Details of the Researcher

PHOTO

Yoshikazu Tanaka
Section
Graduate School of Life Sciences
Job title
Professor
Degree
Profile
High resolution structure give us a lot of information important for understanding molecular mechanism. I use X-ray crystallography to study proteins that play important roles in biology and disease. Our laboratory started since 2017. We are always looking for motivated students who are interested in our research.

Research History 7

  • 2017/04 - Present
    Tohoku University　Graduate School of Life Sciences, Department of Molecular and Chemical Life Sciences

  • 2015/10 - 2019/03
    科学技術振興機構 さきがけ　「超空間制御と革新的機能創成」領域　領域研究者

  • 2016/07 - 2017/03
    マックスプランク研究所（ドイツ）　ゲスト研究員

  • 2012/04 - 2017/03
    Hokkaido University　Faculty of Advanced Life Science

  • 2008/01 - 2012/03
    Hokkaido University

  • 2006/04 - 2008/01
    東京大学大学院　新領域創成科学研究科　博士研究員

  • 2004/04 - 2006/03
    Hokkaido University

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Education 3

  • 東北大学大学院　工学研究科 生物工学専攻　博士課程後期

    2001/04 - 2004/03

  • 東北大学大学院　工学研究科 生物工学専攻　博士課程前期

    1999/04 - 2001/03

  • Tohoku University　Faculty of Engineering

    1995/04 - 1999/03

Committee Memberships 14

  • 自然科学研究機構生命創成探求センター　運営委員会委員

    2022/04 - 2026/03

  • 日本蛋白質科学会　庶務役員

    2023/07 - 2025/07

  • 日本生物物理学会 東北支部　幹事

    2022/05 - 2025/04

  • R022量子構造生物学委員会　委員

    2020/04 - 2025/03

  • 日本蛋白質科学会　会計役員

    2021/06 - 2023/06

  • 文部科学省 学術調査官

    2020/08 - 2022/07

  • 日本生物物理学会　東北支部長

    2020/05 - 2022/04

  • 日本生物物理学会理事

    2021 - 2022

  • クライオ電顕ネットワークユーザーグループ幹事代表

    2019/01 - 2021/03

  • 日本蛋白質科学会　若手育成役員

    2018 - 2019

  • 日本蛋白質科学会　代議員

    2016/06 - 2018/06

  • 日本蛋白質科学会　代議員

    2016/06 - 2018/06

  • トキシンシンポジウム　運営委員会委員

    2015/01 - 2017/12

  • トキシンシンポジウム　運営委員会委員

    2015/01 - 2017/12

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Professional Memberships 6

  • JAPAN SOCIETY FOR BIOSCIENCE, BIOTECHNOLOGY, AND AGROCHEMISTRY

  • PROTEIN SCIENCE SOCIETY OF JAPAN

  • 毒素シンポジウム

  • THE JAPANESE BIOCHEMICAL SOCIETY

  • THE BIOPHYSICAL SOCIETY OF JAPAN

  • THE CRYSTALLOGRAPHIC SOCIETY OF JAPAN

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Research Interests 8

  • 毒素

  • クライオ電子顕微鏡

  • ホストーゲスト化学

  • ヘモシアニン

  • 病原性微生物

  • タンパク質

  • Ｘ線結晶構造解析

  • 構造解析

Research Areas 2

  • Life sciences / Molecular biology /

  • Life sciences / Structural biochemistry /

Awards 2

  1. 2025(令和7)年度総長教育賞

    2026/02　東北大学

  2. 2025年度東北大学全学教育貢献賞

    2025/01　東北大学

Papers 125

  1. Glomerular routing of tumor-derived extracellular vesicles substantiates urinary biopsy. International-journal

    Shota Kawaguchi, Taiga Ajiri, Rina Mitsuya, Reiko Tsuchiya, Koki Kunitake, Yoshikazu Tanaka, Takeshi Yokoyama, Kiichi Sato, Yusuke Sato, Zetao Zhu, Kunanon Chattrairat, Yasuko Kobayashi, Kimiko Inoue, Keisuke Imaeda, Kosei Ueno, Sou Ryuzaki, Akira Kato, Yasuyuki Kimura, Atsushi Natsume, Ryosuke Kojima, Takao Yasui

