Structural study of biological membranes by Cryo-EM

• Project/Area Number: 20H03216
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Basic Section 43040:Biophysics-related
• Research Institution: Yokohama City University
• Principal Investigator: Nishizawa Tomohiro 横浜市立大学, 生命医科学研究科, 教授 (80599077)
• Project Period (FY): 2020-04-01 – 2023-03-31
• Project Status: Completed (Fiscal Year 2022)
• Budget Amount: ¥17,810,000 (Direct Cost: ¥13,700,000、Indirect Cost: ¥4,110,000); Fiscal Year 2022: ¥3,380,000 (Direct Cost: ¥2,600,000、Indirect Cost: ¥780,000); Fiscal Year 2021: ¥6,760,000 (Direct Cost: ¥5,200,000、Indirect Cost: ¥1,560,000); Fiscal Year 2020: ¥7,670,000 (Direct Cost: ¥5,900,000、Indirect Cost: ¥1,770,000)
• Keywords: クライオ電子顕微鏡 / 膜タンパク質 / 輸送体 / 構造生物学 / 膜輸送体 / ポリアミン / 神経変性疾患

Outline of Research at the Start

膜輸送体は、脂質膜を越えて物質を伝搬することで細胞の恒常性維持や細胞間でのコミュニケーションなどに関わる。これらの輸送体は、基質結合部位が細胞内外に開いた二つの異なる状態を含む、複数の中間体を遷移することで機能する。したがって、その輸送機構を理解するためには、動的挙動を含めた解析が必要である。本研究では、クライオ電子顕微鏡と、高速読み出しが可能な電子直接検出器を駆使することで、膜輸送体がはたらく過程を動画として描写することで、これらの分子がどのようにして膜を越えて物質輸送を行うのかを理解することを目的としている。

Outline of Final Research Achievements

Membrane transporters play many essential roles in biological activities, including humans, by transporting various substrates across lipid membranes. In this study, we investigated the molecular mechanisms of several membrane transporters by using cryo-EM. In particular, for the polyamine transporter ATP13A2, which belongs to the P-type transporter family, we revealed the structures of multiple intermediates in the transport cycle, which highlighted its unique mechanism of the N-terminal domain that stabilizes a specific conformation of ATP13A2 and facilitates transport cycle.

Academic Significance and Societal Importance of the Research Achievements

ATP13A2はリソソームではたらくポリアミン輸送体であり、細胞外からのポリアミン取り込み経路において重要な役割を持つ。ATP13A2はパーキンソン病の原因遺伝子としても同定されており、これは老化にともなって細胞内のポリアミン合成が減少すると、ATP13A2のはたらきが相対的に重要になるためであると考えられている。今回の研究から、ATP13A2はNTDと呼ばれる独自のドメインが脂質と相互作用して、特定のコンフォメーションを安定化することで、輸送活性を制御していることが示された。これらの成果は、将来的には、ATP13A2を標的とした、新たなパーキンソン病の治療薬の開発などへつながる可能性がある。

