Molecular mechanisms underlying the selective membrane localization of bacterial lipoproteins

• Project/Area Number: 18K05396
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 38020:Applied microbiology-related
• Research Institution: Morioka College
• Principal Investigator: TOKUDA Hajime 盛岡大学, 盛岡大学顧問, 名誉教授 (40125943)
• Co-Investigator(Kenkyū-buntansha): 垰 和之 東京大学, アイソトープ総合センター, 助教 (00211996) ; 村上 聡 東京工業大学, 生命理工学院, 教授 (30300966) ; 成田 新一郎 山形県立米沢栄養大学, 健康栄養学部, 教授 (30338751)
• Project Period (FY): 2018-04-01 – 2023-03-31
• Project Status: Completed (Fiscal Year 2022)
• Budget Amount: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000); Fiscal Year 2020: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2019: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2018: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: リポ蛋白質 / 細胞質膜 / 外膜 / 蛋白質局在化 / 阻害剤 / Lolシステム / 選別シグナル / 細菌リポ蛋白質 / 蛋白質輸送 / 大腸菌 / ペリプラズム / 化学架橋 / 分子シャペロン / Lol因子 / ABCトランスポーター / 膜局在化 / 細胞膜 / 局在化 / 脂質修飾タンパク質 / 細菌型リポ蛋白質 / 構造・機能相関

Outline of Final Research Achievements

Approximately 100 species of lipoproteins in E. coli are transported to the outer membrane, while some remains in cytoplasmic membranes. Lipoprotein sorting and transport to the outer membrane is catalyzed by a mechanism consisting of five Lol factors. The purpose of this study was to elucidate the molecular mechanism of the Lol system based on its structure. We introduced a light-sensitive amino acid analog into various positions of LolCDE in the cytoplasmic membrane, and analyzed the binding to lipoproteins by light-dependent cross-linking reaction. As a result, we clarified the lipoprotein binding sites of LolC and LolE. We also revealed the interaction site of LolC and LolE. Furthermore, we established an in vitro experimental system to analyze the lipoprotein recognition and release reaction by LolCDE, and clarified the inhibitory mechanism of low-molecular-weight substances that inhibit the ATP-dependent lipoprotein release reaction.

Academic Significance and Societal Importance of the Research Achievements

細菌に広く存在するリポ蛋白質は、細胞表層に存在するさまざまの機能に中心的な役割を果たす蛋白質群である。リポ蛋白質が選別されて外膜に輸送される仕組みは、基礎科学的な面から興味があるだけでなく、細菌感染症の制御という応用面からも重要である。大腸菌におけるリポ蛋白質の選別的外膜局在化機構は、研究代表者らが明らかにしたLolシステムが触媒している。Lolシステムはほかの細菌にも広く存在する機構であることが明らかになりつつある。研究代表者らは、Lolシステムを標的とする薬剤を見出しており、本研究で構造に基づいたLolシステムの機構と、作用する薬剤の機構が解明されたことは社会的にも大きな意義がある。

Research Products

• [Journal Article] Real-time monitoring of polyacrylamide gel electrophoresis by the shadowgraph technique (2022)
  • Author(s): Murakami Satoshi、Adachi Hiroaki、Matsumura Hiroyoshi、Takano Kazufumi、Inoue Tsuyoshi、Mori Yusuke
  • Journal Title: The Journal of Biochemistry Volume: 173 Issue: 2 Pages: 65-72
  • DOI: 10.1093/jb/mvac087
  • Peer Reviewed
• [Journal Article] Structure and function relationship of OqxB efflux pump from Klebsiella pneumoniae (2021)
  • Author(s): Bharatham Nagakumar、Bhowmik Purnendu、Aoki Maho、Okada Ui、Sharma Sreevalli、Yamashita Eiki、Shanbhag Anirudh P.、Rajagopal Sreenath、Thomas Teby、Sarma Maitrayee、Narjari Riya、Nagaraj Savitha、Ramachandran Vasanthi、Katagihallimath Nainesh、Datta Santanu、Murakami Satoshi
  • Journal Title: Nature Communications Volume: 12 Issue: 1 Pages: 1-12
  • DOI: 10.1038/s41467-021-25679-0
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Engineered MATE multidrug transporters reveal two functionally distinct ion-coupling pathways in NorM from Vibrio cholerae (2021)
  • Author(s): Raturi Sagar、Nair Asha V.、Shinoda Keiko、Singh Himansha、Bai Boyan、Murakami Satoshi、Fujitani Hideaki、van Veen Hendrik W.
  • Journal Title: Communications Biology Volume: 4 Issue: 1 Pages: 1-17
  • DOI: 10.1038/s42003-021-02081-6
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Tripartite transporters as mechanotransmitters in periplasmic alternating-access mechanisms. (2020)
  • Author(s): Murakami, Okada, van Veen
  • Journal Title: FEBS Letters Volume: 594 Issue: 23 Pages: 3908-3919
  • DOI: 10.1002/1873-3468.13929
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Reversible auto-inhibitory regulation of Escherichia coli metallopeptidase BepA for selective β-barrel protein degradation. (2020)
  • Author(s): Daimon 他7名
  • Journal Title: Proc. Natl. Acad. Sci. USA. Volume: 117 Issue: 45 Pages: 27989-27996
  • DOI: 10.1073/pnas.2010301117
  • Peer Reviewed / Open Access
• [Journal Article] Structural basis of the function of the β-barrel assembly-enhancing protease BepA (2019)
  • Author(s): Mohammad Shahrizal, Yasushi Daimon, Yoshiki Tanaka, Yugo Hayashi, Shintaro Nakayama, Shigehiro Iwaki, Shin-ichiro Narita, Hironari Kamikubo, Yoshinori Akiyama, Tomoya Tsukazaki
  • Journal Title: Journal of Molecular Biology Volume: 431 Issue: 3 Pages: 625-635
  • DOI: 10.1016/j.jmb.2018.11.024
  • Peer Reviewed / Open Access
• [Journal Article] BpeB, a major resistance-nodulation-cell division transporter from Burkholderia cenocepacia: construct design, crystallization and preliminary structural analysis (2018)
  • Author(s): T. Horikawa, L.-W. Hung, H.-B. Kim, D. Shaya, C.-Y. Kim, T. C. Terwilliger, E. Yamashita, M. Aoki, U. Okada and S. Murakami
  • Journal Title: Acta Crystallogr. F Volume: 74 Issue: 11 Pages: 710-716
  • DOI: 10.1107/s2053230x18013547
  • Peer Reviewed / Open Access / Int'l Joint Research