    Science advances　12　(8)　eaeb0555　2026/02/20

    DOI： 10.1126/sciadv.aeb0555 　

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    Urinary small extracellular vesicles (sEVs), which can reflect systemic conditions, hold great promise for noninvasive cancer diagnostics, yet the mechanism by which tumor-derived sEVs reach urine remains unclear. Here, we demonstrate that the glomerulus actively transcytoses circulating tumor-derived sEVs into urine. Using CRISPR guide RNA-tagged glioma sEVs and bioluminescent/fluorescent green-enhanced nano-lantern (GeNL)-tagged lung and pancreatic cancer sEVs, we tracked their journey from tumors to urine in multiple mouse models. In vivo and in vitro analyses revealed endocytic uptake and transcytotic release by glomerular cells, accompanied by changes in sEV size and surface composition. GeNL-tagged sEVs consistently showed higher signals in urine than plasma, indicating selective excretion. These findings redefine the glomerulus as a dynamic regulator of sEV processing and establish a mechanistic foundation for urinary liquid biopsy.

  2. Synthesis and structure-activity relationship of azithromycin derivatives against non-tuberculous mycobacteria

    Yuka Isozaki, Ali Ahsan Muzahid, Tatsuki Wakata, Maho Fujino, Yoshikazu Tanaka, Chigusa Hayashi, Yoshimasa Ishizaki, Tomokazu Ohishi, Ayumi Morita, Shunichi Ohba, Takeshi Yokoyama, Masayuki Igarashi, Kazunobu Toshima, Daisuke Takahashi

    European Journal of Medicinal Chemistry　304　118520-118520　2026/02

    Publisher: Elsevier BV

    DOI： 10.1016/j.ejmech.2025.118520 　

    ISSN： 0223-5234

  3. Modulation of translational elongation by montanine alkaloids preferentially inhibits RNA viruses

    Ryuichi Sakai, Kyohei Sato, Atsushi Tsugita, Lakkana Thaveepornkul, Ayato Takada, Hiroko Miyamoto, Rumi Kurokawa, Minoru Yoshida, Takeshi Yokoyama, Antonio Evidente, Yuichiro Tsuge, Hiromi Watari, Tomonosuke Sumiya, Ken Matsumoto, Sarin Chimnaronk, Yoshikazu Tanaka

    2025/12/20

    Publisher: openRxiv

    DOI： 10.64898/2025.12.19.695327 　

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    Abstract Montanine, an Amaryllidaceae alkaloid, has demonstrated superior antiviral activity against dengue virus (DENV), SARS-CoV-2, vesicular stomatitis Indiana virus (VSV-G), and vesicular stomatitis virus pseudotyped with Ebola virus glycoprotein (VSV-ZGP), outperforming other antiviral alkaloids tested, including lycorine, narciclasine, tetracetylnarciclasine, and pancracine. We further showed that montanine inhibits translation in mammalian cells, as evidenced by a puromycin incorporation assay. Cryo-EM analysis revealed that montanine binds to the peptidyl transferase center of the human ribosome. A chemical genomics survey indicated that the knockdown of the GCN1 complex, which senses ribosome collisions and triggers the translation quality control process, increased the cytotoxicity of montanine while reducing viral infectivity. Together, these results suggest that the antiviral activity of montanine is closely linked to its impact on translational elongation and GCN1-related stress responses, underscoring the potential of translation control as a targeted mode for RNA virus therapeutics. Significance Statement Montanine, an alkaloid from the Amaryllidaceae family known for its diverse bioactivities, was identified as a potent antiviral compound from a library of natural plant- and fungus-derived substances. Through a combination of biochemical assays and cryo-EM analysis, we discovered that montanine binds to the peptidyl transfer center of the human ribosome, effectively inhibiting protein synthesis. Based on these findings and a chemical genomics approach, we hypothesize that the translational control of montanine triggers a cellular response that impedes viral replication. We propose that molecules capable of modulating translation efficacy could serve as unique lead compounds in the development of broad-spectrum antiviral agents.

