Strctural and Functional Dynamics on Proteins Related to Biometal Dynamics

• Project Area: Integrated Biometal Science: Research to Explore Dynamics of Metals in Cellular System
• Project/Area Number: 19H05761
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Complex systems
• Research Institution: University of Hyogo
• Principal Investigator: SHIRO Yoshitsugu 兵庫県立大学, 理学研究科, 特任教授 (70183051)
• Co-Investigator(Kenkyū-buntansha): 澤井 仁美 長崎大学, 工学研究科, 准教授 (50584851)
• Project Period (FY): 2019-06-28 – 2024-03-31
• Project Status: Completed (Fiscal Year 2023)
• Budget Amount: ¥80,210,000 (Direct Cost: ¥61,700,000、Indirect Cost: ¥18,510,000); Fiscal Year 2023: ¥14,950,000 (Direct Cost: ¥11,500,000、Indirect Cost: ¥3,450,000); Fiscal Year 2022: ¥15,860,000 (Direct Cost: ¥12,200,000、Indirect Cost: ¥3,660,000); Fiscal Year 2021: ¥14,950,000 (Direct Cost: ¥11,500,000、Indirect Cost: ¥3,450,000); Fiscal Year 2020: ¥14,950,000 (Direct Cost: ¥11,500,000、Indirect Cost: ¥3,450,000); Fiscal Year 2019: ¥19,500,000 (Direct Cost: ¥15,000,000、Indirect Cost: ¥4,500,000)
• Keywords: 生体鉄 / 鉄動態 / トランスポーター / 鉄還元酵素 / 鉄センサータンパク質 / 鉄含有タンパク質 / フェリチン / 構造機能解析 / ヘム鉄 / タンパク質複合体 / 構造ダイナミクス / 鉄トランスポーター / センサータンパク質 / 鉄含有酵素 / 構造解析 / 鉄センサー

Outline of Research at the Start

生体にとって重要な鉄の輸送、感知、貯蔵、活用などの生命鉄動態は、様々なタンパク質によって制御されている。その制御が維持できなくなった時には生体は病気となる。この制御は細胞内という様々な生体物質が非常に密に存在する状況で、関連タンパク質同士の相互作用と各々のタンパク質の大きな構造の変化を伴っている。本研究では、時間分解測定の手法を用いて、ヒトと病原菌の生命鉄動態に関わるタンパク質（還元酵素、膜輸送体（トランスポーター）、センサータンパク質、酵素など）を研究対象とし、それらの機能時の動的な構造(構造ダイナミクス)と、それらの細胞内での機能を、分子構造を基に理解する。

Outline of Final Research Achievements

Iron is a bio-metal, which is essential in maintaining the lives of all living system. In this project, we have studies the dynamics of iron in cell (absorption, sensing, transport, storage, usage etc) on the basis of the dynamic structures and interaction of proteins related to the iron dynamics.
In human, the iron in diet is absorbed in duodenum, in which iron reductase (dcytb) and iron transporter (DMT1) play crucial roles. We studies the interaction of Dcytb with the iron by native MASS technique, and with DMT1 in cell to transport the ferrous iron resulted by the reduction of ferric iron by Dcytb by PLA assay.
We also studied the molecular mechanisms of heme inporter, heme sensor protein, and heme exporter in pothogenic bacteria, all of which play crucial roles in acquisition of iron. Especially, we focused on their dynamic structures in functionalization.

Academic Significance and Societal Importance of the Research Achievements

従来の研究は、個別のタンパク質の静的な分子構造を基盤にしたものか、分子生物学あるいは細胞生物学を基盤にした細胞機能の記述が中心であったが、本研究の成功により「鉄の生体内動態に関する細胞機能を、関連するタンパク質群の分子構造・機能を基盤に記述する」ことが可能となった。これにより、鉄が関係する疾病、すなわち鉄の欠乏や過剰が引き起こす重篤な疾病の根本原因の究明や治療に関して、得られた研究成果が直接関与することを目指した。さらに、この研究のために新しく開発した測定解析法は、他の研究分野にも広く応用可能である。

