Shinya Saijo
Tokyo University of Science
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Featured researches published by Shinya Saijo.
Nature | 2013
Satoshi Arai; Shinya Saijo; Kano Suzuki; Kenji Mizutani; Yoshimi Kakinuma; Yoshiko Ishizuka-Katsura; Noboru Ohsawa; Takaho Terada; Mikako Shirouzu; Shigeyuki Yokoyama; So Iwata; Ichiro Yamato; Takeshi Murata
In various cellular membrane systems, vacuolar ATPases (V-ATPases) function as proton pumps, which are involved in many processes such as bone resorption and cancer metastasis, and these membrane proteins represent attractive drug targets for osteoporosis and cancer. The hydrophilic V1 portion is known as a rotary motor, in which a central axis DF complex rotates inside a hexagonally arranged catalytic A3B3 complex using ATP hydrolysis energy, but the molecular mechanism is not well defined owing to a lack of high-resolution structural information. We previously reported on the in vitro expression, purification and reconstitution of Enterococcus hirae V1-ATPase from the A3B3 and DF complexes. Here we report the asymmetric structures of the nucleotide-free (2.8 Å) and nucleotide-bound (3.4 Å) A3B3 complex that demonstrate conformational changes induced by nucleotide binding, suggesting a binding order in the right-handed rotational orientation in a cooperative manner. The crystal structures of the nucleotide-free (2.2 Å) and nucleotide-bound (2.7 Å) V1-ATPase are also reported. The more tightly packed nucleotide-binding site seems to be induced by DF binding, and ATP hydrolysis seems to be stimulated by the approach of a conserved arginine residue. To our knowledge, these asymmetric structures represent the first high-resolution view of the rotational mechanism of V1-ATPase.
Journal of Molecular Biology | 2008
Mayuko Akaboshi; Hiroshi Hashimoto; Hanako Ishida; Shinya Saijo; Nozomu Koizumi; Mamoru Sato; Toshiyuki Shimizu
Calcium signals mediate a multitude of plant responses to external stimuli. Calcineurin B-like (CBL) proteins and their target kinases, CBL-interacting protein kinases (CIPKs), represent important relays in plant calcium signaling. CBL interacts with CIPK through a conserved motif (NAF/FISL motif) within the C-terminal regulatory domain. To better understand the functional role of the CBL-CIPK system, we determined the crystal structure of AtCBL2 in complex with the regulatory domain of AtCIPK14 at 1.2 A resolution. The NAF/FISL motif is inserted into a hydrophobic crevice within AtCBL2, accompanied by a large displacement of the helices and loop on the opposite side of the NAF/FISL motif from the C-terminal region, which shields the hydrophobic crevice in free form. Ca(2+) are coordinated within four EF hands in AtCBL2 in bound form. This calcium coordination pattern differs from that in the structure of the SOS3-SOS2 complex previously reported. Structural comparison of the two structures shows that the recognition of CBL by CIPK is performed in a similar manner, but inherent interactions confer binding affinity and specificity.
Acta Crystallographica Section D-biological Crystallography | 2000
Takao Sato; Yusuke Yamada; Shinya Saijo; Tetsuya Hori; Raita Hirose; Nobuo Tanaka; Gen Sazaki; Kazuo Nakajima; Noriyuki Igarashi; Masahiko Tanaka; Yoshiki Matsuura
Orthorhombic crystals of hen egg-white (HEW) lysozyme were grown in a homogeneous and static magnetic field of 10 T. All crystals grown at 10 T were oriented such that their crystallographic c axes were parallel to the magnetic field direction and showed a narrower average full-width at half-maximum (FWHM) of the rocking curve than those grown at 0 T. Rocking-width measurements were made at the BL-10A station at the Photon Factory, Tsukuba, Japan, using a high-resolution vertical-type four-circle diffractometer. Crystal perfection was evaluated using the FWHM of the rocking curve; the effects of the magnetic field on the quality of the crystals were examined by comparison of the FWHM of seven crystals grown at 10 and 0 T. The FWHMs of the reflections along the a, b and c axes decreased by 23.5, 35.3 and 27.8%, respectively, and those of other general reflections decreased by 17.4-42.2% in the crystals grown at high magnetic field. These results clearly showed that a magnetic field of 10 T improved the crystal perfection of the orthorhombic lysozyme crystals. As a result, the maximum resolution of X-ray diffraction increased from 1.3 A at 0 T to 1.13 A at 10 T. The magnetic field also affected the dimensions of the unit cell, increments being 0.2% for the a and c axes and 0.1% for the b axis, respectively. These facts suggest that the application of a high magnetic field during crystallization might result in remarkable enhancements in the diffraction power of protein crystals having magnetic anisotropy.