  4. RaspL: isolation and characterization of a cell-invading, cytotoxic lectin from the Palauan marine sponge

    Syeda Sobia Nasir, Yuji Sawada, Yoshikazu Tanaka, Yuji Ise, Hiromi Watari, Ryuichi Sakai

    Fisheries Science　2025/12/18

    Publisher: Springer Science and Business Media LLC

    DOI： 10.1007/s12562-025-01954-7 　

    ISSN： 0919-9268

    eISSN： 1444-2906

  5. Zinc finger domains bind low-complexity domain polymers. International-journal

    Naohiko Iguchi, Noriyoshi Isozumi, Yoshikazu Hattori, Tomohiro Imamura, Takeshi Yokoyama, Masatomo So, Hitoki Nanaura, Takao Kiriyama, Nobuyuki Eura, Minako Yamaoka, Naoki Iwasa, Tomo Shiota, Mari Nakanishi, Nanako Konishi, Haruka Ito, Akihito Takeuchi, Masashi Mori, Shinya Ohki, Hiroyuki Kumeta, Hironori Koga, Mai Watabe, Takuya Mabuchi, Shingo Kanemura, Masaki Okumura, Yoshikazu Tanaka, Ken Morishima, Masaaki Sugiyama, Fumika Ide, Hiroyoshi Matsumura, Takuya Yoshizawa, Ichiro Ota, Naoki Suzuki, Masashi Aoki, Yoshito Yamashiro, Tomohide Saio, Kazuma Sugie, Eiichiro Mori

    Nature communications　16　(1)　8922-8922　2025/10/16

    DOI： 10.1038/s41467-025-64382-2 　

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    Self-association of low-complexity protein sequences (LC domains) is important for polymer formation. Several molecular chaperones are involved in the regulation of LC domain polymer formation. However, the mechanisms underlying cell recognition of LC domain polymers remain unclear. Here we show that zinc finger domains (ZnFs) bind LC domains of RNA-binding proteins in a cross-β polymer-dependent manner. ZnFs bound to LC domain hydrogels and suppressed LC domain polymer formation. Moreover, ZnFs preferentially recognize LC domains in the polymeric state. These findings suggest that ZnFs act as physiological regulators of LC domain polymer formation.

  6. Structure-Activity Relationship of an All-α-helical Prenyltransferase Reveals the Mechanism of Indole Prenylation. International-journal

    Takumi Oshiro, Shuta Uehara, Arisa Suto, Yoshikazu Tanaka, Takuya Ito, Yoshio Kodera, Takashi Matsui

    Biochemistry　64　(19)　4196-4205　2025/10/07

    DOI： 10.1021/acs.biochem.5c00329 　

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    Enzymes are involved in the biosynthesis of a variety of secondary metabolites found in nature. The catalytic mechanism is regulated by the three-dimensional structure of the enzyme, particularly at the catalytic site, resulting in the synthesis of natural products with complex conformations derived from a regioselective, chemoselective, or stereoselective preference of the enzyme reaction. Prenyltransferase, which belongs to the prenylsynthase superfamily, catalyzes the condensation of isoprene to an aromatic compound, consequently producing a terpenoid scaffold structure. Prenyltransferase thus plays an important role in expanding the chemical diversity of the terpenoids. Although the three-dimensional structures of prenylsynthases categorized in the same superfamily have been resolved, the catalytic mechanism of prenyltransferase has been veiled. In this study, we determined the X-ray crystal structure of a novel prenyltransferase, Ord1, which is derived from Streptomyces. Here, we report the enzymatic characteristics of the Ord1 and discuss its catalytic mechanism.

