Investigation of the mechanism of conversion from proton motive force to rotational energy in ATP synthase.

• Project/Area Number: 20K06514
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 43020:Structural biochemistry-related
• Research Institution: Osaka University
• Principal Investigator: Kishikawa Jun-ichi 大阪大学, 蛋白質研究所, 助教 (80599241)
• Project Period (FY): 2020-04-01 – 2023-03-31
• Project Status: Completed (Fiscal Year 2022)
• Budget Amount: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000); Fiscal Year 2022: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2021: ¥780,000 (Direct Cost: ¥600,000、Indirect Cost: ¥180,000); Fiscal Year 2020: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: 回転分子モーター / V-ATPase / クライオ電子顕微鏡 / 単粒子解析 / 膜タンパク質 / ATP 合成酵素 / 低温電子顕微鏡

Outline of Research at the Start

ATP 合成酵素はプロトンの濃度勾配を回転エネルギーに変換することで、その機能を発揮している。エネルギーの変換を担う膜内在性部分の構造解析をすることで、そのメカニズムを明らかにする。
調製した ATP 合成酵素の膜内在性部分の単粒子解析によって、その詳細構造の解明を目指す。また、複数状態の構造を明らかにすることで、ATP 合成酵素の反応メカニズムに迫る。

Outline of Final Research Achievements

ATP synthase plays a crucial role in synthesizing ATP by converting the proton-motive force across inside and outside of membrane into rotational energy. We investigated the energy conversion mechanism of ATP synthase by analyzing the high-resolution structure of the transmembrane domain from a thermophilic bacterium. Through single particle analysis using cryo-electron microscopy, we determined the protonation state of key residues involved in proton transport. Molecular simulations were then conducted to explore the energy conversion process. Our findings provide insights into how ATP synthase converts proton-motive force into rotational energy, contributing to a deeper understanding of this fundamental process in bioenergetics. These insights have potential applications in bioengineering.

Academic Significance and Societal Importance of the Research Achievements

ATP合成酵素は、ATPの合成とプロトン輸送を回転によって共役するユニークなタンパク質である。親水性部分でATPの合成、膜内在性部分でイオンの輸送を行う。親水性部分は、一分子回転観察などにより詳細に調べられている。一方で、膜内在性でのプロトン輸送機構は不明な点が多かった。本研究で得られた知見は、この輸送機構の原子レベルでの解明に光を当てるものであり、学術的な意義が深い。また、回転分子モーターは非常に精密な機械と捉えることもでき、その機構が分かることは、生物工学的にも興味深い。

Research Products

• [Journal Article] Cryo-EM analysis of V/A-ATPase intermediates reveals the transition of the ground-state structure to steady-state structures by sequential ATP binding (2023)
  • Author(s): Nakanishi Atsuko、Kishikawa Jun-ichi、Mitsuoka Kaoru、Yokoyama Ken
  • Journal Title: Journal of Biological Chemistry Volume: 299 Issue: 2 Pages: 102884-102884
  • DOI: 10.1016/j.jbc.2023.102884
  • Peer Reviewed
• [Journal Article] Structure of MotA, a flagellar stator protein, from hyperthermophile (2023)
  • Author(s): Tatsuro Nishikino, Norihiro Takekawa, Duy Phuoc Tran, Jun-ichi Kishikawa, Mika Hirose, Sakura Onoe, Seiji Kojima, Michio Homma, Akio Kitao, Takayuki Kato and Katsumi Imada
  • Journal Title: Biochemical and Biophysical Research Communications Volume: 631 Pages: 78-85
  • DOI: 10.1016/j.bbrc.2022.09.072
  • Peer Reviewed
• [Journal Article] Cryo-EM structures of Na+-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae (2022)
  • Author(s): Kishikawa Jun-ichi、Ishikawa Moe、Masuya Takahiro、Murai Masatoshi、Kitazumi Yuki、Butler Nicole L.、Kato Takayuki、Barquera Blanca、Miyoshi Hideto
  • Journal Title: Nature Communications Volume: 13 Issue: 1 Pages: 4082-4082
  • DOI: 10.1038/s41467-022-31718-1
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Structures of multisubunit membrane complexes with the CRYO ARM 200 (2022)
  • Author(s): Gerle Christoph、Kishikawa Jun-ichi、Yamaguchi Tomoko、Nakanishi Atsuko、?oruh Orkun、Makino Fumiaki、Miyata Tomoko、Kawamoto Akihiro、Yokoyama Ken、Namba Keiichi、Kurisu Genji、Kato Takayuki
  • Journal Title: Microscopy Volume: 71 Issue: 5 Pages: 249-261
  • DOI: 10.1093/jmicro/dfac037
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Structural basis of unisite catalysis of bacterial F0F1-ATPase (2022)
  • Author(s): Nakano Atsuki、Kishikawa Jun-ichi、Nakanishi Atsuko、Mitsuoka Kaoru、Yokoyama Ken
  • Journal Title: PNAS Nexus Volume: 1 Issue: 3
  • DOI: 10.1093/pnasnexus/pgac116
• [Journal Article] Structural snapshots of V/A-ATPase reveal the rotary catalytic mechanism of rotary ATPases (2022)
  • Author(s): Kishikawa J.、Nakanishi A.、Nakano A.、Saeki S.、Furuta A.、Kato T.、Mistuoka K.、Yokoyama K.
  • Journal Title: Nature Communications Volume: 13 Issue: 1 Pages: 1213-1213
  • DOI: 10.1038/s41467-022-28832-5
  • Peer Reviewed / Open Access
• [Presentation] Single particle cryo-EM of prokaryotic V/A-ATPase during catalytic cycle (2022)
  • Author(s): Jun-ichi Kishikawa
  • Organizer: 48th Indian Biophysical Society Meeting
  • Int'l Joint Research / Invited
• [Presentation] Structural snapshots of V/A-ATPase reveal the rotary catalytic mechanism of rotary ATPases (2022)
  • Author(s): Jun-ichi Kishikawa, Atsuko Nakanishi, Atsuki Nakano, Ken Yokoyama
  • Organizer: 第60回 日本生物物理学会年会
• [Presentation] Mechanical inhibition of isolated Vo from V/A-ATPase for proton conductance. (2020)
  • Author(s): 岸川淳一、中西温子、古田綾、加藤貴之、難波啓一、玉腰雅忠、光岡薫、横山謙
  • Organizer: 第58回日本生物物理学会年会
• [Presentation] 好熱菌 V 型 ATPase の 単粒子解析から分かってきたこと (2020)
  • Author(s): 岸川淳一
  • Organizer: 第１０回分子モーター討論会
  • Invited