Research Products

• [Int'l Joint Research] Ubiversity of Bristrol/University of Liverpool(英国)
• [Int'l Joint Research] University of Liverpool/University of Leeds(英国)
• [Journal Article] Trapping of a Mononitrosyl Nonheme Intermediate of Nitric Oxide Reductase by Cryo-Photolysis of Caged Nitric Oxide (2023)
  • Author(s): Takeda Hanae、Shimba Kanji、Horitani Masaki、Kimura Tetsunari、Nomura Takashi、Kubo Minoru、Shiro Yoshitsugu、Tosha Takehiko
  • Journal Title: The Journal of Physical Chemistry B Volume: 127 Issue: 4 Pages: 846-854
  • DOI: 10.1021/acs.jpcb.2c05852
  • Peer Reviewed / Open Access
• [Journal Article] Structural basis for heme detoxification by an ATP-binding cassette?type efflux pump in gram-positive pathogenic bacteria (2022)
  • Author(s): Nakamura Hiro、Hisano Tamao、Rahman Md. Mahfuzur、Tosha Takehiko、Shirouzu Mikako、Shiro Yoshitsugu
  • Journal Title: Proceedings of the National Academy of Sciences Volume: 119 Issue: 27
  • DOI: 10.1073/pnas.2123385119
  • Peer Reviewed / Open Access
• [Journal Article] Identifying antibiotics based on structural differences in the conserved allostery from mitochondrial heme-copper oxidases. (2022)
  • Author(s): Nishida Y, Yanagisawa S, Morita R, Shigematsu H, Shinzawa-Itoh K, Yuki H, Ogasawara S, et al
  • Journal Title: Nat Commun. Volume: 13 Issue: 1 Pages: 7591-7591
  • DOI: 10.1038/s41467-022-34771-y
  • Peer Reviewed / Open Access
• [Journal Article] Capacity of extracellular globins to reduce liver fibrosis via scavenging reactive oxygen species and promoting MMP-1 secretion. (2022)
  • Author(s): Hieu VN, Thuy LTT, Hai H, Dat NQ, Hoang DV, Hanh NV, Phuong DM, Hoang TH, Sawai H, Shiro Y, Sato-Matsubara M, Oikawa D, Tokunaga F, Yoshizato K, Kawada N.
  • Journal Title: Redox Biol. Volume: 52 Pages: 102286-102286
  • DOI: 10.1016/j.redox.2022.102286
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] XFEL Crystal Structures of Peroxidase Compound II (2021)
  • Author(s): Kwon, H., Basran, J., Pathak, C., Hussain, M., Freeman, S. L., Fielding, A. J., Bailey, A. J., Stefanou, N., Sparkes, H. A., Tosha, T., Yamashita, K., Hirata, K., Murakami, H., Ueno, G., Ago, H., Tono, K., Yamamoto, M., Sawai, H., Shiro, Y., Sugimoto, H., Raven, E., Moody, P. C. E.
  • Journal Title: Angewandte Chemie International Edition Volume: - Issue: 26 Pages: 14578-14585
  • DOI: 10.1002/anie.202103010
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Short-lived intermediate in N2O generation by P450 NO reductase captured by time-resolved IR spectroscopy and XFEL crystallography (2021)
  • Author(s): Nomura, T., Kimura, T., Kanematsu, Y., Yamada, D., Yamashita, K., Hirata, K., Ueno, G., Murakami, H., Hisano, T., Yamagiwa, R., Takeda, H., Gopalasingam, C., Kousaka, R., Yanagisawa, S., Shoji, O., Kumasaka, T., Yamamoto, M., Takano, Y., Sugimoto, H., Tosha, T., Kubo, M., Shiro, Y
  • Journal Title: Proceedings of the National Academy of Sciences Volume: 118 Issue: 21
  • DOI: 10.1073/pnas.2101481118
  • Peer Reviewed / Open Access
• [Journal Article] Spatially restricted substrate-binding site of cortisol-synthesizing CYP11B1 limits multiple hydroxylations and hinders aldosterone synthesis (2021)