  7. Structure reveals a regulation mechanism of plant outward-rectifying K+ channel GORK by structural rearrangements in the CNBD-Ankyrin bridge. International-journal

    Taro Yamanashi, Yuki Muraoka, Tadaomi Furuta, Tsukasa Kume, Natsuko Sekido, Shunya Saito, Shota Terashima, Takeshi Yokoyama, Yoshikazu Tanaka, Atsushi Miyamoto, Kanane Sato, Tomoyuki Ito, Hikaru Nakazawa, Mitsuo Umetsu, Ellen Tanudjaja, Masaru Tsujii, Ingo Dreyer, Julian I Schroeder, Yasuhiro Ishimaru, Nobuyuki Uozumi

    Proceedings of the National Academy of Sciences of the United States of America　122　(30)　e2500070122　2025/07/29

    DOI： 10.1073/pnas.2500070122 　

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    Guard cells, which regulate stomatal apertures in plants, possess a sophisticated mechanism for regulating turgor pressure. The outward-rectifying "K+out" channel GORK, expressed in guard cells of the plant Arabidopsis thaliana, is a central component that promotes stomatal closure by releasing K+ to the extracellular space, thereby lowering turgor pressure. To date, the structural basis underlying the regulation of the K+ transport activity of GORK is unclear. Using cryo-EM, we determined the structures of the GORK outward-rectifying K+ channel with a resolution of 3.16 to 3.27 Å in five distinct conformations that differ significantly in their C-terminal cyclic nucleotide binding domain (CNBD) and ankyrin repeat (ANK) domain. The C-linker connects the transmembrane domains to the C-terminal domains, i.e., CNBD, CNBD-Ankyrin bridge, and ANK. The structural changes and interactions in the C-linker determine whether the closed state of GORK is closer to the preopen state or in a more removed state from the open state of the channel. In particular, interconversion in the short sequence within the CNBD-Ankyrin bridge plays a decisive role in this determination. This region forms an α-helix in the preopened state, while it adopts a nonhelical structure in further distant closed states. The dynamics of the cytosolic region strongly suggest that the K+ channel activity of GORK is regulated by cytosolic signaling factors during stomatal closure.

  8. Bispecific antibody-antigen complex structures reveal activity enhancement by domain rearrangement. International-journal

    Kyohei Sato, Shiro Uehara, Atsushi Tsugita, Mayuka Ishii, Shieru Ishiyama, Atsushi Maejima, Ishin Nakahara, Misae Nazuka, Takashi Matsui, Gatsogiannis Christos, Takeshi Yokoyama, Izumi Kumagai, Koki Makabe, Ryutaro Asano, Yoshikazu Tanaka

    Cell reports　44　(7)　115965-115965　2025/07/22

    DOI： 10.1016/j.celrep.2025.115965 　

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    Bispecific antibodies (BsAbs) have been developed as anti-cancer drugs that accumulate activated T cells on cancer cells by bridging the antigens present in each cell. Ex3 is a diabody-type BsAb composed of an anti-epidermal growth factor receptor (EGFR) antibody and an anti-CD3 antibody. In the design of Ex3, the LH-type domain order (Ex3LH) is shown to have more than 100-fold greater anti-cancer activity than the HL-type domain order (Ex3HL). To understand this phenomenon of activity enhancement by domain-order rearrangement, we report here cryoelectron microscopy (cryo-EM) structures of both Ex3HL and Ex3LH in complex with EGFR and CD3. A structural comparison of the HL and LH types reveals that the domain rearrangement leads to drastic structural changes and that the avoidance of steric hindrance by a favorable bridging angle on the cell surface is the fundamental mechanism for this activity enhancement.

