radiation at the BL-6A beamline station of the Photon Factory, KEK, Tsukuba, Japan. The X-ray beam was monochromatized to 1.00 Å with an Si (111) monochromator, and an aperture collimator of 0.1-mm diameter was used. Oscillation photographs were taken by the ADSC Quantum 4R CCD detector. The oscillation range was 5° during a 10-min exposure and the crystal-to-CCD detector distance was 300 mm. The data were processed using DPS/ MOSFLM (Leslie, 1992; Rossman and van Beek, 1999) and programs from the CCP4 suite (Collaborative Computing Project No. 4, 1994). Preliminary intensity data were collected in which the diffraction extended beyond 3.4-Å resolution (Fig. 2). The data indicated that the crystals belong to the trigonal space group P3. The unit cell dimensions were determined as a ⫽ b ⫽ 89.0 Å, c ⫽ 179.2 Å, and ␥ ⫽ 120°. Assuming that the unit cell con-
CRYSTALLIZATION NOTE
tains one molecule of V1-ATPase with a molecular mass of 404 kDa, the V m value is calculated as 3.0 Å3/Da, which is within the range observed for protein crystals (Matthews, 1968), and corresponds to a solvent content of 59.6%. The statistics for the initial data collection are summarized in Table I. The initial processing of data has been restricted to the diffraction range of 20 – 6.5 Å, due to the anisotropic nature of the diffraction data obtained. There still remain several possibilities to assign the space group within the hexagonal system, such as P6, P63, or P6322, as well as to the trigonal system P3. The peripheral portion (hexagon) composed of the large subunits, A and B, may strongly dominate the diffraction data, especially if the small ␥ and ␦ subunits are located with some disorder, as has been described for the atomic model of mitochondrial F1 (Abrahams et al., 1994). Recognizing that the A and B subunits are highly homologous but not identical, and that there are only single copies of the ␥ and ␦ subunits in the V1 moiety, we have to be aware that the (pseudo-) symmetrical axis just suggested may be limited to only the low- and middle-resolution range. We thus conclude that the space group of P3 is the one that is most likely to describe the crystal at the current resolution range (20 – 6.5 Å). The unit cell dimensions seem reasonable from the view of the molecular size of V1-ATPase and from a comparison with the previously reported dimensions for the crystals of F1-ATPases (Abrahams et al., 1994; Lutter et al., 1993; Shirakihara et al., 1997). According to the report by Yokoyama et al. (1998), the crystals that we obtained and described in this article are likely to contain V1-ATPase molecules in the Mg-ADP-inhibited form. Thus, the structure analysis of this crystal form may help to elucidate the manner in which Mg-ADP associates with and inactivates V1-ATPase during ATP hydrolysis. The crystals obtained under these conditions can be used for structure determination at higher resolution. The further optimization of crystallization conditions and of the techniques for cryo-preservation is in progress, along with intensity data collection and structure determination. The data collection was performed under the approval of Photon Factory Advisory Committee (Proposal No. 00P003 and 01G339). We also thank Dr. R. M. Glaeser for useful discussion and for critically reading the manuscript. REFERENCES 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. Bianchet, M. A., Hullihen, J., Pedersen, P., and Amzel, L. M. (1998) The 2.8-Å structure of rat liver F1-ATPase: Configura-
