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P型イオンポンプによる能動輪送機構の構造的解明

Research Project

Project/Area Number 14GS0308
Research Category

Grant-in-Aid for Creative Scientific Research

Allocation TypeSingle-year Grants
Research InstitutionThe University of Tokyo

Principal Investigator

豊島 近  The University of Tokyo, 分子細胞生物学研究所, 教授 (70172210)

Co-Investigator(Kenkyū-buntansha) 杉田 有冶 (杉田 有治)  独立行政法人理化学研究所, 中央研究所, 准主任研究員 (80311190)
津田 岳夫  東京大学, 分子細胞生物学研究所, 助教 (10345233)
木寺 詔紀  横浜市立大学, 大学院・国際総合科学研究科, 教授 (00186280)
鈴木 裕  旭川医科大学, 医学部, 教授 (50183421)
Project Period (FY) 2002 – 2006
Project Status Completed (Fiscal Year 2006)
Budget Amount *help
¥406,900,000 (Direct Cost: ¥339,430,000、Indirect Cost: ¥67,470,000)
Fiscal Year 2006: ¥56,550,000 (Direct Cost: ¥43,500,000、Indirect Cost: ¥13,050,000)
Fiscal Year 2005: ¥66,690,000 (Direct Cost: ¥51,300,000、Indirect Cost: ¥15,390,000)
Fiscal Year 2004: ¥76,830,000 (Direct Cost: ¥59,100,000、Indirect Cost: ¥17,730,000)
Fiscal Year 2003: ¥92,300,000 (Direct Cost: ¥71,000,000、Indirect Cost: ¥21,300,000)
Fiscal Year 2002: ¥114,530,000 (Direct Cost: ¥114,530,000)
Keywords生物物理 / 能動輸送 / 膜蛋白質 / イオンポンプ / X線結晶 / ATPase / X線結晶解析
Research Abstract

本研究の最大の目標は筋小胞体カルシウムポンプ(ATPase)の反応中間体を結晶化し、原子構造に基づいてポンプの作動機構を明らかにすることであった。分解能が十分でなかったE2・AlFx複合体の結晶性を改良し、反応過程ほぼ全体をカバーする6つの状態に関し2.6Åを越える分解能で構造を決定出来た。しかし、真のE2Pアナログの結晶であるE2・BeFxに関しては不満足なものしか得られていなかった。そこで、研究期間を延長し、強力阻害剤タプシガーギン存在下ではあるが、この状態に関しても2.4Å分解能で構造を決定でき、燐酸化アスパラギン酸の処理と膜内イオン通路の開閉がどう連携しているかをある程度明らかにすることが出来た。第2の柱である計算構造生物学では、特に心筋における調節蛋白として重要なフォスフォランバンの燐酸化による制御に関し徹底的な計算を行い、燐酸化の構造的意味を明らかにした。第3の柱である、発現系に関しては、筋小胞体カルシウムポンプを一残基改変することによって、抗マラリア薬に反応することが判明したので、大量生産・精製を行い結晶化可能な標品を得ることが出来た。また、重金属ポンプに関しては、好熱菌銅イオンポンプCopAに関し、蛋白質分解酵素による限定分解によって、蛋白質のトポロジーは大きく異なるにもかかわらず、カルシウムポンプと同様の構造変化を示すことを明らかにし、未知であったN端末金属結合ドメインの役割をも明らかにした。さらに、細胞質領域の2つのドメインと核酸複合体の高分解能結晶解析に成功した。このように、研究のすべての面で大きく前進した。

Report

(5 results)
  • 2006 Annual Research Report
  • 2005 Annual Research Report
  • 2004 Annual Research Report
  • 2003 Annual Research Report
  • 2002 Annual Research Report
  • Research Products

    (22 results)

All 2007 2006 2005 2004 Other

All Journal Article (13 results) Publications (9 results)

  • [Journal Article] Domain organization and movements in heavy metal ion pumps: Papain digestion of CopA, a Cu^+-transporting ATPase.2007

    • Author(s)
      Y. Hatori
    • Journal Title

      J. Biol. Chem. 282

      Pages: 25213-25221

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Interdomain communication in calcium pump as revealed in the crystal structures with transmembrane inhibitors.2007

    • Author(s)
      M. Takahashi
    • Journal Title

      Proc. Nat. Acad. Sci. USA. 104

      Pages: 5800-5805

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Concerted conformational effects of Ca^<2+> and ATP are required for activation of sequential reactions in the Ca^<2+> ATPase (SERCA) catalytic cycle.2006