  9. Mitochondria-Homing Drug Mitochonic Acid 5 Improves Barth Syndrome Myopathy in a Human-Induced Pluripotent Stem Cell Model and Barth Syndrome Drosophila Model. International-journal

    Yoshiyasu Tongu, Tomoko Kasahara, Tetsuro Matsuhashi, Yoshitsugu Oikawa, Ryota Akimoto, Yuhan Luo, Sayaka Sekine, Momoka Suzuki, Hitomi Kashiwagi, Shinichiro Kanno, Yoshikazu Tanaka, Kyohei Sato, Yusuke Okubo, Akihiko Muto, Hidetaka Tokuno, Chitose Suzuki, Chiharu Kawabe, Takamasa Ishikawa, Shun Watanabe, Koichi Kikuchi, Shun Itai, Takeya Sato, Takehiro Suzuki, Kazuhiro Igarashi, Shinji Fukuda, Tomoyoshi Soga, Kei Murayama, Erina Kuranaga, Takafumi Toyohara, Takaaki Abe

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology　39　(12)　e70739　2025/06/30

    DOI： 10.1096/fj.202401856RRR 　

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    Barth syndrome (BTHS) is a rare disease caused by mutations in the tafazzin gene that affects the heart and muscles; however, to date, no clinically effective drugs are available. In BTHS, mitochondrial function is reduced owing to changes in cardiolipin metabolism. We developed mitochonic acid 5 (MA-5), a small-molecule compound that increases ATP levels, improves mitochondrial dynamics, and is effective in treating mitochondrial and muscle diseases. Therefore, this study examined the effectiveness of MA-5 in treating BTHS. The mitochondrial functions of four isolated BTHS skin fibroblasts were examined. Human BTHS induced pluripotent stem cell (iPSC) were differentiated into myoblasts and cardiolipin metabolism and mitochondrial functions were analyzed. RNA-seq was performed to clarify the metabolic changes. Using a Drosophila melanogaster model of BTHS, the effects of MA-5 on motor performance and cardiac phenotype were examined. MA-5 improved mitochondrial function and reduced cell death due to oxidative stress in skin fibroblasts of patients with BTHS. MA-5 promoted ATP production and reduced oxidative stress in human BTHS iPS cell-derived myoblasts. RNA-seq analysis revealed that MA-5 alleviated endoplasmic reticulum stress in BTHS cells. Administration of MA-5 to BTHS Drosophila improved locomotor ability and tachycardia observed in patients with BTHS. Protein interaction analyses suggested colocalization of ATPase and the MA-5-binding protein mitofilin. These data suggested that MA-5 improves BTHS dysfunction and may serve as a novel therapeutic agent for BTHS.

  10. Creation of a macrolide antibiotic against non-tuberculous Mycobacterium using late-stage boron-mediated aglycon delivery. International-journal

    Yuka Isozaki, Takumi Makikawa, Kosuke Kimura, Daiki Nishihara, Maho Fujino, Yoshikazu Tanaka, Chigusa Hayashi, Yoshimasa Ishizaki, Masayuki Igarashi, Takeshi Yokoyama, Kazunobu Toshima, Daisuke Takahashi

    Science advances　11　(10)　eadt2352　2025/03/07

    DOI： 10.1126/sciadv.adt2352 　

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    Non-tuberculous mycobacteria (NTM) is gaining clinical recognition as a recently emerging pulmonary pathogen. Mycobacterium avium complex (MAC), the most common NTM, is the cause of pulmonary MAC disease. Currently, the macrolide azithromycin (AZM) is the standard first-line antibiotic for treatment of the disease. However, the rise of drug-resistant MAC necessitates the development of alternative therapeutics. Here, we present a late-stage boron-mediated aglycon delivery strategy for selective modification of AZM, generating a library of potential anti-MAC drugs designated KU01 to KU13. Screening of KU01 to KU13 revealed that KU13 exhibited enhanced antimicrobial activity against wild-type and macrolide-resistant MAC compared to AZM. Cryo-electron microscopy analysis indicated that the inserted tercyclic moiety of KU13 formed a robust anchor on the bacterial ribosome, creating a binding pocket with base flipping of U2847, potentially bypassing the standard mechanism of macrolide resistance. These results position KU13 as a promising lead for therapeutics against macrolide-resistant MAC.

