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X線結晶解析による金属蛋白質の構造生物化学

Research Project

Project/Area Number 12147206
Research Category

Grant-in-Aid for Scientific Research on Priority Areas

Allocation TypeSingle-year Grants
Review Section Biological Sciences
Research InstitutionNara Institute of Science and Technology

Principal Investigator

箱嶋 敏雄  奈良先端科学技術大学院大学, 情報科学研究科, 教授 (00164773)

Co-Investigator(Kenkyū-buntansha) 北野 健  奈良先端科学技術大学院大学, 情報科学研究科, 助手 (40346309)
岡田 健吾  奈良先端科学技術大学院大学, 情報科学研究科, 助手 (60304169)
清水 敏之  奈良先端科学技術大学院大学, バイオサイエンス研究科, 助手 (30273858)
Project Period (FY) 2000 – 2004
Project Status Completed (Fiscal Year 2004)
Budget Amount *help
¥51,400,000 (Direct Cost: ¥51,400,000)
Fiscal Year 2004: ¥10,000,000 (Direct Cost: ¥10,000,000)
Fiscal Year 2003: ¥9,900,000 (Direct Cost: ¥9,900,000)
Fiscal Year 2002: ¥14,500,000 (Direct Cost: ¥14,500,000)
Fiscal Year 2001: ¥17,000,000 (Direct Cost: ¥17,000,000)
KeywordsX線 / 構造生物学 / 金属 / ジストニア / アロステリック
Research Abstract

X線結晶解析により金属蛋白質、およびその制御蛋白質やシグナル分子(あるいは基質)との複合体の結晶構造を決定し、得られた三次元構造をもとに金属の果たす役割とその制御メカニズムを解明することを目的とした。特に、制御蛋白質などとの相互作用を通して、金属を含むセンサーあるいは活性部位の配向や構造、蛋白質内部での金属周辺の環境や金属の配位状態がどのように変化して、活性等の切り換え(スウィッチ)機構が達成されるかを主眼にして、研究を推進した。
MgイオンあるいはMnイオンを必須として(2-metal center)、枝分かれしたDNAを切りそろえるFEN-1 (flap endonuclease 1)の構造解析と機能解析を完了して、論文発表した。
このFEN-1は、5'末端突出DNA (flap DNA)を特異的に切断する構造特異的ヌクレアーゼであり、DNA複製ではラギング鎖の岡崎フラグメントのRNAプライマーを、塩基除去修復では損傷塩基を含むオリゴヌクレオチドを切断除去する。FEN-1の酵素活性は、この酵素をDNAに固定するDNAクランプであるproliferating cell nuclear antigen (PCNA)と結合することによって、大幅に促進される。ヒト由来のFEN-1とPCNAとの複合体結晶構造をもとに、両者間の相互作用には、ドメイン-ドメインのものと、ドメイン-ペプチド性のものがあり、FEN-1のコアドメインとPCNAリングとの相互作用がFEN-1を不活性な、しかしDNA上の移動には都合のよい、状態に保つというメカニズムを提唱し、変異実験による解析によって、証明した。
また、FEN-1、PCNA、DNAの三者複合体の結晶化については、今後の展開に期待できるところまで、推進することができた。

Report

(5 results)
  • 2004 Annual Research Report
  • 2003 Annual Research Report
  • 2002 Annual Research Report
  • 2001 Annual Research Report
  • 2000 Annual Research Report
  • Research Products

    (26 results)

All 2005 2004 Other

All Journal Article (5 results) Publications (21 results)

  • [Journal Article] Structural basis for recruitment of human flap endonuclease 1 to PCNA.2005

    • Author(s)
      S.Sakurai
    • Journal Title

      EMBO J 24・4

      Pages: 683-693

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Crystal-structure of the protein histidine phosphatase SixA in the multistep His-Asp phospharelay.2005

    • Author(s)
      K.Hamada
    • Journal Title

      Genes to Cells 10・1

      Pages: 1-11

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Structural basis of biopterin-induced inhibition of GTP cyclohydrolase I by GFRP, its feedback regulatory proteins.2004

    • Author(s)
      N.Maita
    • Journal Title

      J.Biol.Chem. 279・49

      Pages: 51534-51540

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Crystal structure of the inhibitory GTP Cyclohydrolase I and GFRP complex2004

