Structural and functional analysis of mechanosensitive channel TMC proteins

• Project/Area Number: 20K15754
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 43040:Biophysics-related
• Research Institution: The University of Tokyo
• Principal Investigator: Kusakizako Tsukasa 東京大学, 大学院理学系研究科(理学部), 助教 (80815316)
• 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 2021: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000); Fiscal Year 2020: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
• Keywords: チャネル / 膜タンパク質 / クライオ電子顕微鏡 / 単粒子解析

Outline of Research at the Start

音すなわち空気の振動によって機械刺激を受けた有毛細胞では，膜タンパク質を含むMechanotransduction (MET) channel複合体が，機械刺激を電気的シグナルへと情報を変換することで，有毛細胞から聴神経へシグナルが伝達すると考えられている。MET channel複合体のチャネル透過経路を形成するサブユニットの最有力候補がTMC1及びTMC2である。本研究では，クライオ電子顕微鏡単粒子解析によりTMC1またはTMC2の立体構造を決定し，構造情報に基づいた電気生理学解析をおこなうことで，TMC1/TMC2のイオン透過機構・機械刺激感受性チャネル開閉機構の解明を目指す。

Outline of Final Research Achievements

Mechanosensitive channel TMC1/TMC2 proteins interact with auxiliary subunits to form the mechanoelectrical transduction (MET) channel complex in hair cells. In this study, we aimed to elucidate the molecular mechanisms of ion permeation and mechanosensitive gating of TMC1/2 using cryo-EM single particle analysis. Expanding from the original plan, we successfully prepared complexes of TMC and CIB proteins, which are the component of the MET channel complex, and performed the structural analysis of the TMC-CIB complex.

Academic Significance and Societal Importance of the Research Achievements

TMCタンパク質を含む複合体解析の基盤が整ったことで、イオン透過機構のみならず、補助サブユニットによるMETチャネル複合体の活性調節機構の理解にも繋がる点で学術的意義がある。今後、補助サブユニットも含めたMETチャネル複合体の構造機能研究へと展開していく。また、METチャネル複合体は聴覚に関わることから、本研究の成果と今後の展開は難聴の病態理解を深め、構造情報に基づいた薬剤開発の可能性を提供すると期待される。