  11. Construction of bispecific antibodies by specific pairing between the heavy chain and the light chain using removable SpyCatcher/SnoopCatcher units. International-journal

    Jyunna Yoshida, Yuki Kato, Ai Isogawa, Yoshikazu Tanaka, Izumi Kumagai, Ryutaro Asano, Takeshi Nakanishi, Koki Makabe

    Journal of biological engineering　18　(1)　57-57　2024/10/14

    DOI： 10.1186/s13036-024-00454-z 　

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    During the production of bispecific antibodies (bsAbs), nonspecific pairing results in low yields of target bsAb molecules, an issue known as the "mispairing problem." Several antibody engineering techniques have been developed to overcome mispairing issues. Here, we introduce "bsAb by external pairing and excision" (BAPE), a novel chain pairing method that induces specific chain pairing by fusing external SpyCatcher/Tag and SnoopCatcher/Tag units. These tags are then removed via protease cleavage. In this study, we applied this method to force the correct pairings of heavy and light chains while the heavy-chain pairing was achieved by the Knobs-into-Holes mutation. We then confirmed the formation of interchain bridges with covalent isopeptide bonds. Both anti-CD3/anti-Her2 and anti-CD3/anti-EGFR bsAbs displayed satisfactory target binding activities and in vitro cell-killing activity with activated T-cells.

  12. Design of Cyborg Proteins by Loop Region Replacement with Oligo(ethylene glycol): Exploring Suitable Mutations for Cyborg Protein Construction Using Machine Learning Peer-reviewed

    Wijak Yospanya, Akari Matsumura, Yukihiro Imasato, Tomoyuki Itou, Yusuke Aoki, Hikaru Nakazawa, Takashi Matsui, Takeshi Yokoyama, Mihoko Ui, Mitsuo Umetsu, Satoru Nagatoishi, Kouhei Tsumoto, Yoshikazu Tanaka, Kazushi Kinbara

    Bulletin of the Chemical Society of Japan　2024/09/02

    Publisher: Oxford University Press (OUP)

    DOI： 10.1093/bulcsj/uoae090 　

    ISSN： 0009-2673

    eISSN： 1348-0634

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    Abstract We synthesized a “cyborg protein,” wherein a synthetic molecule partially substitutes the main peptide chain by linking two protein domains with a synthetic oligomer. Green fluorescent protein (GFP) served as the model for constructing the cyborg proteins. We prepared circularly permuted GFP (cpGFP) with new termini between β10 and β11, where the original N- and C-termini were linked by a cleavable peptide loop. The cyborg GFP was constructed from cpGFP by linking the β10 and β11 with oligo(ethylene glycol) using maleimide-cysteine couplings, followed by the enzymatic cleavage of the N- and C-termini linking loop by thrombin. With the help of machine learning, we were able to obtain the cpGFP mutants that significantly alter the fluorescence activity (53% increase) by thrombin treatment, which splits cpGFP into two fragments (fragmented-GFP), and by heat shock. When the cyborg GFP was constructed using this mutant, the fluorescence intensity increased by 13% after heat treatment, similar to cpGFP (33% increase), and the behavior was significantly different from that of the fragmented-GFP. This result suggests the possibility that the oligo(ethylene glycol) chain in the cyborg protein plays a similar role to the peptide in the main chain of the protein.