Proceedings of the National Academy of Sciences of the United States of America | 2011
Shinya Saijo; Satoshi Arai; Khandoker Mohammad Mozaffor Hossain; Ichiro Yamato; Kano Suzuki; Yoshimi Kakinuma; Yoshiko Ishizuka-Katsura; Noboru Ohsawa; Takaho Terada; Mikako Shirouzu; Shigeyuki Yokoyama; So Iwata; Takeshi Murata
V-ATPases function as ATP-dependent ion pumps in various membrane systems of living organisms. ATP hydrolysis causes rotation of the central rotor complex, which is composed of the central axis D subunit and a membrane c ring that are connected by F and d subunits. Here we determined the crystal structure of the DF complex of the prokaryotic V-ATPase of Enterococcus hirae at 2.0-Å resolution. The structure of the D subunit comprised a long left-handed coiled coil with a unique short β-hairpin region that is effective in stimulating the ATPase activity of V1-ATPase by twofold. The F subunit is bound to the middle portion of the D subunit. The C-terminal helix of the F subunit, which was believed to function as a regulatory region by extending into the catalytic A3B3 complex, contributes to tight binding to the D subunit by forming a three-helix bundle. Both D and F subunits are necessary to bind the d subunit that links to the c ring. From these findings, we modeled the entire rotor complex (DFdc ring) of V-ATPase.
Acta Crystallographica Section D-biological Crystallography | 2004
Takao Sato; Shusaku Hara; Takuro Matsui; Gen Sazaki; Shinya Saijo; Tadashi Ganbe; Nobuo Tanaka; Yasushi Sugano; Makoto Shoda
The dye-decolorizing peroxidase DyP is a key enzyme in the decolorizing fungus Thanatephorus cucumeris Dec 1 that degrades azo and antraquinone dyes. The gene dyp from T. cucumeris Dec 1, which has low homology to other peroxidase genes, was cloned and transformed into Aspergillus oryzae and glycosylated DyP was expressed at high levels. Purified DyP was deglycosylated using GST Endo F1 and then crystallized in a strong magnetic field (10 T) at 283 K using ammonium sulfate as precipitant. X-ray diffraction data to 2.96 A resolution collected from a native crystal at the Photon Factory (Tsukuba, Japan) showed that the crystal belonged to the hexagonal space group P6(5)22, with unit-cell parameters a = b = 136.15, c = 363.46 A. The asymmetric unit of the crystal contained four DyP molecules, with a corresponding Matthews coefficient (V(M)) of 2.50 A(3) Da(-1) and a solvent content of 51%. Heavy-atom derivatives of DyP have been obtained and electron-density maps have been calculated. The haem is visible and continuous electron density between the haem and protein clearly indicates the location of the proximal histidine ligand.