  • Author(s): Mukai Kuniaki、Sugimoto Hiroshi、Kamiya Katsumasa、Suzuki Reiko、Matsuura Tomomi、Hishiki Takako、Shimada Hideo、Shiro Yoshitsugu、Suematsu Makoto、Kagawa Norio
  • Journal Title: Current Research in Structural Biology Volume: 3 Pages: 192-205
  • DOI: 10.1016/j.crstbi.2021.08.001
  • Peer Reviewed / Open Access
• [Journal Article] Regulatory Switching by Concerted Motions on the Microsecond Time Scale of the Oxygen Sensor Protein FixL (2021)
  • Author(s): Yamawaki Takeo、Mizuno Misao、Ishikawa Haruto、Takemura Kazuhiro、Kitao Akio、Shiro Yoshitsugu、Mizutani Yasuhisa
  • Journal Title: The Journal of Physical Chemistry B Volume: 125 Issue: 25 Pages: 6847-6856
  • DOI: 10.1021/acs.jpcb.1c01885
  • Peer Reviewed
• [Journal Article] NO Dynamics in Microbial Denitrification System (2021)
  • Author(s): Tosha Takehiko、Yamagiwa Raika、Sawai Hitomi、Shiro Yoshitsugu
  • Journal Title: Chemistry Letters Volume: 50 Issue: 2 Pages: 280-288
  • DOI: 10.1246/cl.200629
  • NAID: 130007986360
  • Peer Reviewed / Open Access
• [Journal Article] Impact of membrane protein-lipid interactions on formation of bilayer lipid membranes on SAM-modified gold electrode (2021)
  • Author(s): Kato Masaru、Masuda Yuya、Yoshida Narumi、Tosha Takehiko、Shiro Yoshitsugu、Yagi Ichizo
  • Journal Title: Electrochimica Acta Volume: 373 Pages: 137888-137888
  • DOI: 10.1016/j.electacta.2021.137888
  • Peer Reviewed / Open Access
• [Journal Article] Heme controls the structural rearrangement of its sensor protein mediating the hemolytic bacterial survival (2021)
  • Author(s): Nishinaga Megumi、Sugimoto Hiroshi、Nishitani Yudai、Nagai Seina、Nagatoishi Satoru、Muraki Norifumi、Tosha Takehiko、Tsumoto Kouhei、Aono Shigetoshi、Shiro Yoshitsugu、Sawai Hitomi
  • Journal Title: Communications Biology Volume: 4 Issue: 1 Pages: 467-467
  • DOI: 10.1038/s42003-021-01987-5
  • Peer Reviewed / Open Access
• [Journal Article] The active form of quinol-dependent nitric oxide reductase from Neisseria meningitidis is a dimer (2020)
  • Author(s): Jamali M. Arif M.、Gopalasingam Chai C.、Johnson Rachel M.、Tosha Takehiko、Muramoto Kazumasa、Muench Stephen P.、Antonyuk Svetlana V.、Shiro Yoshitsugu、Hasnain Samar S.
  • Journal Title: IUCrJ Volume: 7 Issue: 3 Pages: 404-415
  • DOI: 10.1107/s2052252520003656
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Timing of NO Binding and Protonation in the Catalytic Reaction of Bacterial Nitric Oxide Reductase as Established by Time-Resolved Spectroscopy. (2020)
  • Author(s): Hanae Takeda, Tatsunari Kimura, Takashi Nomura, Masaki Horitani, Azusa Yokota, Akiko Matsubayashi, Shoko Ishii, Yoshitsugu Shiro, Minoru Kubo and Takehiko Tosha
  • Journal Title: Bulletin of the Chemical Society of Japan Volume: - Issue: 7 Pages: 825-833
  • DOI: 10.1246/bcsj.20200038
  • NAID: 130007879554
  • Peer Reviewed / Open Access
• [Journal Article] 分子構造を基盤にしたヘムタンパク質・ヘム酵素の機能解明 (2020)
  • Author(s): 城 宜嗣
  • Journal Title: BJSCC Volume: 75 Pages: 51-56
  • NAID: 130007879402
  • Peer Reviewed
• [Journal Article] UV Resonance Raman Characterization of a Substrate Bound to Human Indoleamine 2,3-Dioxygenase 1 (2019)
  • Author(s): Sachiko Yanagisawa , Kure’e Kayama , Masayuki Hara , Hiroshi Sugimoto , Yoshitsugu Shiro , Takashi Ogura
  • Journal Title: Biophysical Journal Volume: 117 Issue: 4 Pages: 706-716
