膜結合型AAA-ATPase p47/p97複合体による細胞膜認識とその制御

• Project/Area Number: 15083206
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Yokohama City University
• Principal Investigator: JEREMYR.H. Tame (TAME J.R.H. / J・R・H Tame / JEREMY R・H Tame / JEREMY R.H.Tame) Yokohama City University, 国際総合科学研究科, 教授 (00336588)
• Co-Investigator(Kenkyū-buntansha): 朴 三用 横浜市立大学, 国際総合科学研究科, 准教授 (20291932) ; 雲財 悟 横浜市立大学, 国際総合科学研究科, 助教 (60336592)
• Project Period (FY): 2003 – 2008
• Project Status: Completed (Fiscal Year 2008)
• Budget Amount: ¥80,600,000 (Direct Cost: ¥80,600,000); Fiscal Year 2008: ¥14,800,000 (Direct Cost: ¥14,800,000); Fiscal Year 2007: ¥14,800,000 (Direct Cost: ¥14,800,000); Fiscal Year 2006: ¥14,400,000 (Direct Cost: ¥14,400,000); Fiscal Year 2005: ¥14,400,000 (Direct Cost: ¥14,400,000); Fiscal Year 2004: ¥14,800,000 (Direct Cost: ¥14,800,000); Fiscal Year 2003: ¥7,400,000 (Direct Cost: ¥7,400,000)
• Keywords: X線結晶構造解析 / タンパク質 / 膜輸送 / 膜蛋白質 / 蛋白質間相互作用 / 生物物理 / 蛋白質

Research Abstract

(1)TRAPチューブ
E50CのみTRAPに変異導入したものとV69Cのみに変異導入したものをチューブの最小単位となるように作製した。これはTRAP同士がどのように結合しチューブとなるかをX線結晶構造解析により解明するためである。D8H、F32D、E50C、R66H、V69C(以下HCCDH)と、D8H、F32A、E50C、R66H、V69C(以下HCCAH)というように5箇所に変異を導入し、E50とV69に導入したシステインによりS-S結合を形成することを予想し設計した。
(2)TRAP-AT複合体
Bacillus subtilisの細胞質の中では、TRAP活性はanti-TRAPというタンパク質に制御されており、トリプトファンの不足している状態で、TRAPとantiTRAPは複合体を作る。したがって、RNA結合が阻害される。今まではどのようにTRAPとantiTRAPが相互作用しているか明らかではなかった。そのため、我々はこの複合体の結晶化を行い、結晶構造を決定した。
TRAP-antiTRAP複合体のX線結晶構造を決定した。結晶化とともに、TRAPは11量体から12量体になった。結晶対称軸とTRAPリング軸を合わせて、非対称単位が数個の分子しか含まない。それから、通常では大きい複合体は十分に分解能回折データを測ることができないが、今回は高い分解能のデータを測ることができた。