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] Structure and engineering of the minimal type VI CRISPR-Cas13bt3 (2022)
  • Author(s): Nakagawa Ryoya、Kannan Soumya、Altae-Tran Han、Takeda Satoru N.、Tomita Atsuhiro、Hirano Hisato、Kusakizako Tsukasa、Nishizawa Tomohiro、Yamashita Keitaro、Zhang Feng、Nishimasu Hiroshi、Nureki Osamu
  • Journal Title: Molecular Cell Volume: 82 Issue: 17 Pages: 3178-3192
  • DOI: 10.1016/j.molcel.2022.08.001
  • 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] Endogenous ligand recognition and structural transition of a human PTH receptor (2022)
  • Author(s): Kobayashi Kazuhiro、Kawakami Kouki、Kusakizako Tsukasa、Miyauchi Hirotake、Tomita Atsuhiro、Kobayashi Kan、Shihoya Wataru、Yamashita Keitaro、Nishizawa Tomohiro、Kato Hideaki E.、Inoue Asuka、Nureki Osamu
  • Journal Title: Molecular Cell Volume: 82 Issue: 18 Pages: 3468-3483
  • DOI: 10.1016/j.molcel.2022.07.003
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Structure of the Dicer-2-R2D2 heterodimer bound to a small RNA duplex (2022)
  • Author(s): Yamaguchi Sonomi、Naganuma Masahiro、Nishizawa Tomohiro、Kusakizako Tsukasa、Tomari Yukihide、Nishimasu Hiroshi、Nureki Osamu
  • Journal Title: Nature Volume: 607 Issue: 7918 Pages: 393-398
  • DOI: 10.1038/s41586-022-04790-2
  • Peer Reviewed / Open Access
• [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] Structure of the type V-C CRISPR-Cas effector enzyme (2022)
  • Author(s): Kurihara Nina、Nakagawa Ryoya、Hirano Hisato、Okazaki Sae、Tomita Atsuhiro、Kobayashi Kan、Kusakizako Tsukasa、Nishizawa Tomohiro、Yamashita Keitaro、Scott David A.、Nishimasu Hiroshi、Nureki Osamu
  • Journal Title: Molecular Cell Volume: 82 Issue: 10 Pages: 1865-1877
  • DOI: 10.1016/j.molcel.2022.03.006
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Structural basis for channel conduction in the pump-like channelrhodopsin ChRmine. (2022)
  • Author(s): Koichiro E. Kishi et al.
  • Journal Title: Cell Volume: 185 Issue: 4 Pages: 672-689
  • DOI: 10.1016/j.cell.2022.01.007
  • Peer Reviewed / Open Access / Int'l Joint Research
• [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] 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] Cryo-EM structure of the human MT1-Gi signaling complex. (2021)
  • Author(s): Okamoto, H. H., Miyauchi, H., Inoue, A., Raimondi, F., Tsujimoto, H., Kusakizako, T., Shihoya, W., Yamashita, K., Suno, R., Nomura, N., Kobayashi, T., Iwata, S., Nishizawa, T. and Nureki, O.
  • Journal Title: Nat. Struct. Mol. Biol. Volume: 28 Issue: 8 Pages: 694-701
  • DOI: 10.1038/s41594-021-00634-1
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Structural Biology of MATE Proteins (2021)
  • Author(s): 草木迫 司
  • Journal Title: Seibutsu Butsuri Volume: 61 Issue: 3 Pages: 183-185
  • DOI: 10.2142/biophys.61.183
  • NAID: 130008045146
  • ISSN: 0582-4052, 1347-4219
  • Peer Reviewed / Open Access
• [Journal Article] The structure of MgtE in the absence of magnesium provides new insights into channel gating. (2021)
  • Author(s): Jin F, Sun M, Fujii T, Yamada Y, Wang J, Maturana AD, Wada M, Su S, Ma J, Takeda H, Kusakizako T, Tomita A, Nakada-Nakura Y, Liu K, Uemura T, Nomura Y, Nomura N, Ito K, Nureki O, Namba K, Iwata S, Yu Y, Hattori M.
  • Journal Title: PLoS Biology Volume: 19 Issue: 4 Pages: e3001231-e3001231
  • DOI: 10.1371/journal.pbio.3001231
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Time-resolved serial femtosecond crystallography reveals early structural changes in channelrhodopsin (2021)
  • Author(s): Oda Kazumasa、Nomura Takashi、Nakane Takanori、Yamashita Keitaro、Inoue Keiichi、Ito Shota、Vierock Johannes、Hirata Kunio、Maturana Andres D、Katayama Kota、( 32 authers )、Kandori Hideki、Hegemann Peter、Iwata So、Kubo Minoru、Nishizawa Tomohiro、Nureki Osamu
  • Journal Title: eLife Volume: 10 Pages: 1-21
  • DOI: 10.7554/elife.62389
  • NAID: 120006996980
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Conserved binding site in the N-lobe of prokaryotic MATE transporters suggests a role for Na+ in ion-coupled drug efflux (2021)