Research Products

• [Journal Article] Cryo-EM structures of thermostabilized prestin provide mechanistic insights underlying outer hair cell electromotility (2022)
  • Author(s): Futamata Haon、Fukuda Masahiro、Umeda Rie、Yamashita Keitaro、Tomita Atsuhiro、Takahashi Satoe、Shikakura Takafumi、Hayashi Shigehiko、Kusakizako Tsukasa、Nishizawa Tomohiro、Homma Kazuaki、Nureki Osamu
  • Journal Title: Nature Communications Volume: 13 Issue: 1 Pages: 6208-6208
  • DOI: 10.1038/s41467-022-34017-x
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Structural insights into inhibitory mechanism of human excitatory amino acid transporter EAAT2 (2022)
  • Author(s): Kato Takafumi、Kusakizako Tsukasa、Jin Chunhuan、Zhou Xinyu、Ohgaki Ryuichi、Quan LiLi、Xu Minhui、Okuda Suguru、Kobayashi Kan、Yamashita Keitaro、Nishizawa Tomohiro、Kanai Yoshikatsu、Nureki Osamu
  • Journal Title: Nature Communications Volume: 13 Issue: 1 Pages: 4714-4714
  • DOI: 10.1038/s41467-022-32442-6
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Structural insights into the HBV receptor and bile acid transporter NTCP (2022)
  • Author(s): Park JH, Iwamoto M, Yun JH, Uchikubo-Kamo T, Son D, Jin Z, Yoshida H, Ohki M, Ishimoto N, Mizutani K, Oshima M, Muramatsu M, Wakita T, Shirouzu M, Liu K, Uemura T, Nomura N, Iwata S, Watashi K, Tame JRH, Nishizawa T, Lee W, Park SY.
  • Journal Title: Nature Volume: 606 Issue: 7916 Pages: 1027-1031
  • DOI: 10.1038/s41586-022-04857-0
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Cryo-EM structures of thylakoid-located voltage-dependent chloride channel VCCN1 (2022)
  • Author(s): Hagino Tatsuya、Kato Takafumi、Kasuya Go、Kobayashi Kan、Kusakizako Tsukasa、Hamamoto Shin、Sobajima Tomoaki、Fujiwara Yuichiro、Yamashita Keitaro、Kawasaki Hisashi、Maturana Andr?s D.、Nishizawa Tomohiro、Nureki Osamu
  • Journal Title: Nature Communications Volume: 13 Issue: 1 Pages: 2505-2505
  • DOI: 10.1038/s41467-022-30292-w
  • Peer Reviewed / Open Access
• [Journal Article] Cryo-EM of the ATP11C flippase reconstituted in Nanodiscs shows a distended phospholipid bilayer inner membrane around transmembrane helix 2 (2022)
  • Author(s): Nakanishi Hanayo、Hayashida Kenichi、Nishizawa Tomohiro、Oshima Atsunori、Abe Kazuhiro
  • Journal Title: Journal of Biological Chemistry Volume: 298 Issue: 1 Pages: 101498-101498
  • DOI: 10.1016/j.jbc.2021.101498
  • Peer Reviewed / Open Access
• [Journal Article] Cryo-EM reveals mechanistic insights into lipid-facilitated polyamine export by human ATP13A2. (2021)
  • Author(s): Tomita, A., Daiho, T., Kusakizako, T., Yamashita, K., Ogasawara, S., Murata, T., Nishizawa, T. and Nureki, O.
  • Journal Title: Mol. Cell Volume: 81 Issue: 23 Pages: 4799-4809
  • DOI: 10.1016/j.molcel.2021.11.001
  • Peer Reviewed / Open Access
• [Journal Article] Gastric proton pump with two occluded K+ engineered with sodium pump-mimetic mutations (2021)
  • Author(s): Abe Kazuhiro、Yamamoto Kenta、Irie Katsumasa、Nishizawa Tomohiro、Oshima Atsunori
  • Journal Title: Nature Communications Volume: 12 Issue: 1 Pages: 5709-5709
  • DOI: 10.1038/s41467-021-26024-1
  • Peer Reviewed / Open Access
• [Journal Article] Structural basis of gating modulation of Kv4 channel complexes. (2021)
  • Author(s): Kise Y, Kasuya G, Okamoto HH, Yamanouchi D, Kobayashi K, Kusakizako T, Nishizawa T, Nakajo K, Nureki O.
  • Journal Title: Nature Volume: 599 Issue: 7883 Pages: 158-164
  • DOI: 10.1038/s41586-021-03935-z
  • Peer Reviewed / Open Access
• [Journal Article] Transport Cycle of Plasma Membrane Flippase ATP11C by Cryo-EM (2020)
  • Author(s): Nakanishi Hanayo、Nishizawa Tomohiro、Segawa Katsumori、Nureki Osamu、Fujiyoshi Yoshinori、Nagata Shigekazu、Abe Kazuhiro
  • Journal Title: Cell Reports Volume: 32 Issue: 13 Pages: 108208-108208
  • DOI: 10.1016/j.celrep.2020.108208
  • Peer Reviewed / Open Access
• [Journal Article] Consensus mutagenesis approach improves the thermal stability of system xc- transporter, xCT, and enables cryo‐EM analyses (2020)
  • Author(s): Oda Kazumasa、Lee Yongchan、Wiriyasermkul Pattama、Tanaka Yoko、Takemoto Mizuki、Yamashita Keitaro、Nagamori Shushi、Nishizawa Tomohiro、Nureki Osamu
  • Journal Title: Protein Science Volume: 29 Issue: 12 Pages: 2398-2407
  • DOI: 10.1002/pro.3966
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] 「non-conventional」なP型ATPase輸送体の基質認識機構 (2023)
  • Author(s): 西澤知宏
  • Organizer: 日本薬学会
  • Invited
• [Presentation] クライオ電顕で見えてくるトランスポーターとリガンドの相互作用 (2023)
  • Author(s): 西澤知宏
  • Organizer: 日本医学総会
• [Presentation] Protein Dynamics Visualized by Cryo-EM (2022)
  • Author(s): 西澤知宏
  • Organizer: 日本生化学大会
  • Int'l Joint Research / Invited
• [Presentation] クライオ電子顕微鏡によって見えてきた P4-ATPaseによる脂質輸送メカニズム (2020)
  • Author(s): 西澤知宏
  • Organizer: 第15回トランスポーター研究会
• [Presentation] Time-resolved serial femtosecond crystallography reveals early structural changes in channelrhodopsin (2020)
  • Author(s): 西澤知宏
  • Organizer: 第58回日本生物物理学会年会
  • Invited