    • Author(s)
      T.Hatakeyama
    • Journal Title

      Pterins, Folates and Neurotransmitters in Molecular Medicine

      Pages: 16-19

    • Related Report
      2004 Annual Research Report
  • [Journal Article] 核酸分子認識とその機能を中心とした構造生物学2004

    • Author(s)
      箱嶋 敏雄
    • Journal Title

      日本結晶学会誌 46

      Pages: 201-208

    • NAID

      10013129619

    • Related Report
      2004 Annual Research Report
  • [Publications] Sakurai, S.: "Preparation and crystallization of human flap endonuclease FEN-1 in complex with proliferating-cell nuclear antigen, PCNA."Acta Crystallogr.D. D59・5. 933-935 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Hakoshima, T.: "Structural basis of the Rho GTPase signaling."J.Biochem.(Tokyo). 134・3. 327-331 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Shimizu, T.: "Parallel coiled-coil association of the RhoA-binding domain in Rho-kinase."J.Biol.Chem.. 278・46. 46046-46051 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Hakoshima, T.: "Nature Encyclopedia of the Human Genome, Vol.3"Nature Pub. Group. 1023 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] S.Terawaki: "Crystallographic characterization of the radixin FERM domain bound to the C-terminal region of the human Na^+/H^+ exchanger regulatory factor (NHERF)"Acta Crystallogr. D. 59・1. 177-179 (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] K.Hamada: "Structural basis of adhesion protein recognition by ERM proteins revealed by the crystal structure of the FERM/ICAM-2 complex"EMBO J.. 22・3. 502-514 (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] S.Sakurai: "Preparation and crystalization of human flap endonuclease FEN-1 in complex with proliferating cell nuclear antigen, PCNA"Acta Crystallogr. D. (印刷中). (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] 箱嶋 敏雄: "Rhoシグナリングの構造生物学"生化学「特集:構造生物学の最前線」(稲垣冬彦・箱崎敏雄編). 74・10. 1237-1242 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] T.Shimizu: "Structural basis for neurofibromatosis Type 2: Crystal structure the Merlin FERM domain of Merlin"J. Biol. Chem.. 277・12. 10332-10336 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] 北野 健: "ポストシーケンス タンパク質実験法 第3巻 構造機能解析の基礎(上)"東京化学同人. 23 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Maita, N.: "Crystal structure of the stimulatory complex of GTP cyclohydrolase I and its feedback regulatory protein GFRP"Proc. Natl. Acad. Sci. USA. 99・3. 1213-1217 (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] Shimizu, T.: "Crystal strucuture of the FERM domain of Merlin, the neurofibromatosis2(NF-2)tumor suppressor protein"J. Biol. Chem.. (印刷中). (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] Hamada, K.: "Crystallization and preliminary crystallographic studies RhoGDI in complex with the radixin FERM domain"Acta Cryst. D. Biol. Crystallogr.. 57・6. 889-890 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Hamada, K.: "Crystallographic characterization of the radixin FERM domain bound to the cytosolic tail of the adhesion protein ICAM-2"Acta Cryst. D. Biol. Crystallogr.. 57・6. 891-892 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Ishikawa, H.: "Structural conversion between open and closed forms of radixin : low-angle shadowing electron microscopy"J. Mol. Biol.. 310・5. 973-978 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Maita, N.: "Crystallographic characterization of the stimulatory and inhibitory complexes of GTP cyclohydrase I and its feedback reguratory protein, GFRP"Acta Cryst. D. Biol. Crystallogr.. 57・8. 1153-1156 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] K.Hamada: "Structural basis of the membrane-targeting"EMBO J.. 19(17). 4449-4462 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Y.Fuju: "Structural basis for the DNA recognition"Nature Struct.Biol.. 7(10). 889-893 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] T.Shimizu: "An open conformation of switch I"J.Biol.Chem.. 275(24). 18311-18317 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] K.Hamada: "Crystallographic characterization of the "Acta Crystallegr.D. 56. 922-923 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] K.Ihara: "Crystallization and prelimiuarg"Acta Crgstallogr.D. 56. 1042-1044 (2000)

    • Related Report
      2000 Annual Research Report

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Published: 2001-04-01   Modified: 2018-03-28  

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