Journal of Biological Chemistry | 2008
Misaki Yamamoto; Satoru Unzai; Shinya Saijo; Kazuki Ito; Kenji Mizutani; Chiyo Suno-Ikeda; Yukako Yabuki-Miyata; Takaho Terada; Mitsutoshi Toyama; Mikako Shirouzu; Takuya Kobayashi; Yoshimi Kakinuma; Ichiro Yamato; Shigeyuki Yokoyama; So Iwata; Takeshi Murata
The vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain and an integral membrane domain connected by a central stalk and a few peripheral stalks. The number and arrangement of the peripheral stalk subunits remain controversial. The peripheral stalk of Na+-translocating V-ATPase from Enterococcus hirae is likely to be composed of NtpE and NtpF (corresponding to subunit G of eukaryotic V-ATPase) subunits together with the N-terminal hydrophilic domain of NtpI (corresponding to subunit a of eukaryotic V-ATPase). Here we purified NtpE, NtpF, and the N-terminal hydrophilic domain of NtpI (NtpINterm) as separate recombinant His-tagged proteins and examined interactions between these three subunits by pulldown assay using one tagged subunit, CD spectroscopy, surface plasmon resonance, and analytical ultracentrifugation. NtpINterm directly bound NtpF, but not NtpE. NtpE bound NtpF tightly. NtpINterm bound the NtpE-F complex stronger than NtpF only, suggesting that NtpE increases the binding affinity between NtpINterm and NtpF. Purified NtpE-F-INterm complex appeared to be monodisperse, and the molecular masses estimated from analytical ultracentrifugation and small-angle x-ray scattering (SAXS) indicated that the ternary complex is formed with a 1:1:1 stoichiometry. A low resolution structure model of the complex produced from the SAXS data showed an elongated “L” shape.
Acta Crystallographica Section D-biological Crystallography | 2005
Shinya Saijo; Yusuke Yamada; Takao Sato; Nobuo Tanaka; Takuro Matsui; Gen Sazaki; Kazuo Nakajima; Yoshiki Matsuura
A novel method has been developed to improve protein-crystal perfection during crystallization in a high magnetic field and structural studies have been undertaken. The three-dimensional structure of orthorhombic hen egg-white (HEW) lysozyme crystals grown in a homogeneous and static magnetic field of 10 T has been determined and refined to a resolution of 1.13 angstroms and an R factor of 17.0%. The 10 T crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 56.54 (3), b = 73.86 (6), c = 30.50 (2) angstroms and one molecule per asymmetric unit. A comparison of the structures of the 0 T and 10 T crystals has been carried out. The magnitude of the structural changes, with a root-mean-square deviation value of 0.75 angstroms for the positions of all protein atoms, is similar to that observed when an identical protein structure is resolved in two different crystalline lattices. The structures remain similar, with the exception of a few residues e.g. Arg68, Arg73, Arg128 and Gln121. The shifts of the arginine residues result in very significant structural fluctuations, which can have large effects on a proteins crystallization properties. The high magnetic field contributed to an improvement in diffraction intensity by (i) the displacement of the charged side chains of Arg68 and Arg73 in the flexible loop and of Arg128 at the C-terminus and (ii) the removal of the alternate conformations of the charged side chains of Arg21, Lys97 or Arg114. The improvement in crystal perfection might arise from the magnetic effect on molecular orientation without structural change and differences in molecular interactions. X-ray diffraction and molecular-modelling studies of lysozyme crystals grown in a 10 T field have indicated that the field contributes to the stability of the dihedral angle. The average difference in conformational energy has a value of -578 kJ mol(-1) per charged residue in favour of the crystal grown in the magnetic field. For most protein atoms, the average B factor in the 10 T crystal shows an improvement of 1.8 angstroms(2) over that for the 0 T control; subsequently, the difference in diffraction intensity between the 10 T and 0 T crystals corresponds to an increase of 22.6% at the resolution limit. The mosaicity of the 10 T crystal was better than that of the 0 T crystal. More highly isotropic values of 0.0065, 0.0049 and 0.0048 degrees were recorded along the a, b and c axes, respectively. Anisotropic mosaicity analysis indicated that crystal growth is most perfect in the direction that corresponds to the favoured growth direction of the crystal, and that the crystal grown in the magnetic field had domains that were three times the volume of those of the control crystal. Overall, the magnetic field has improved the quality of these crystals and the diffracted intensity has increased significantly with the magnetic field, leading to a higher resolution.
