Structural analysis of SecA-SecYE complex from Thermus thermophilus

• Project/Area Number: 19370085
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Cell biology
• Research Institution: Kyoto University
• Principal Investigator: MORI Hiroyuki Kyoto University, ウイルス研究所, 准教授 (10243271)
• Project Period (FY): 2007 – 2009
• Project Status: Completed (Fiscal Year 2009)
• Budget Amount: ¥13,650,000 (Direct Cost: ¥10,500,000、Indirect Cost: ¥3,150,000); Fiscal Year 2009: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000); Fiscal Year 2008: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000); Fiscal Year 2007: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000)
• Keywords: 生体膜 / SecA / SecYEG / トランスロコン / タンパク質膜透過膜 / ATPase / X線結晶構造解析 / バクテリア / 化学架橋 / SecYE / Fabフラグメント / タンパク質膜透過 / 膜タンパク質 / 膜タンパク質複合体 / モノクローナル抗体 / タンパク質膜透過装置 / Translocon / SecY / E / 好熱菌

Research Abstract

Evolutionarily conserved membrane protein complex, the Sec translocon, functions as a protein-conducting channel to facilitate secretory protein export and membrane protein integration. In this project, we determined the first atomic resolution crystal structure of the SecYE translocon from a SecA (bacterial translocation motor)-containing organism, Thermus thermophilus, in complex with an anti-SecY Fab fragment. The structure has revealed a significant conformational change from the previously determined archaeal SecYEβ structure, in that several SecY transmembrane helices are shifted to create a hydrophobic crevasse open to the cytoplasm. Disulfide mapping indicate that this "pre-open" state of the bacterial SecYE complex was induced by the Fab fragment and SecA, which in common bind to SecY residues at the tip of the cytoplasmic domain. Our disulfide crosslinking experiments revealed that some of these SecY residues contact specific residues of SecA that are otherwise buried and unavailable in the isolated SecA molecule. These results suggest that the motor components of the Sec machinery undergo cooperative conformational changes upon their interaction, presumably as important steps for the entrance of the preprotein-SecA complex and enhancement of the ATPase activity of SecA.