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tion of a critical intermediate in ATP synthesis/hydrolysis, Proc. Natl. Acad. Sci. USA 95, 11065–11070. Bowman, B. J., Dscida, W. J., Harris, T., and Bowman, E. J. (1989) The vacuolar ATPase of Neurospola crassa contains an F1-like structure, J. Biol. Chem. 264, 15606 –15612. Boyer, P. D. (1993) The binding change mechanism for ATP synthase—Some probabilities and possibilities, Biochim. Biophys. Acta 1140, 215–240. Collaborative Computing Project No. 4 (1994) The CCP4 suites: Programs for protein crystallography, Acta Crystallogr. Sect. D 50, 760 –763. Forgac, M. (1989) Structure and function of vacuolar class of ATP-driven proton pumps, Physiol. Rev. 69, 765–796. Futai, M., Noumi, T., and Maeda, M. (1988) ATP synthase (H⫹ATPase): Results by combined biochemical and molecular biological approaches, Annu. Rev. Biochem. 58, 111–136. Inatomi, K. (1986) Characterization and purification of the membrane-bound ATPase of the archaebacterium Methanosarcina barkeri, J. Bacteriol. 167, 837– 841. Jancarik, J., and Kim, S.-H. (1991) Sparse matrix sampling: A screening method for crystallization of proteins, J. Appl. Crystallogr. 24, 409 – 411. Kakinuma, Y., and Igarashi, K. (1990) Some features of the Streptococcus faecalis Na⫹-ATPase resemble those of the vacuolar-type ATPases, FEBS Lett. 271, 97–101. Konishi, J., Wakagi, T., Oshima, T., and Yoshida, M. (1987) Purification and properties of the ATPase solubilized from membranes of an acidothermophilic archaebacterium, Sulfolobus acidocaldarius, J. Biochem. 102, 1379 –1387. Leslie, A. G. W. (1992) Joint CCP4 and ESF-EACMB Newsletter on Protein Crystallography No. 26, Daresbury Laboratory, Warrington, UK. Lutter, R., Abrahams, J. P., van Raaij, M. J., Todd, R. J., Lundqvist, T., Buchanan, S. K., Leslie, A. G. W., and Walker, J. E. (1993) Crystallization of F1-ATPase from bovine heart mitochondria, J. Mol. Biol. 229, 787–790. Matthews, B. W. (1968) Solvent content of protein crystals, J. Mol. Biol. 33, 491– 497. Nanba, T., and Mukohata, Y. (1987) A membrane-bound ATPase from Halobacterium halobium: Purification and characterization, J. Biochem. 102, 591–598. Nelson, N. (1992a) Evolution of organellar proton-ATPases, Biochim. Biophys. Acta 1100, 109 –124. Nelson, N. (1992b) The vacuolar H⫹-ATPase—One of the most fundamental ion pump in nature, J. Exp. Biol. 172, 19 –27. Nogi, T., Fukami, T. A., Ishida, M., Yoshida, M., and Miki, K. (1999) Purification, crystallization, and preliminary X-ray crystallographic analysis of Thermus thermophilus V1-ATPase B subunit, J. Struct. Biol. 127, 79 – 82. Noji, H., Yasuda, R., Yoshida, M., and Kinoshita, K., Jr. (1997) Direct observation of the rotation of F1-ATPase, Nature 386, 299 –302. Reidlinger, J., Mayer, F., and Muller, V. (1994) The molecular structure of Na⫹-translocating F1F0-ATPase of Acetobacterium woodii, as revealed by electron microscopy, resembles that of H⫹-translocating ATPases, FEBS Lett. 356, 17–20. Rossman, M. G., and van Beek, C. G. (1999) Data processing, Acta Crystallogr. Sect. D 55, 1631–1640. Senior, A. E. (1988) ATP synthesis by oxidative phosphorylation, Physiol. Rev. 68, 177–231. Shirakihara, Y., Leslie, A. G., Abrahams, J. P., Walker, J. E., Ueda, T., Sekimoto, Y., Kambara, M., Saika, K., Kagawa, Y., and Yoshida, M. (1997) The crystal structure of the nucleotide-
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free ␣33 subcomplex of F1-ATPase from the thermophilic Bacillus PS3 is a symmetric trimer, Structure 5, 825– 836. Stock, D., Leslie, A. G. W., and Walker, J. E. (1999) Molecular architecture of the rotary motor in ATP synthase, Science 286, 1700 –1705. Yasuda, R., Noji, H., Kinoshita, K., Jr., and Yoshida, M. (1998) F1-ATPase is a highly efficient molecular motor that rotates with discrete 120 degree steps, Cell 93, 1117–1124. Yokoyama, K., Oshima, T., and Yoshida, M. (1990) Thermus thermophilus membrane associated ATPase: Indication of a eubacterial V-type ATPase, J. Biol. Chem. 265, 21946 –21950.
Yokoyama, K., Akabane, Y., Ishii, N., and Yoshida, M. (1994) Isolation of prokaryotic V0V1-ATPase from a thermophilic eubacterium Thermus thermophilus, J. Biol. Chem. 269, 12248 – 12253. Yokoyama, K., Muneyuki, E., Amano, T., Mizutani, S., Yoshida, M., Ishida, M., and Ohkuma, S. (1998) V-ATPase of Thermus thermophilus is inactivated during ATP hydrolysis but can synthesize ATP, J. Biol. Chem. 273, 20504 –20510. Yokoyama, K., Ohkuma, S., Taguchi, H., Yasunaga, T., Wakabayashi, T., and Yoshida, M. (2000) V-type H⫹-ATPase/synthase from a thermophilic eubacterium, Thermus thermophilus, J. Biol. Chem. 275, 13955–13961.