Journal of Crystal Growth | 2001
Takao Sato; Yusuke Yamada; Shinya Saijo; Tetsuya Hori; Raita Hirose; Nobuo Tanaka; Gen Sazaki; Kazuo Nakajima; Noriyuki Igarashi; Masahiko Tanaka; Yoshiki Matsuura
Orthorhombic crystals of hen egg-white lysozyme (HEWL) were grown under a homogeneous magnetic field of 10 T (B = 10 T) In the magnetic field, crystals were oriented such that their crystallographic c-axes were parallel to the magnetic field, and gave narrower average rocking widths than those grown under 0 T. Crystal quality was evaluated using the full-width at half-maximum (FWHM) of the rocking curve, and the effects of a magnetic field were examined by comparing FWHM of seven crystals grown at 10 T with an equal number grown at OT. FWHM values in (1000), (080), and (006) reflections decreased by 25%, 38%, and 32%, respectively, and those of general reflections decreased by 7-45% in crystals grown under high magnetic field. These results show that a magnetic field of 10 T improved the crystal perfection of the orthorhombic lysozyme crystals. At the same time, the maximum resolution limit of X-ray diffraction increased from 1.33 A for 0 T to 1.13 A for 10 T. These facts suggest that the application of magnetic field for crystallization might generally have a striking effect in enhancing the diffraction power of protein crystals.
Biochemical and Biophysical Research Communications | 2009
Satoshi Arai; Ichiro Yamato; Asuka Shiokawa; Shinya Saijo; Yoshimi Kakinuma; Yoshiko Ishizuka-Katsura; Mitsutoshi Toyama; Takaho Terada; Mikako Shirouzu; Shigeyuki Yokoyama; So Iwata; Takeshi Murata
Enterococcus hirae vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain (V(1); NtpA(3)-B(3)-D-G) and an integral membrane domain (V(0); NtpI-K(10)) connected by a central and peripheral stalk(s) (NtpC and NtpE-F). Here we examined the nucleotide binding of NtpA monomer, NtpB monomer or NtpD-G heterodimer purified by using Escherichia coli expression system in vivo or in vitro, and the reconstitution of the V(1) portion with these polypeptides. The affinity of nucleotide binding to NtpA was 6.6 microM for ADP or 3.1 microM for ATP, while NtpB or NtpD-G did not show any binding. The NtpA and NtpB monomers assembled into NtpA(3)-B(3) heterohexamer in nucleotide binding-dependent manner. NtpD-G bound NtpA(3)-B(3) forming V(1) (NtpA(3)-B(3)-D-G) complex independent of nucleotides. The V(1) formation from individual NtpA and NtpB monomers with NtpD-G heterodimer was absolutely dependent on nucleotides. The ATPase activity of reconstituted V(1) complex was as high as that of native V(1)-ATPase purified from the V(0)V(1) complex by EDTA treatment of cell membrane. This in vitro reconstitution system of E. hirae V(1) complex will be valuable for characterizing the subunit-subunit interactions and assembly mechanism of the V(1)-ATPase complex.
Acta Crystallographica Section D-biological Crystallography | 2001
Yusuke Yamada; Shinya Saijo; Takao Sato; Noriyuki Igarashi; Hideo Usui; Taketomo Fujiwara; Nobuo Tanaka
Catalase-peroxidases are bifunctional enzymes found in many microorganisms. Crystals of catalase-peroxidase from the halophilic archaeon Haloarcula marismortui were obtained using the hanging-drop vapour-diffusion method. The rhombic plate-shaped crystals were grown from purified protein solution using (NH(4))(2)SO(4) as precipitant at 293 K. The crystal belongs to the monoclinic system, space group C2, and diffracted beyond 2.0 A resolution.