Research Products

• [Journal Article] 細菌型Secトランスロコンの構造から明らかとなった蛋白質膜透過装置の構造変化 (2009)
  • Author(s): 塚崎智也、森博幸
  • Journal Title: 生物物理 49 Pages: 288-289
• [Journal Article] 立体構造解析からみえてきたSecAによる蛋白質膜透過の分子メカニズム (2009)
  • Author(s): 森博幸、塚崎智也
  • Journal Title: 蛋白質核酸酵素 54 Pages: 685-695
• [Journal Article] 細菌型Secトランスロコンの構造から明らかとなった蛋白質膜透過装置の構造変化 (2009)
  • Author(s): 塚崎智也, 森博幸
  • Journal Title: 生物物理 49 Pages: 288-289
• [Journal Article] 立体構造解析からみえてきたSecAによる蛋白質膜透過の分子メカニズム (2009)
  • Author(s): 森博幸, 塚崎智也
  • Journal Title: 蛋白質核酸酵素 54 Pages: 685-695
• [Journal Article] (2009)
  • Author(s): Ito K., Mori H.
  • Journal Title: Bacterial Secreted Proteins in The Sec protein secretion system(Caister Academic Press, Norfolk, UK) Pages: 3-22
• [Journal Article] Conformational transition of Sec machinery inferred from bacterial SecYE structures. (2008)
  • Author(s): Tsukazaki, T. *, Mori, H. *, Fukai, S., Ishitani, R., Mori, T., Dohmae, N., Perederina, A., Sugita, Y., Vassylyev, D.G., Ito, K., Nureki O.
  • Journal Title: Nature 455 Pages: 988-991
  • NAID: 10025975982
  • Peer Reviewed
• [Journal Article] Conformational transition of Sec machinery inferred from bacterial SecYE structures. (2008)
  • Author(s): Tsukazaki, T., Mori, H., et al. (計11名)
  • Journal Title: Nature 455 Pages: 988-991
  • NAID: 10025975982
  • Peer Reviewed
• [Presentation] Structure and function of bacterial protein translocation machinery (2010)
  • Author(s): Mori H., Tsukazaki T., Echizen, Y., Nureki, O., Ito, K.
  • Organizer: 第83回日本細菌学会総会W5「細菌のタンパク質分泌系」
  • Place of Presentation: パシフィコ横浜
  • Year and Date: 2010-03-27
• [Presentation] タンパク質膜透過補助因子SecDFの構造と機能. (2010)
  • Author(s): 森博幸
  • Organizer: 2009年度国立遺伝学研究所共同研究会「ゲノム情報を活用した単細胞細胞増殖システムの研究」
  • Place of Presentation: 三島
• [Presentation] Structure and function of bacterial protein translocation machinery (2010)
  • Author(s): Mori H., Tsukazaki T., Echizen, Y., Nureki, O., Ito, K.
  • Organizer: 第83回日本細菌学会総会W5「細菌のタンパク質分泌系」
  • Place of Presentation: 横浜
• [Presentation] Molecular mechanisms of SecA mediated protein translocation (2009)
  • Author(s): 森博幸, 塚崎智也, 濡木理, 伊藤維昭
  • Organizer: 第47回日本生物物理学会年会 シンポジウム「タンパク質の膜透過輸送の最前線」
  • Place of Presentation: 徳島文理大学
  • Year and Date: 2009-10-30
• [Presentation] バクテリアのタンパク質膜透過装置の構造と機能 (2009)
  • Author(s): 森博幸, 塚崎智也, 越前友香, 濡木理, 伊藤維昭
  • Organizer: 第82回日本生化学会大会 シンポジウム「タンパク質膜透過装置の構造とダイナミックな機能」
  • Place of Presentation: 神戸ポートピアホテル
  • Year and Date: 2009-10-21
• [Presentation] Functional dissection of bacterial Sec translocation machinery (2009)
  • Author(s): Mori H., Tsukazaki T., Echizen, Y., Ishitani, R., Nureki, O., Ito, K.
  • Organizer: International Symposium 'Innovative Nanoscience of Supermolecular Motor Proteins Working in Biomembranes'
  • Place of Presentation: 芝蘭会館 (京都)
  • Year and Date: 2009-09-08
• [Presentation] Molecular mechanisms of SecA mediated protein translocation. (2009)
  • Author(s): 森博幸、塚崎智也、濡木理、伊藤維昭
  • Organizer: 第47回日本生物物理学会年会シンポジウム「タンパク質の膜透過輸送の最前線」
  • Place of Presentation: 徳島
• [Presentation] バクテリアのタンパク質膜透過装置の構造と機能. (2009)
  • Author(s): 森博幸、塚崎智也、越前友香、濡木理、伊藤維昭
  • Organizer: 第82回日本生化学会大会シンポジウム「タンパク質膜透過装置の構造とダイナミックな機能」
  • Place of Presentation: 神戸
• [Presentation] Structural analysis of bacterial Sec translocation machinery. (2009)
  • Author(s): Tsukazaki T., Mori H., Echizen, Y., Fukai, S., Ishitani, R., Mori, T., Dohmae, N., Perederina, A., Sugita, Y., Vassylyev, D.G., Ito, K., Nureki, O.
  • Organizer: International Symposium‘Innovative Nanoscience of Supermolecular Motor Proteins Working in Biomembranes'
  • Place of Presentation: Kyoto, Japan
• [Presentation] Functional dissection of bacterial Sec translocation machinery. (2009)
  • Author(s): Mori H., Tsukazaki T., Echizen, Y., Ishitani, R., Nureki, O., Ito, K.
  • Organizer: International Symposium‘Innovative Nanoscience of Supermolecular Motor Proteins Working in Biomembranes'
  • Place of Presentation: Kyoto, Japan
• [Presentation] Crystal structure of T. thermophilus Sec translocon with an Fab fragment implies a translocation-initiating state of SecY. (2008)
  • Author(s): Tsukazaki, T., Mori, H., et al. (計11名)
  • Organizer: 第31回日本分子生物学会年会、第81回日本生化学会大会 合同大会
  • Place of Presentation: 神戸国際会議場
  • Year and Date: 2008-12-12
• [Presentation] How does SecA ATPase interact with translocon and drive protein translocation? (2008)
  • Author(s): Mori, H., Tsukazaki, T., Nureki, O. and Ito, K.
  • Organizer: G-COE International Symposium Frontier of Organelle Dynamics and Protein Functions
  • Place of Presentation: 名古屋(メルパルク名古屋)
  • Year and Date: 2008-03-13
• [Presentation] Crystal structure of T. thermophilus Sec translocon with an anti-SecY Fab fragment implies a translocation-initiating state of SecY (2008)
  • Author(s): Tsukazaki T., Mori H., Fukai, S., Ishitani, R., Mori, T., Dohmae, N., Perederina, A., Sugita, Y., Vassylyev, D.G., Ito, K., Nureki, O.
  • Organizer: 第31回日本分子生物学会年会、第81回日本生化学会大会合同大会シンポジウム「タンパク質機能発現システム-シャペロンからトランスロケーターまで」
  • Place of Presentation: 神戸
• [Presentation] 細菌型Secトランスロコン複合体の結晶構造解析 (2008)
  • Author(s): Tsukazaki T., Mori H., Fukai, S., Ishitani, R., Perederina, A., Vassylyev, D.G., Ito, K., Nureki, O.
  • Organizer: 第46回日本生物物理学会シンポジウム「膜を介した物質輸送・シグナル変換の構造基盤」
  • Place of Presentation: 福岡
• [Presentation] タンパク質膜透過装置の構造と駆動メカニズム (2008)
  • Author(s): 森博幸
  • Organizer: 国立遺伝学研究所研究会「細胞周期制御をめぐる単細胞システム分子生物学」
  • Place of Presentation: 三島
• [Presentation] How does SecA ATPase interact with translocon and drive protein translocation? (2008)
  • Author(s): Mori H.
  • Organizer: G-COE in Chemistry "Frontier of Organelle Dynamics and Protein Functions""
  • Place of Presentation: Nagoya, Japan
• [Presentation] SecA ATPaseはどのようにtransloconと相互作用し、タンパク質膜透過を駆動しているのか? (2008)
  • Author(s): 森博幸
  • Organizer: 大阪大学蛋白質研究所セミナー「蛋白質の膜透過と膜挿入の分子メカニズム-その核心に迫る」
  • Place of Presentation: 大阪
• [Presentation] How does SecA ATPase drive protein translocation? (2007)
  • Author(s): Mori H., Ito K.
  • Organizer: The 14th East Asia Joint Symposium on Biomedical Research
  • Place of Presentation: Tokyo
  • Year and Date: 2007-11-14
• [Presentation] How does SecA ATPase drive protein translocation? (2007)
  • Author(s): 森博幸
  • Organizer: 第40回日本発生生物学会・第59回日本細胞生物学会合同大会
  • Place of Presentation: 福岡
• [Book] The Sec protein secretion system. in Bacterial Secreted Proteins, Ed. K. Wooldridge (2009)
  • Author(s): Ito, K., Mori, H.
  • Publisher: Academic Press
• [Remarks]
  • URL: http://www.virus.kyoto-u.ac.jp/Lab/akiyama/index.html