CRYSTALLIZATION NOTE
Crystallization and Preliminary X-ray Studies of V1-ATPase of Thermus thermophilus HB8 Complexed with Mg-ADP Noriyuki Ishii,*,1,2 Shinya Saijo,† Takao Sato,† Nobuo Tanaka,† and Kazuaki Harata*,1 *Biophysical Chemistry Laboratory, National Institute of Bioscience and Human Technology, 1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan; and †Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Kanagawa 226-8501, Japan Received January 29, 2001, and in revised form April 9, 2001; published online June 13, 2001
sociated with a fairly small integral membrane sector, F0, which functions as a proton channel. In prokaryotes, for example, F1 consists of ␣33␥1␦1⑀1 subunits, and F0 of a 1 b 2 c 12 , though there are some more additional subunits in eukaryotic counterparts. On the other hand, V-type, or V0V1-, ATPase resides in endomembrane systems such as endosomes, lysosomes, Golgi-related vesicles, chromaffin granules, and the vacuolar membranes of plants, yeast, and Neurospora (Nelson, 1992b). V-type ATPase belongs to a class of ATP-driven proton pumps responsible for acidification of intracellular compartments in the cells. Although it had been believed that all H⫹-translocating ATPases in prokaryotic cells are F0F1-ATPases, some kinds of eubacteria as well as most archea have been found to hold V0V1-ATPases whose role is ATP synthesis coupled with proton flux across membranes (Inatomi, 1986; Kakinuma and Igarashi, 1990; Konishi et al., 1987; Nanba and Mukohata, 1987; Yokoyama et al., 1994). V0V1-ATPases consist of two functional sectors, as does the F0F1-ATPase, in this case designated as a peripheral V1 moiety and an integral membrane V0 moiety. The V1 moiety has an ATP synthesis/hydrolysis activity and is composed of two large subunits, A and B, and other smaller subunits. From electron microscopic studies (Bowman et al., 1989; Reidlinger et al., 1994; Yokoyama et al., 1994, 2000), the gross features of V1 have been shown to consist of a roughly spherical assembly with a 90- to 100-Å diameter, containing three copies each of the A and B subunits, which alternate in a hexagonal arrangement around an aqueous cavity in which the ␥ and ␦ subunits are located. The amino acid sequence analysis has shown that the A subunit in V1-ATPases is homologous to the  subunit in F1-
Crystals have been grown of the V1-ATPase sector of the V-type ATP synthase complex (V0V1) from the thermophilic eubacterium Thermus thermophilus HB8. These crystals are grown by the vapor diffusion method in the presence of 5 mM Mg-ADP, from solutions containing 100 mM sodium acetate and 2 M sodium formate, pH 5.5. The crystals diffracted X rays beyond 3.4 Å in resolution on a synchrotron radiation source. The crystals belong to the trigonal space group P3, with unit cell dimensions of a ⴝ b ⴝ 89.0 Å, c ⴝ 179.2 Å, and ␥ ⴝ 120°. The unit cell presumably contains one molecule of V1-ATPase and the Vm value is calculated as 3.0 Å3/Da. © 2001 Academic Press
Key Words: V1-ATPase; Thermus thermophilus; Xray crystallography.
Vacuolar (V)-type ATPase and F0F1-ATPase are two major subclasses, related by evolution, of a superfamily of proton translocating ATPases that do not involve the formation of phosphorylated enzymes as an intermediate (Forgac, 1989; Nelson, 1992a). F0F1-ATPase is the well-known ATP synthase. This important enzyme is present in the inner membranes of mitochondria, thylakoid membranes of chloroplasts, and plasma membranes of prokaryotic cells (Boyer, 1993; Senior, 1988; Futai et al., 1988). F-type ATPase is composed of several kinds of subunits and exists as a huge (⬃500 kDa) complex in which a water-soluble catalytic sector, F1, is as1 Present address: Biological Information Research Center, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan. 2 To whom correspondence should be addressed. E-mail: [email protected]. Fax: ⫹81-298-61-6194.
1047-8477/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. Crystals of Thermus thermophilus V1-ATPase grown in the presence of Mg-ADP. The approximate dimensions are 36 ⫻ 36 ⫻ 143 m. The inset shows an enlarged crystal having a typical shape.