Research Products

• [Journal Article] The nature of the TRAP-Anti-TRAP complex (2009)
  • Author(s): Watanabe M, et.al.
  • Journal Title: Proc Natl Acad Sci USA 106(7) Pages: 2176-81
  • Peer Reviewed
• [Journal Article] The structural basis for an essential subunit interaction in influenza virus RNA polymerase (2008)
  • Author(s): Obayashi E, et.al.
  • Journal Title: Nature 454(7208) Pages: 1127-31
  • Peer Reviewed
• [Journal Article] Intersubunit linker length as a modifier of protein stability : crvstal structures and thermostability of mutant TRAP (2008)
  • Author(s): Watanabe M, et.al.
  • Journal Title: ProteinSci 17(3) Pages: 518-26
  • Peer Reviewed
• [Journal Article] Intersubunit linker length as a modifier of protein stability:crystal structures and thermostability of mutant TRAP (2008)
  • Author(s): Watanabe M, Mishima Y, Yamashita I, Park SY, Tame JR, Heddle JG
  • Journal Title: Protin Sci. 17(3) Pages: 518-26
  • Peer Reviewed
• [Journal Article] EFFect of N-terminal reidues on the structural stability of recmbinant horse L-chain apoferritin in an acidic environment (2007)
  • Author(s): Yoshizwa K, Mishima Y, Park SY, Heddle JG, Tame JR, Iwahori K, Kobayashi M, Yamashita I
  • Journal Title: J Biochem 142(6) Pages: 707-13
  • Peer Reviewed
• [Journal Article] Using the ringshaped protein TRAP to capture and confine gold nanod ots on a surface (2007)
  • Author(s): Heddle JG, Fujiwara I, Yamadaki H, Yoshii S, Nishio K, Addy C, Yamsdhita I, Tame JR
  • Journal Title: Small 3(11) Pages: 1950-6
  • Peer Reviewed
• [Journal Article] Crystallication and preliminary crystallographic studies of the metallogl ycoprotein esterase A4 using a baculovirus expressio system (2007)
  • Author(s): Hiraki T, Shibayama N, Yoon YH, Yun KM, Hamamoto T, Tame JR, Park SY
  • Journal Title: Acta Crystallogr sect F struct Biol Cryst Commun 63(Pt9) Pages: 734-6
  • Peer Reviewed
• [Journal Article] Structure and mechanism of HpcG,a hydratase in the homoprotocatech uate degradation pathway of Escherichia coli (2007)
  • Author(s): Izumi A, Rea D, Adachi T, Unzai S, Park SY, Roper DI, Tame JR
  • Journal Title: J Mol Biol 370(5) Pages: 899-911
  • Peer Reviewed
• [Journal Article] Dynamic allostery in the ring protein TRAP (2007)
  • Author(s): Heddle JG, Okajima T, Scott DJ, Akashi S, Psrk SY, Tame JR
  • Journal Title: J Mol Biol 371(1) Pages: 154-67
  • Peer Reviewed
• [Journal Article] Limited tolerance towards folded elements during secretion of the autotransporter Hbp. (2007)
  • Author(s): Jong WS, ten Hagen-Jongman CM, den Blaauwen T, Jan Slotboom D, Tame JR, Wickstrom D, de Gier JW, Otto BR, Luinnk J.
  • Journal Title: Mol Microbiol. 63(5) Pages: 1524-1536
• [Journal Article] Nickel binding to NikA : an additional binding site reconciles spectroscopy, calorimetry and crystallography. (2007)
  • Author(s): Addy C, Chara M, Kawai F, Kidera A, Ikeguchi M, Fuchigami S, Osawa M, Shimada I, Park SY, Tame JR, Heddle JG
  • Journal Title: Acta Crystallogr D Biol Crystallogr. 63(Pt 2) Pages: 221-229
• [Journal Article] 1.25 A resolution crystal structures of human haemoglobin in the oxy, deoxy and carbonmonoxy forms. (2006)
  • Author(s): Park SY, Yokoyama T, Shibayama N, Shiro Y, Tame JR
  • Journal Title: J Mol Biol. 360(3) Pages: 690-701
• [Journal Article] Rounding up : Engineering 12-membered rings from the cyclic 11-mer TRAP. (2006)
  • Author(s): Heddle JG, Yokoyama T, Yamashita I, Park SY, Tame JR
  • Journal Title: Structure. 14(5) Pages: 925-933
• [Journal Article] The Swi5-Sfrl complex stimulates Rhp51/Rad51- and Dmcl-mediated DNA strand exchange in vitro. (2006)
  • Author(s): Haruta N, Kurokawa Y, Murayama Y, Akamatsu Y, Unzai S, Tsutsui Y, Iwasaki H
  • Journal Title: Nat Struct Mol Biol 13(9) Pages: 823-830
• [Journal Article] Structural basis for protein recognition by B30.2/SPRY domains (2006)
  • Author(s): J-S Woo, H-Y Suh, S-Y Park, B-H Oh
  • Journal Title: Mol. Cell 24 Pages: 967-976
• [Journal Article] Crystal Structure of penicillin Binding Protein4(dacB)from Escherichia coli, both in the Native Form and Covalently Linked to Various Antibiotics. (2006)
  • Author(s): Kishida, H., Unzai, S., Roper, D.I., Lloyd, A., Park, S.Y., Tame, J.R.
  • Journal Title: Biochemistry 45(3) Pages: 783-792
• [Journal Article] R-state Haemoglobin with Low Oxygen Affinity : Crystal Structures of Deoxy Human and Carbonmonoxy Horse Haemoglobin Bound to the Effector Molecule L35. (2006)
  • Author(s): Yokoyama, T., Neya, S., Tsuneshige, A., Yonetani, T., Park, S.Y., Tame, J.R.
  • Journal Title: Journal of Molecular Biology 356(3) Pages: 790-801
• [Journal Article] Scoring functions--the first 100 years. (2005)
  • Author(s): Tame, J.R
  • Journal Title: J.Comput.Aided.Mol.Des 19(6) Pages: 445-451
• [Journal Article] Characterization of an iron-regulated alpha-enolase of Bacteroides fragilis. (2005)