  13. Discrimination of extracellular miRNA sources for the identification of tumor-related functions based on nanowire thermofluidics

    Kunanon Chattrairat, Akira Yokoi, Min Zhang, Mikiko Iida, Kosuke Yoshida, Masami Kitagawa, Ayuka Niwa, Masatoshi Maeki, Takeshi Hasegawa, Takeshi Yokoyama, Yoshikazu Tanaka, Yusuke Miyazaki, Wataru Shinoda, Manabu Tokeshi, Kazuki Nagashima, Takeshi Yanagida, Hiroaki Kajiyama, Yoshinobu Baba, Takao Yasui

    Device　2　(6)　100363　2024/06

    Publisher: Elsevier BV

    DOI： 10.1016/j.device.2024.100363 　

    ISSN： 2666-9986

  14. Characterization of a novel format scFv×VHH single-chain biparatopic antibody against metal binding protein MtsA. International-journal

    Risa Asano, Miyu Takeuchi, Makoto Nakakido, Sho Ito, Chihiro Aikawa, Takeshi Yokoyama, Akinobu Senoo, Go Ueno, Satoru Nagatoishi, Yoshikazu Tanaka, Ichiro Nakagawa, Kouhei Tsumoto

    Protein science : a publication of the Protein Society　33　(6)　e5017　2024/06

    DOI： 10.1002/pro.5017 　

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    Biparatopic antibodies (bpAbs) are engineered antibodies that bind to multiple different epitopes within the same antigens. bpAbs comprise diverse formats, including fragment-based formats, and choosing the appropriate molecular format for a desired function against a target molecule is a challenging task. Moreover, optimizing the design of constructs requires selecting appropriate antibody modalities and adjusting linker length for individual bpAbs. Therefore, it is crucial to understand the characteristics of bpAbs at the molecular level. In this study, we first obtained single-chain variable fragments and camelid heavy-chain variable domains targeting distinct epitopes of the metal binding protein MtsA and then developed a novel format single-chain bpAb connecting these fragment antibodies with various linkers. The physicochemical properties, binding activities, complex formation states with antigen, and functions of the bpAb were analyzed using multiple approaches. Notably, we found that the assembly state of the complexes was controlled by a linker and that longer linkers tended to form more compact complexes. These observations provide detailed molecular information that should be considered in the design of bpAbs.

  15. "Rich arginine and strong positive charge" antimicrobial protein protamine: From its action on cell membranes to inhibition of bacterial vital functions. International-journal

    Momoka Ookubo, Yuka Tashiro, Kosuke Asano, Yoshiharu Kamei, Yoshikazu Tanaka, Takayuki Honda, Takeshi Yokoyama, Michiyo Honda

    Biochimica et biophysica acta. Biomembranes　1866　(5)　184323-184323　2024/06

    DOI： 10.1016/j.bbamem.2024.184323 　

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    Protamine, an antimicrobial protein derived from salmon sperm with a molecular weight of approximately 5 kDa, is composed of 60-70 % arginine and is a highly charged protein. Here, we investigated the mechanism of antimicrobial action of protamine against Cutibacterium acnes (C. acnes) focusing on its rich arginine content and strong positive charge. Especially, we focused on the attribution of dual mechanisms of antimicrobial protein, including membrane disruption or interaction with intracellular components. We first determined the dose-dependent antibacterial activity of protamine against C. acnes. In order to explore the interaction between bacterial membrane and protamine, we analyzed cell morphology, zeta potential, membrane permeability, and the composition of membrane fatty acid. In addition, the localization of protamine in bacteria was observed using fluorescent-labeled protamine. For investigation of the intracellular targets of protamine, bacterial translation was examined using a cell-free translation system. Based on our results, the mechanism of the antimicrobial action of protamine against C. acnes is as follows: 1) electrostatic interactions with the bacterial cell membrane; 2) self-internalization into the bacterial cell by changing the composition of the bacterial membrane; and 3) inhibition of bacterial growth by blocking translation inside the bacteria. However, owing to its strong electric charge, protamine can also interact with DNA, RNA, and other proteins inside the bacteria, and may inhibit various bacterial life processes beyond the translation process.

