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Structural and Functional Study of Binary Toxin Translocon by Cryo-EM and X-ray Crystallography

Research Project

Project/Area Number 18K06170
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeMulti-year Fund
Section一般
Review Section Basic Section 43040:Biophysics-related
Research InstitutionKyoto Sangyo University

Principal Investigator

TSUGE Hideaki  京都産業大学, 生命科学部, 教授 (40299342)

Co-Investigator(Kenkyū-buntansha) 吉田 徹  京都産業大学, 総合生命科学部, 研究員 (30724546)
Project Period (FY) 2018-04-01 – 2020-03-31
Project Status Completed (Fiscal Year 2019)
Budget Amount *help
¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000)
Fiscal Year 2020: ¥650,000 (Direct Cost: ¥500,000、Indirect Cost: ¥150,000)
Fiscal Year 2019: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2018: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
Keywordsウェルシュ菌 / 二成分毒素 / タンパク質膜透過 / ADPリボシル化 / ディフィシル菌 / クライオ電子顕微鏡 / 単粒子解析 / 超分子複合体 / イオタ毒素 / 膜結合 / アンフォールド / 膜孔 / トランスロコン / 複合体構造解析 / ADPリボシルトランスフェラーゼ / フォールディング / 膜孔形成毒素
Outline of Final Research Achievements

The iota toxin produced by Clostridium perfringens type E is a binary toxin comprising two independent polypeptides: Ia, an ADP-ribosyltransferase, and Ib, which is involved in cell binding and translocation of Ia across the cell membrane. Here we report cryo-EM structures of the translocation channel Ib-pore and its complex with Ia. The high-resolution Ib-pore structure demonstrates a similar structural framework to that of the catalytic phi-clamp of the anthrax protective antigen pore. However, the Ia-bound Ib-pore structure shows a unique binding mode of Ia: one Ia binds to the Ib-pore, and the Ia amino-terminal domain forms multiple weak interactions with two additional Ib-pore constriction sites. Furthermore, Ib-binding induces tilting and partial unfolding of the Ia N-terminal α-helix, permitting its extension to the phi-clamp gate. This new mechanism of N-terminal unfolding is crucial for protein translocation.

Academic Significance and Societal Importance of the Research Achievements

ウェルシュ菌の二成分毒素はアクチンをADPリボシル化する酵素成分のIaと、Iaを膜を介して細胞内に透過させる膜結合成分Ibからなる。Ibは中央に孔を持つIb膜孔を形成し、Iaがこの中を通過する。この二成分毒素複合体の構造を明らかにした。このことより、タンパク質膜透過の仕組みの一端を明らかにした。似た二成分毒素はディフィシル菌(ヒト感染症)や炭疽菌でもみられ、膜孔の阻害剤創薬の重要な知見となる。

Report

(3 results)
  • 2019 Annual Research Report   Final Research Report ( PDF )
  • 2018 Research-status Report
  • Research Products

    (18 results)

All 2020 2019 2018 Other

All Journal Article (2 results) (of which Peer Reviewed: 2 results,  Open Access: 1 results) Presentation (11 results) (of which Int'l Joint Research: 5 results,  Invited: 3 results) Remarks (4 results) Funded Workshop (1 results)

  • [Journal Article] Cryo-EM structures reveal translocational unfolding in the clostridial binary iota toxin complex2020

    • Author(s)
      Yamada Tomohito、Yoshida Toru、Kawamoto Akihiro、Mitsuoka Kaoru、Iwasaki Kenji、Tsuge Hideaki
    • Journal Title

      Nature Structural & Molecular Biology

      Volume: 27 Issue: 3 Pages: 288-296

    • DOI

      10.1038/s41594-020-0388-6

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Substrate N2 atom recognition mechanism in pierisin family DNA-targeting, guanine-specific ADP-ribosyltransferase ScARP2018

    • Author(s)
      Yoshida Toru、Tsuge Hideaki
    • Journal Title

      Journal of Biological Chemistry

      Volume: 293 Issue: 36 Pages: 13768-13774

    • DOI

      10.1074/jbc.ac118.004412

    • Related Report
      2018 Research-status Report
    • Peer Reviewed
  • [Presentation] Cryo-EM structure of clostridial binary toxin translocon Ib-pore2019

    • Author(s)
      Yamada T, Yoshida T, Kawamoto A, Mitsuoka K, Iwasaki K, and Tsuge H
    • Organizer
      European Workshops on Bacterial Protein Toxins - ETOX19
    • Related Report
      2019 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Diarrheagenic activity of each component of CPILE as a new enterotoxin produced by Clostridium perfringens isolated from foodborne outbreaks2019