  • Author(s): Castellano Steven、Claxton Derek P.、Ficici Emel、Kusakizako Tsukasa、Stix Robyn、Zhou Wenchang、Nureki Osamu、Mchaourab Hassane S.、Faraldo-Gomez Jose D.
  • Journal Title: Journal of Biological Chemistry Volume: 296 Pages: 100262-100262
  • DOI: 10.1016/j.jbc.2021.100262
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Structure of the miniature type V-F CRISPR-Cas effector enzyme (2021)
  • Author(s): Takeda Satoru N.、Nakagawa Ryoya、Okazaki Sae、Hirano Hisato、Kobayashi Kan、Kusakizako Tsukasa、Nishizawa Tomohiro、Yamashita Keitaro、Nishimasu Hiroshi、Nureki Osamu
  • Journal Title: Molecular Cell Volume: 81 Issue: 3 Pages: 558-570.e3
  • DOI: 10.1016/j.molcel.2020.11.035
  • Peer Reviewed
• [Journal Article] Taste transduction and channel synapses in taste buds (2021)
  • Author(s): Taruno Akiyuki、Nomura Kengo、Kusakizako Tsukasa、Ma Zhongming、Nureki Osamu、Foskett J. Kevin
  • Journal Title: Pflugers Archiv - European Journal of Physiology Volume: 473 Issue: 1 Pages: 3-13
  • DOI: 10.1007/s00424-020-02464-4
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Structure of the human secretin receptor coupled to an engineered heterotrimeric G protein (2020)
  • Author(s): Fukuhara Satoshi、Kobayashi Kazuhiro、Kusakizako Tsukasa、Iida Wataru、Kato Masahiko、Shihoya Wataru、Nureki Osamu
  • Journal Title: Biochemical and Biophysical Research Communications Volume: 533 Issue: 4 Pages: 861-866
  • DOI: 10.1016/j.bbrc.2020.08.042
  • Peer Reviewed / Open Access
• [Journal Article] Cryo-EM structures of calcium homeostasis modulator channels in diverse oligomeric assemblies (2020)
  • Author(s): Demura Kanae*、Kusakizako Tsukasa*、Shihoya Wataru*、Hiraizumi Masahiro*、Nomura Kengo、Shimada Hiroto、Yamashita Keitaro、Nishizawa Tomohiro、Taruno Akiyuki、Nureki Osamu (*equal contribution)
  • Journal Title: Science Advances Volume: 6 Issue: 29
  • DOI: 10.1126/sciadv.aba8105
  • Peer Reviewed / Open Access
• [Journal Article] The structure of lipid nanodisc-reconstituted TRPV3 reveals the gating mechanism (2020)
  • Author(s): Shimada Hiroto*、Kusakizako Tsukasa*、Dung Nguyen T. H.、Nishizawa Tomohiro、Hino Tomoya、Tominaga Makoto、Nureki Osamu (*equal contribution)
  • Journal Title: Nature Structural & Molecular Biology Volume: 27 Issue: 7 Pages: 645-652
  • DOI: 10.1038/s41594-020-0439-z
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] 多剤排出輸送体MATEの構造および機能解析 (2020)
  • Author(s): 田中 良樹、草木迫 司
  • Journal Title: Farumashia Volume: 56 Issue: 6 Pages: 514-518
  • DOI: 10.14894/faruawpsj.56.6_514
  • NAID: 130007850170
  • ISSN: 0014-8601, 2189-7026
  • Peer Reviewed
• [Journal Article] Cryo-EM structure of the volume-regulated anion channel LRRC8D isoform identifies features important for substrate permeation (2020)
  • Author(s): Nakamura Ryoki、Numata Tomohiro、Kasuya Go、Yokoyama Takeshi、Nishizawa Tomohiro、Kusakizako Tsukasa、Kato Takafumi、Hagino Tatsuya、Dohmae Naoshi、Inoue Masato、Watanabe Kengo、Ichijo Hidenori、Kikkawa Masahide、Shirouzu Mikako、Jentsch Thomas J.、Ishitani Ryuichiro、Okada Yasunobu、Nureki Osamu
  • Journal Title: Communications Biology Volume: 3 Issue: 1 Pages: 240-240
  • DOI: 10.1038/s42003-020-0951-z
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] ChRの機能発現機構 (2022)
  • Author(s): 草木迫 司
  • Organizer: 第35回日本放射光学会年会・放射光科学合同シンポジウム
  • Invited
• [Presentation] クライオ電子顕微鏡による膜タンパク質の単粒子解析 (2021)
  • Author(s): 草木迫 司
  • Organizer: 大阪大学蛋白質研究所セミナー
  • Invited
• [Presentation] MATEトランスポーターの構造生物学 (2020)
  • Author(s): 草木迫 司、宮内 弘剛、石谷 隆一郎、濡木 理
  • Organizer: 第5回トランスポーター研究会関東部会
  • Invited
• [Remarks] 骨代謝に関わるPTH1受容体のシグナル伝達複合体を可視化（東京大学プレスリリース）
  • URL: https://www.s.u-tokyo.ac.jp/ja/press/2022/8014/
• [Remarks] 新規光駆動型イオンチャネルの構造解明と高性能分子ツールの創出～神経科学に光を当てる～
  • URL: https://www.u-tokyo.ac.jp/focus/ja/press/z0109_00034.html
• [Remarks] 細胞内へのポリアミン輸送メカニズムの解明（東京大学プレスリリース）
  • URL: https://www.s.u-tokyo.ac.jp/ja/press/2021/7644/
• [Remarks] 制御サブユニットによるイオンチャネル巨大複合体のモジュレーション機構を解明（東京大学プレスリリース）
  • URL: https://www.s.u-tokyo.ac.jp/ja/press/2021/7525/
• [Remarks] 甘味・うま味・苦味物質の認識に必要なATPチャネルの構造可視化に成功（東京大学理学部プレスリリース）
  • URL: https://www.s.u-tokyo.ac.jp/ja/press/2020/6936/