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ATPases, and the B subunit in V1-ATPases is homologous to the ␣ subunits in F1-ATPases. Thus one can expect that the active sites for ATP synthesis/ hydrolysis for V1-ATPases should be located on the A subunit. The V0 moiety has a proton pump activity. Remarkable achievements have been reported recently in the structural studies of F0F1-ATPases (Abrahams et al., 1994; Bianchet et al., 1998; Shirakihara et al., 1997; Stock et al., 1999) as well as in kinetic mechanisms, including experimental demonstration of the rotation of the centrally located ␥ subunit in the ␣33 hexagon of the F1 moiety, coupled to ATP hydrolysis (Noji et al., 1997; Yasuda et al., 1998). The structure and mechanism reported for F0F1-ATPases are thought to be basically similar to those for V0V1-ATPases. However, information obtained about the enzymatic mechanism of ATP synthesis by F0F1-ATPases cannot tell us what is different about the mechanism used by V0V1ATPases to generate a proton gradient. A structure determination at high resolution is indispensable in order to understand these differences. Comparison
FIG. 2. Oscillation photograph of a crystal of Thermus thermophilus V1-ATPase taken with the use of the synchrotron radiation source. The oscillation range during a 10-min exposure was 5° and the distance from the crystal to the CCD detector was 300 mm. The resolution at the edge of the detector is 3.3 Å.
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of structures between F0F1-ATPases and V0V1ATPases would be expected to explain the known differences in enzymatic properties, such as the low specific ATPase activity, high K m values, and resistance to azide inhibition of V0V1-ATPases. A structural comparison at high resolution would also be expected to elucidate the common features involved in biological energy by which the ATP synthesis/ hydrolysis reaction is coupled to proton translocation across a membrane. The proton translocating ATPase found in the plasma membrane of an aerobic thermophilic eubacterium, Thermus thermophilus, belongs to the class of V0V1-ATPases (Yokoyama et al., 1990). The V1 moiety from T. thermophilus HB8 consists of four subunits with a stoichiometry of A 3 B 3 ␥ 1 ␦ 1 . The molecular masses of the individual subunits are approximately 66 ( A), 55 (B), 30 (␥), and 11 kDa (␦) (Yokoyama et al., 1998). The crystallization of the noncatalytic B subunit has been reported (Nogi et al., 1999). We here report the crystallization of V1-ATPase from T. thermophilus HB8. The protein was purified, as reported previously, without further modification (Yokoyama et al., 1990, 1994). Prior to the crystallization, the concentration of V1-ATPase was adjusted as required, and the protein solution was then incubated in the presence of 5 mM Mg-ADP at 328 K for 10 min in the buffer containing 20 mM Tris–HCl, pH 7.0. Crystallization conditions were screened by use of the sparse-matrix approach (Jancarik and Kim, 1991). The hanging drop vapor diffusion method was employed throughout the crystallization study. Crystals of V1-ATPase were initially observed for several different conditions, all containing sodium acetate. The final crystallization procedure was as follows: 2 l of a solution containing 16.2 mg/ml Thermus V1-ATPase in 20 mM Tris– HCl, pH 7.2, and the same volume of a reservoir solution were equilibrated against 500 l of reservoir at 298 K for 2–3 weeks. The reservoir solution contained 100 mM sodium acetate, 2 M sodium formate, and 5 mM Mg-ADP, adjusting pH to 5.5. Almost no difference was seen in the appearance and growth manner of the crystals at incubation temperatures between 288 and 298 K. Crystals of Thermus V1-ATPase with approximate dimensions of 36 ⫻ 36 ⫻ 143 m were frequently obtained as shown in Fig. 1. Larger crystals were rare, probably due to the tendency of V1-ATPase to unfold at the interfaces, forming a film of unstructured polypeptides that inhibited further equilibration. The crystals were analyzed with polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate after being gathered by centrifugation. No difference was seen between the mobilities of each subunit from the
TABLE I Crystallographic Data for the Initial Data Collection Source Beamline Mode Detector Wavelength (Å) Maximum resolution (Å) Distance (mm) Oscillation angle (degrees) Frame Exposure time (s) Dezinger Mosaicity (degrees) Number of crystals Crystal dimensions Crystal system Cell dimensions (Å) a ⫽ b ⫽ c ⫽ Space group Resolution range (Å) Measured reflection Unique reflection Multiplicity 具I/ 典 Rmergea Completeness (%)
PF BL-6A 3 GeV Multi-bunch ADSC Quantum 4R CCD detector 1.000 3.4 300 5 18 600 Used 0.220–0.440 1 0.036 mm ⫻ 0.036 mm ⫻ 0.143 mm Trigonal 89.0 179.2 P3 20–6.5 8563 2970 2.8 4.5 0.142 96.5
Note. The unit cell is a ⫽ b ⫽ 89.0 Å, c ⫽ 179.2 Å, ␣ ⫽  ⫽ 90°, ␥ ⫽ 120°, and the space group is P3. a Rmerge ⫽ ¥兩I i ⫺ 具I典兩/¥I i, where I i is the measured intensity of an individual reflection, and 具I典 is the mean intensity of symmetry-related measurements of this reflection.