    • Author(s)
      Monma C, Saiki D, Shimojima Y, Suzuki J, Sadamasu K, Yamada T, Tsuge H, Kamata Y
    • Organizer
      CLOSPATH 11
    • Related Report
      2019 Annual Research Report
    • Int'l Joint Research
  • [Presentation] ウェルシュ菌2成分毒素 膜孔複合体の単粒子解析2019

    • Author(s)
      山田等仁、津下英明
    • Organizer
      生理研研究会 クライオ電子顕微鏡によるタンパク質の高分解能単粒子構造解析~みんなのクライオ電顕~
    • Related Report
      2019 Annual Research Report
    • Invited
  • [Presentation] タンパク質膜透過チャネルの単粒子構造解析2019

    • Author(s)
      津下英明
    • Organizer
      第453回ビタミンB 研究協議会
    • Related Report
      2019 Annual Research Report
  • [Presentation] Cryo-EM structure of clostridial binary toxin translocation channel Ib-pore2019

    • Author(s)
      山田等仁、吉田徹、川本暁大、光岡薫、岩崎憲司、津下英明
    • Organizer
      第57回日本生物物理学会
    • Related Report
      2019 Annual Research Report
  • [Presentation] Clostridium Binary Toxin:膜孔形成タンパク質トロンスロコンの作用機構について2019

    • Author(s)
      津下英明
    • Organizer
      京都薬科大学セミナー
    • Related Report
      2018 Research-status Report
    • Invited
  • [Presentation] Membrane transport and ADP-ribosylation mechanism of binary toxin C. perfringens iota toxin2018

    • Author(s)
      Yoshida T, Yamada T, and Tsuge H
    • Organizer
      International Symposium on “Proteins; from the Cradle to the Grave”
    • Related Report
      2018 Research-status Report
    • Int'l Joint Research / Invited
  • [Presentation] Substrate recognition and reaction mechanism of DNA-targeting guanine-specific ADP-ribosyltransferase2018

    • Author(s)
      Yoshida T, and Tsuge H
    • Organizer
      International Symposium on “Proteins; from the Cradle to the Grave”
    • Related Report
      2018 Research-status Report
    • Int'l Joint Research
  • [Presentation] Cryo-EM single particle analysis of C. perfringens binary toxin2018

    • Author(s)
      Yamada T, Yoshida T, and Tsuge H
    • Organizer
      International Symposium on “Proteins; from the Cradle to the Grave”
    • Related Report
      2018 Research-status Report
    • Int'l Joint Research
  • [Presentation] DNAを標的としたADPリボシル化酵素の基質認識機構2018

    • Author(s)
      津下英明
    • Organizer
      第453回ビタミンB 研究協議会
    • Related Report
      2018 Research-status Report
  • [Presentation] From Protein to DNA Modification: Substrate recognition mechanism in pierisin family DNA-targeting guanine-specific ADP-ribosyltransferase ScARP2018

    • Author(s)
      津下英明
    • Organizer
      日本分子生物学会 ワークショップADP-ribosylation in intra- & extracellular communication and diseases
    • Related Report
      2018 Research-status Report
  • [Remarks] クライオ電子顕微鏡により明らかにした細菌毒素タンパク質の膜透過機構

    • URL

      https://www.kyoto-su.ac.jp/news/20200303_400a_thnews.html

    • Related Report
      2019 Annual Research Report
  • [Remarks] Press Release:クライオ電子顕微鏡により明らかにした細菌毒素タンパク質の膜透過機構

    • URL

      http://www.protein.osaka-u.ac.jp/mibunrui/press-release20200310_cryo

    • Related Report
      2019 Annual Research Report
  • [Remarks] 細菌毒素タンパク質の膜透過機構の一端を解明

    • URL

      http://www.tsukuba.ac.jp/attention-research/p202003101400.html

    • Related Report
      2019 Annual Research Report
  • [Remarks] Posing just before protein translocation

    • URL

      https://naturemicrobiologycommunity.nature.com/users/352568-hideaki-tsuge/posts/61288-posing-just-before-protein-translocation-in-bacterial-binary-toxin

    • Related Report
      2019 Annual Research Report
  • [Funded Workshop] 京都産業大学生命科学セミナー2018

    • Related Report
      2018 Research-status Report

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Published: 2018-04-23   Modified: 2021-02-19  

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