Time-resolved serial femtosecond crystallography at an XFEL to reveal conformational changes in Cl- pump rhodopsin, NM-R3

• Project/Area Number: 17K07324
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Structural biochemistry
• Research Institution: Institute of Physical and Chemical Research
• Principal Investigator: Hosaka Toshiaki 国立研究開発法人理化学研究所, 生命機能科学研究センター, 研究員 (40462725)
• Project Period (FY): 2017-04-01 – 2021-03-31
• Project Status: Completed (Fiscal Year 2020)
• Budget Amount: ¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000); Fiscal Year 2019: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2018: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2017: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
• Keywords: 時分割構造解析 / X線自由電子レーザー / 膜タンパク質 / クロライドイオンポンプロドプシン / クロライドイオンポンプ / X線結晶構造解析 / SACLA / 時分割結晶構造解析 / クロライドポンプ / イオンポンプ / 結晶構造解析

Outline of Final Research Achievements

The marine bacterium derived Nonlabens marinus rhodopsin-3 (NM-R3) is a light-driven chloride ion-pump. Although we previously determined the crystal structure of NM-R3, its detailed ion transporting mechanism remains unknown. In this study, we used time resolved serial femtosecond crystallography with an X-ray free electron laser to observe the conformational changes during ion pumping process in NM-R3. After photoisomerization of retinal chromophore, causes simultaneous migration of anion from base position. In addition, the side chain of Asn98 that associates with anion in the ground state moves to the empty space created by the anion transfer, causing helix C to distort. Consequently, an ion transport pathway is created in the cytoplasmic interhelical space. In addition, we speculated that the location of the anion moved from the ground state to the cytoplasmic side of the NM-R3. A series of conformational changes explains how NM-R3 transports the anion across the protein.

Academic Significance and Societal Importance of the Research Achievements

SACLAでの時分割実験の研究成果は、中間体のような「静的な構造」ではなく、そのタンパク質が作動中の「動的な構造」変化を実際に目に見える形でとらえることができることを示している。今回のNM-R3での研究の特徴は、タンパク質の動作を見るだけではなく基質の動きを同時に見えることを示せたことである。今後は、コンピューターシミュレーションでの研究などを用いて、より詳細なイオン輸送機構を調べるなど光駆動ポンプの統合的な理解につながることが期待できる研究になったと考えている。

Research Products

• [Journal Article] A distinct lineage of giant viruses brings a rhodopsin photosystem to unicellular marine predators (2019)
  • Author(s): Needham David M.、Yoshizawa Susumu、Hosaka Toshiaki et. al.
  • Journal Title: Proceedings of the National Academy of Sciences Volume: 116 Issue: 41 Pages: 20574-20583
  • DOI: 10.1073/pnas.1907517116
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Crystal structural characterization reveals novel oligomeric interactions of human voltage-dependent anion channel 1. (2017)
  • Author(s): Hosaka T, Okazaki M, Kimura-Someya T, Ishizuka-Katsura Y, Ito K, Yokoyama S, Dodo K, Sodeoka M, Shirouzu M.
  • Journal Title: Protein Science Volume: 9 Issue: 9 Pages: 1749-1758
  • DOI: 10.1002/pro.3211
  • Peer Reviewed
• [Presentation] Cl-イオンポンプロドプシンの時分割XFEL結晶構造解析 (2019)
  • Author(s): 保坂俊彰
  • Organizer: 第19回日本蛋白質科学会年会
• [Presentation] Structural changes in Cl- pump rhodopsin by time-resolved serial femtosecond crystallography (2019)
  • Author(s): 保坂俊彰
  • Organizer: CBI学会2019年大会
• [Presentation] Nonlabens marinus由来新規光駆動性クロライドポンプの結晶構造解析 (2017)
  • Author(s): 保坂俊彰, 吉澤晋, 中島悠, 大沢登, 羽藤正勝, Edward F. DeLong, 木暮 一啓, 横山茂之, 染谷友美, 岩崎渉, 白水美香子
  • Organizer: 日本蛋白質科学会