crystals and from the intact V1-ATPase solution (data not shown). The crystals obtained were mounted in cryo-loops followed by rapid dipping through a buffer containing the same components as the reservoir solution plus 30% glycerol as a cryo-protectant. Intensity data were collected at 105 K using synchrotron radiation at the BL-6A beamline station of the Photon Factory, KEK, Tsukuba, Japan. The X-ray beam was monochromatized to 1.00 Å with an Si (111) monochromator, and an aperture collimator of 0.1-mm diameter was used. Oscillation photographs were taken by the ADSC Quantum 4R CCD detector. The oscillation range was 5° during a 10-min exposure and the crystal-to-CCD detector distance was 300 mm. The data were processed using DPS/ MOSFLM (Leslie, 1992; Rossman and van Beek, 1999) and programs from the CCP4 suite (Collaborative Computing Project No. 4, 1994). Preliminary intensity data were collected in which the diffraction extended beyond 3.4-Å resolution (Fig. 2). The data indicated that the crystals belong to the trigonal space group P3. The unit cell dimensions were determined as a ⫽ b ⫽ 89.0 Å, c ⫽ 179.2 Å, and ␥ ⫽ 120°. Assuming that the unit cell con-
CRYSTALLIZATION NOTE
tains one molecule of V1-ATPase with a molecular mass of 404 kDa, the V m value is calculated as 3.0 Å3/Da, which is within the range observed for protein crystals (Matthews, 1968), and corresponds to a solvent content of 59.6%. The statistics for the initial data collection are summarized in Table I. The initial processing of data has been restricted to the diffraction range of 20 – 6.5 Å, due to the anisotropic nature of the diffraction data obtained. There still remain several possibilities to assign the space group within the hexagonal system, such as P6, P63, or P6322, as well as to the trigonal system P3. The peripheral portion (hexagon) composed of the large subunits, A and B, may strongly dominate the diffraction data, especially if the small ␥ and ␦ subunits are located with some disorder, as has been described for the atomic model of mitochondrial F1 (Abrahams et al., 1994). Recognizing that the A and B subunits are highly homologous but not identical, and that there are only single copies of the ␥ and ␦ subunits in the V1 moiety, we have to be aware that the (pseudo-) symmetrical axis just suggested may be limited to only the low- and middle-resolution range. We thus conclude that the space group of P3 is the one that is most likely to describe the crystal at the current resolution range (20 – 6.5 Å). The unit cell dimensions seem reasonable from the view of the molecular size of V1-ATPase and from a comparison with the previously reported dimensions for the crystals of F1-ATPases (Abrahams et al., 1994; Lutter et al., 1993; Shirakihara et al., 1997). According to the report by Yokoyama et al. (1998), the crystals that we obtained and described in this article are likely to contain V1-ATPase molecules in the Mg-ADP-inhibited form. Thus, the structure analysis of this crystal form may help to elucidate the manner in which Mg-ADP associates with and inactivates V1-ATPase during ATP hydrolysis. The crystals obtained under these conditions can be used for structure determination at higher resolution. The further optimization of crystallization conditions and of the techniques for cryo-preservation is in progress, along with intensity data collection and structure determination. The data collection was performed under the approval of Photon Factory Advisory Committee (Proposal No. 00P003 and 01G339). We also thank Dr. R. M. Glaeser for useful discussion and for critically reading the manuscript. REFERENCES 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. Bianchet, M. A., Hullihen, J., Pedersen, P., and Amzel, L. M. (1998) The 2.8-Å structure of rat liver F1-ATPase: Configura-
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