  • Author(s): Sijbrandi, R., Den Blaauwen, T., Tame, J.R., Oudega, B., Luirink, J., Otto, B.R.
  • Journal Title: Microbes.Infect. 7(1) Pages: 9-18
• [Journal Article] Backbone 1H,13C, and 15N assignments of a 56 kDa E.coli nickel binding protein NikA. (2005)
  • Author(s): Rajesh, S., Heddle, J.G, Kurashima-Ito, K., Nietlispach, D., Shirakawa, M., Tame, J.R., Ito, Y.
  • Journal Title: J.Biomol.NMR 32(2) Pages: 177-177
• [Journal Article] Crystal structure of hemoglobin protease, a heme binding autotransporter protein from pathogenic Escherichia coli. (2005)
  • Author(s): Otto, B.R., Sijbrandi, R., Luirink, J., Oudega, B., Heddle, J.G., Mizutani, K., Park, S.Y., Tame, J.R.
  • Journal Title: J.Biol.Chem. 280(17) Pages: 17339-17345
• [Journal Article] Crystal structure of heme binding protein, an autotransporter hemoglobin protease from pathogenic escherichia coli. (2005)
  • Author(s): Otto, B.R., et al.
  • Journal Title: Journal of Biological Chemistry (in press)
• [Journal Article] Crystal structure of a novel polyisoprenoid-binding protein from Thermus thermophilus HB8. (2005)
  • Author(s): Handa, N., et al.
  • Journal Title: Protein Science 14(4) Pages: 1004-1010
• [Journal Article] Characterization of an iron-regulated alpha-enolase of Bacteroides fragilis. (2004)
  • Author(s): Sijbrandi, R., et al.
  • Journal Title: Microbes and infection 7(1) Pages: 9-18
• [Journal Article] Crystal structure of a conserved hypothetical protein TT1751 from Thermus thermophilus HB8. (2004)
  • Author(s): Kishishita, S., et al.
  • Journal Title: Proteins 57(4) Pages: 883-887
• [Journal Article] Crystal structures of unliganded and half-liganded human hemoglobin derivatives cross-linked between Lys 82betal and Lys 82beta2. (2004)
  • Author(s): Park, S.Y., et al.
  • Journal Title: Biochemistry 43(27) Pages: 87111-87117
• [Journal Article] Crystal structure of elongation factor P from Thermus thermophilus HB8. (2004)
  • Author(s): Hanawa-Suetsugu K., et al.
  • Journal Title: Proc.Natl.Acad.Sci.USA. 101(26) Pages: 9595-9600
• [Journal Article] Novel mechanisms of pH sensitivity in tuna hemoglobin : a structural explanation of the root effect. (2004)
  • Author(s): Yokoyama, T., et al.
  • Journal Title: Journal of Biological Chemistry 279(27) Pages: 28632-28640
• [Journal Article] Crystal structures of catalytic domains of pseudouridine synthases RluC and RluD from Escherichia coli. (2004)
  • Author(s): Mizutani, K., et al.
  • Journal Title: Biochemistry 43(15) Pages: 4454-4463
• [Presentation] What a crystal structure doesn't tell you (2008)
  • Author(s): Jeremy R.H. Tame
  • Organizer: よこはまNMR構造生物学研究会国際シンポジウム
  • Place of Presentation: 横浜
• [Presentation] Structural insights into serine protease autotoransporter HBP rfom path ogentic Escherichia coli (2008)
  • Author(s): J.Tame
  • Organizer: Society for General Microbiology
  • Place of Presentation: Edinburgh,UK
• [Presentation] X-ray Crystallography of Autotransporters from Pathogenic Bacteria (2007)
  • Author(s): Kaoru Nishimura, Sam-Yog Park, and Jeremy Tame
  • Organizer: Molecular Microbiology Young Microbiologists Minisymposium Prokaryotic Membranes
  • Place of Presentation: Paris,France
  • Year and Date: 2007-10-12
• [Presentation] Dynamic allosteRy in the tRyptohan-binding pRotein TRAP (2007)
  • Author(s): Heddle JG, Akashi S, Psrk SY, Tame JR
  • Organizer: Biophysice of ligand binding to drug tagets
  • Place of Presentation: Strasbourg,France
• [Book] 「生命秩序を担う生体超分子」蛋白質核酸酵素増刊 (2005)
  • Author(s): 雲財 悟, 朴 三用, Jeremy R.H.Tame
  • Total Pages: 273
  • Publisher: 共立出版
• [Publications] Heddle J, Scott DJ, Unzai S, Park SY, Tame JR.: "Crystal structures of the liganded and unliganded nickel-binding protein NikA from Escherichia coli."Journal of Biological Chemistry.. 278(50). 50322-50329 (2003)
• [Publications] Wada, T., Kuzuyama, T., Satoh, S., Kuramitsu, S., Yokoyama, S., Unzai, S., Tame, J.R.H., Park, S.Y.: "Crystal structure of 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase, an enzyme in the non-mevalonate pathway of isoprenoid synthesis"Journal of Biological Chemistry. 278. 30022-30027 (2003)
• [Publications] Handa, N., et al.: "Crystal structure of the conserved protein TT1542 from Thermus thermophilus HB8."Protein Science. 12. 1621-1632 (2003)
• [Publications] Adachi, S., Park, S.Y., Tame, J.R.H., Shiro, Y., Shibayama, N.: "Direct observation of photolysis-induced tertiary structural changes in hemoglobin."Proc.Natl.Ac.Sci.USA. 100. 7039-7044 (2003)
• [Publications] H.Kishida, et al.: "The crystal structure and catalytic mechanism of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MECDP) synthase, an enzyme in the nonmevalonate pathway of isoprenoid synthesis"Acta Crystallog.. D59. 23-31 (2003)
• [Publications] T.Wada, et al.: "Structure of a conserved CoA-binding protein synthesized a cell-free system"Acta Crystallog.. D59. 1213-1218 (2003)