  • DOI: 10.1016/j.bpj.2019.07.017
  • Peer Reviewed
• [Journal Article] Dimeric structures of quinol-dependent nitric oxide reductases (qNORs) revealed by cryo-electron microscopy (2019)
  • Author(s): Gopalasingam Chai C.、Johnson Rachel M.、Chiduza George N.、Tosha Takehiko、Yamamoto Masaki、Shiro Yoshitsugu、Antonyuk Svetlana V.、Muench Stephen P.、Hasnain S. Samar
  • Journal Title: Science Advances Volume: 5 Issue: 8
  • DOI: 10.1126/sciadv.aax1803
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Chemomechanical Coupling in the Transport Cycle of a Type II ABC Transporter (2019)
  • Author(s): K. Tamura, H. Sugimoto, Y. Shiro, Y. Sugita
  • Journal Title: J. Phys. Chem. B Volume: 123 Issue: 34 Pages: 7270-7281
  • DOI: 10.1021/acs.jpcb.9b04356
  • Peer Reviewed
• [Journal Article] Missing Piece of Two-Component Signal Transduction Systems Unveiled by SEC-SAXS (2019)
  • Author(s): H. Sawai, Y. Shiro
  • Journal Title: SPring-8/SACLA Research Frontiers 2018 Volume: - Pages: 15-16
• [Journal Article] Observation of Enzymatic Reactions by Time-resolved X-ray Crystallography Using Photosensitive Caged Substrate (2019)
  • Author(s): T. Tosha, M. Kubo
  • Journal Title: SPring-8/SACLA Research Frontiers 2018 Volume: - Pages: 28-29
• [Presentation] Thinking about Iron: the Molecular and Cellular Mechanisms of Dietary Iron Absorption (2023)
  • Author(s): Hitomi Sawai
  • Organizer: 20th International Conference on Biological Inorganic Chemistry (ICBIC20)
  • Int'l Joint Research / Invited
• [Presentation] Food for thought: the Protein Interactions for Dietary Iron Absorption in Humans (2023)
  • Author(s): Hitomi Sawai
  • Organizer: UK/Japan Meeting and Workshop: Dynamic Crystallography;XFELs and Synchrotrons to study enzyme reactions
  • Int'l Joint Research / Invited
• [Presentation] Elucidation of Mechanisms for Catalytic Reaction of Nitric Oxide Reductases by Time-resolved Techniques (2022)
  • Author(s): T. Tosha, H. Takeda, Y. Shiro and M. Kubo
  • Organizer: International Conference on Porphyrins and Phthalocyanines (ICPP-12)
  • Int'l Joint Research / Invited
• [Presentation] Molecular Mechanism of NO Reduction Catalyzed by Fe-containing Nitric Oxide Reductases (2022)
  • Author(s): Y. Shiro
  • Organizer: 8th Asian Conference on Coordination Chemistry (ACCC8)
  • Int'l Joint Research / Invited
• [Presentation] Heme Controls the Structural Rearrangement of Its Sensor Protein Mediating Survival of Pathogenic Bacteria (2022)
  • Author(s): H. Sawai
  • Organizer: 10th Asian Biological Inorganic Chemistry Conference (AsBIC10)
  • Int'l Joint Research / Invited
• [Presentation] 鉄の生体内動態の分子科学 (2022)
  • Author(s): 城 宜嗣
  • Organizer: 第２回レドックスR&D戦略委員会 春のシンポジウム 最先端技術が切り拓くレドックスバイオロジー
  • Invited
• [Presentation] Structural Basis for the Multifunctionality of the Heme-responsive Sensor Protein for Heme Detoxification in Hemolytic Bacteria (2021)
  • Author(s): H. Sawai
  • Organizer: 11th International Conference on Porphyrins and Phthalocyanines (ICPP-11)
  • Int'l Joint Research / Invited
• [Presentation] Structural Insights into the Multifunctionality of the Heme-responsive Sensor Protein for Detoxification in Hemolytic Bacteria (2021)