    • Author(s)
      G. Inesi
    • Journal Title

      Biochemistry 46

      Pages: 13769-13778

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Structural changes of the cytoplasmic domain of phospholamban by phosphorylation at Ser16: A molecular dynamics study.2006

    • Author(s)
      Y. Sugita
    • Journal Title

      Biochemistry 45

      Pages: 11752-11761

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Effects of inhibitors on luminal opening of Ca^<2+> binding sites in an E2P-like complex of sarcoplasmic reticulum Ca^<2+>-ATPase with Be^<2+>-fluoride.2006

    • Author(s)
      M. Picard
    • Journal Title

      J. Biol. Chem. 281

      Pages: 3360-3369

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Comprehensive Analysis of Expression and Function of 51 Sarco (endo) plasmic Reticulum Ca^<2+>-ATPase Mutants Associated with Darier Disease.2006

    • Author(s)
      Y. Miyauchi
    • Journal Title

      J. Biol. Chem. 281

      Pages: 22882-22895

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Structural role of countertransport revealed in Ca^<2+> pump crystal structure in the absence of Ca^<2+>.2005

    • Author(s)
      K.Obara
    • Journal Title

      Proc. Nat. Acad. Sci. USA 102

      Pages: 14489-14496

    • Related Report
      2005 Annual Research Report
  • [Journal Article] The average conformation at micromolar [Ca^<2+>] of Ca^<2+>-ATPase with bound nucleotide differs from that adopted with the transition state analog ADP.AIFx or with AMPPCP under crystallization conditions at millimolar [Ca^<2+>].2005

    • Author(s)
      M.Picard
    • Journal Title

      J. Biol. Chem. 280

      Pages: 18745-18754

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Protonation of the acidic residues in the transmembrane cation-binding sites of the Ca^<2+> pump.2005

    • Author(s)
      Y.Sugita
    • Journal Title

      J. Am. Chem. Soc. 127

      Pages: 6150-6451

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Lumenal gating mechanism revealed in calcium pump crystal structures with phosphate analogues.2004

    • Author(s)
      C.Toyoshima
    • Journal Title

      Nature 432

      Pages: 361-368

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Crystal structure of the calcium pump wlth a bound ATP analogue.2004

    • Author(s)
      C.Toyoshima
    • Journal Title

      Nature 430

      Pages: 529-535

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Specific structural requirement for the inhibitory effect of thapsigargin on the Ca^<2+> ATPase SERCA2004

    • Author(s)
      C.Xu
    • Journal Title

      J.Biol.Chem. 279

      Pages: 17973-17979

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Structural basis of ion pumping by Ca^<2+> -ATPase of the sarcoplasmic reticulum2004

    • Author(s)
      C.Toyoshima
    • Journal Title

      Ann.Rev.Biochem. 73

      Pages: 269-92

    • Related Report
      2004 Annual Research Report
  • [Publications] C.Toyoshima: "Structural basis of ion pumping by Ca^<2+>-ATPase of sarcoplasmic reticulum."FEBS Letters. 555. 106-110 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] H.Ma: "Substrate-induced conformational fit and headpiece closure in the Ca^<2+>-ATPase (SERCA)."J.Biol.Chem.. 278. 28938-28943 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] C.Toyoshima: "Modeling of the inhibitory interaction of phospholamban with the Ca^<2+>-ATPase."Proc.Nat.Acad.Sci.USA. 100. 467-472 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] C.Toyoshima: "Modeling of the inhibitory interaction of phospholamban with the Ca^<2+>ATPase"Proc.Nat.Acad.Sci. USA. 100. 467-472 (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] C.Toyoshima: "Structural changes in the calcium pump accompanying the dissociation of calcium"Nature. 418. 605-611 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] H.Ogawa: "Homology modeling of the cation binding sites of Na^+ K^+ -ATPase"Proc.Nat.Acad.Sci. USA. 99. 15977-15982 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] S.Hua: "Functional role of "N" (Nucleotide) and "P" (Phosphorylation) domain interactions in the sarcoplasmic reticulum (SERCA) ATPase"Biochemistry. 41. 2264-2272 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] S.Hua: "Fe^<2+>-catalyzed oxidation and cleavage of sarcoplasmic reticulum ATPase reveals Mg^<2+> and Mg^<2+>-ATP site"Biochemistry. 41. 11405-11410 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] J.V.Moller: "Calcium transport by sarcoplasmic reticulum Ca^<2+>-ATPase : role of the A-domain and its C-terminal link with the transmembrane region"J.Biol.Chem.. 41. 38647-38659 (2002)

    • Related Report
      2002 Annual Research Report

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Published: 2002-04-01   Modified: 2016-04-21  

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