  • Author(s): H. Sawai
  • Organizer: The International Chemical Congress of Pacific Basin Societies 2021 (Pacifichem 2021)
  • Int'l Joint Research / Invited
• [Presentation] 分子構造を基盤にした鉄結合タンパク質の機能解明：生命金属科学への展開をめざして (2021)
  • Author(s): 城 宜嗣
  • Organizer: 第15回バイオ関連化学シンポジウム
  • Invited
• [Presentation] 鉄イオンの吸収に関わる膜タンパク質の構造と腸管モデル細胞を用いた機能解析 (2021)
  • Author(s): 澤井仁美
  • Organizer: 第１回トランスポーター研究会関西部会
  • Invited
• [Presentation] ヘムABCトランスポーターの基質輸送機構に関する分光学的解析 (2021)
  • Author(s): 木村哲就, 浅田拓也, 仲絢香, 林沙英, 城宜嗣, 杉本宏
  • Organizer: 第47回生体分子科学討論会
• [Presentation] ナノディスク再構成型BhuUV-Tによる段階的な基質輸送の分光学的観察 (2021)
  • Author(s): 木村哲就, 浅田拓也, 林沙英, 鍔木基成, 城宜嗣, 杉本宏
  • Organizer: 第21回蛋白質科学会年会
• [Presentation] Structural Analyses of ABC Transporters in Nucleotide Bound States Investigated by CW-ESR Spectroscopy (2021)
  • Author(s): 仲絢香、小堀康博、鍔木基成、城宜嗣、杉本宏、木村哲就
  • Organizer: 第59回日本生物物理学会年会
• [Presentation] 酸素バリア性フィルムを用いた嫌気条件下での一酸化窒素還元酵素反応中間体の構造解析への挑戦 (2021)
  • Author(s): 榛葉幹治、松浦滉明、平田邦生、山本雅貴、城宜嗣、當舎武彦
  • Organizer: 第47回生体分子科学討論会
• [Presentation] ヒト腸管細胞モデル系を用いた鉄イオンの吸収を向上させる食品成分の探索とそれらの成分が鉄輸送関連タンパク質に作用する分子機構 (2021)
  • Author(s): 髙原教代、杉本宏、神戸大朋、城宜嗣、澤井仁美
  • Organizer: 第94回日本生化学会大会
• [Presentation] 分子構造を基盤にした鉄結合タンパク質の機能解明：Functional Studies of Iron-Related Proteins on Molecular Basis (2021)
  • Author(s): 城 宜嗣
  • Organizer: 日本化学会第１０１春季年会
• [Presentation] 『生命金属科学』のめざすところ (2020)
  • Author(s): 城 宜嗣
  • Organizer: CSJ化学フェスタ
• [Presentation] Dynamics of Nitric Oxide in Bacterial Cellular Systems: NO Generation and Decomposition (2019)
  • Author(s): Shiro, Y.
  • Organizer: Japan-BIOCEV Symposium: Macromolecules; Structure, Function and Beyond
  • Int'l Joint Research / Invited
• [Presentation] Dynamics of Nitric Oxide in Cellular Systems: NO Generation and Decomposition (2019)
  • Author(s): Shiro Y.
  • Organizer: KTJ Bioinorganic Chemistry Symposium 2019 (KTJ-BICS 2019)
  • Int'l Joint Research / Invited
• [Presentation] 分子構造を基盤にしたヘムタンパク質の機能解明～生命金属科学への展開をめざして～ (2019)
  • Author(s): 城 宜嗣
  • Organizer: 錯体化学会第69回討論会
  • Invited
• [Presentation] ヘム蛋白質の機能を司る構造・ダイナミクスとエネルギー流：実験と理論 (2019)
  • Author(s): 城 宜嗣
  • Organizer: 第57回日本生物物理学会年会
  • Invited
• [Presentation] Elucidation of NO Reduction Mechanism in Soluble NO Reductase by Time-Resolved Crystallography with Photosensitive Caged Compound (2019)
  • Author(s): T. Tosha
  • Organizer: 32nd European Crystallographic Meeting (ECM32),
  • Invited
• [Presentation] 原子から細胞レベルの研究による鉄イオンの吸収メカニズムの理解と有効な鉄栄養強化食品成分の探索 (2019)
  • Author(s): 澤井仁美
  • Organizer: 第92回日本生化学会大会
  • Invited
• [Presentation] 病原菌の鉄獲得システムで機能するヘムセンサー蛋白質の多機能性とその構造的機序 (2019)
  • Author(s): 澤井仁美
  • Organizer: 第19回日本蛋白質科学会年会／第71回日本細胞生物学会大会合同年次大会
  • Invited
• [Book] ヘムタンパク質の科学 (2022)
  • Author(s): 城 宜嗣、青野 重利、齋藤 正男
  • Total Pages: 472
  • Publisher: エヌ・ティー・エス
  • ISBN: 9784860437787
• [Book] 生命金属ダイナミクス 生体内における金属の挙動と制御 (2021)
  • Author(s): 城 宜嗣・津本浩平
  • Total Pages: 511
  • Publisher: NTS
  • ISBN: 9784860437060
• [Remarks] 兵庫県立大学大学院理学研究科細胞制御Ⅱ講座
  • URL: https://www.sci.u-hyogo.ac.jp/life/regulation/index-j.html
• [Remarks] 兵庫県立大学大学院理学研究科細胞膜超分子複合体機能解析学講座
  • URL: http://www2.riken.jp/biometal/index.htm
• [Remarks] 兵庫県立大大学院生命理学研究科細胞制御Ⅱ講座
  • URL: https://www.sci.u-hyogo.ac.jp/life/regulation/index-j.html