  16. Direct visualization of ribosomes in the cell-free system revealed the functional evolution of aminoglycoside. International-journal Peer-reviewed

    Junta Tomono, Kosuke Asano, Takuma Chiashi, Masato Suzuki, Masayuki Igarashi, Yoshiaki Takahashi, Yoshikazu Tanaka, Takeshi Yokoyama

    Journal of biochemistry　175　(6)　587-598　2024/05/31

    Publisher: Oxford University Press (OUP)

    DOI： 10.1093/jb/mvae002 　

    ISSN： 0021-924X

    eISSN： 1756-2651

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    Abstract The rapid emergence of multi-drug-resistant bacteria has raised a serious public health concern. Therefore, new antibiotic developments have been highly desired. Here, we propose a new method to visualize antibiotic actions on translating ribosomes in the cell-free system under macromolecular crowding conditions by cryo-electron microscopy, designated as the DARC method: the Direct visualization of Antibiotic binding on Ribosomes in the Cell-free translation system. This new method allows for acquiring a more comprehensive understanding of the mode of action of antibiotics on the translation inhibition without ribosome purification. Furthermore, with the direct link to biochemical analysis at the same condition as cryo-EM observation, we revealed the evolution of 2-DOS aminoglycosides from dibekacin (DBK) to arbekacin (ABK) by acquiring the synthetic tailored anchoring motif to lead to stronger binding affinity to ribosomes. Our cryo-EM structures of DBK and ABK bound ribosomes in the cell-free environment clearly depicted a synthetic tailored γ-amino-α-hydroxybutyryl (HABA) motif formed additional interactions with the ribosome enhancing antibiotic bindings. This new approach would be valuable for understanding the function of antibiotics for more efficient drug development.

  17. Multistep conformational changes leading to the gate opening of light-driven sodium pump rhodopsin. International-journal

    Yukino Sato, Tsubasa Hashimoto, Koji Kato, Akiko Okamura, Kaito Hasegawa, Tsukasa Shinone, Yoshikazu Tanaka, Yoshiki Tanaka, Tomoya Tsukazaki, Takashi Tsukamoto, Makoto Demura, Min Yao, Takashi Kikukawa

    The Journal of biological chemistry　299　(12)　105393-105393　2023/12

    DOI： 10.1016/j.jbc.2023.105393 　

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    Membrane transport proteins require a gating mechanism that opens and closes the substrate transport pathway to carry out unidirectional transport. The "gating" involves large conformational changes and is achieved via multistep reactions. However, these elementary steps have not been clarified for most transporters due to the difficulty of detecting the individual steps. Here, we propose these steps for the gate opening of the bacterial Na+ pump rhodopsin, which outwardly pumps Na+ upon illumination. We herein solved an asymmetric dimer structure of Na+ pump rhodopsin from the bacterium Indibacter alkaliphilus. In one protomer, the Arg108 sidechain is oriented toward the protein center and appears to block a Na+ release pathway to the extracellular (EC) medium. In the other protomer, however, this sidechain swings to the EC side and then opens the release pathway. Assuming that the latter protomer mimics the Na+-releasing intermediate, we examined the mechanism for the swing motion of the Arg108 sidechain. On the EC surface of the first protomer, there is a characteristic cluster consisting of Glu10, Glu159, and Arg242 residues connecting three helices. In contrast, this cluster is disrupted in the second protomer. Our experimental results suggested that this disruption is a key process. The cluster disruption induces the outward movement of the Glu159-Arg242 pair and simultaneously rotates the seventh transmembrane helix. This rotation resultantly opens a space for the swing motion of the Arg108 sidechain. Thus, cluster disruption might occur during the photoreaction and then trigger sequential conformation changes leading to the gate-open state.

  18. Synthesis of epitope-targeting nanobody based on native protein-protein interactions for FtsZ filamentation suppressor. International-journal

    Hikaru Nakazawa, Taiji Katsuki, Takashi Matsui, Atsushi Tsugita, Takeshi Yokoyama, Tomoyuki Ito, Sakiya Kawada, Yoshikazu Tanaka, Mitsuo Umetsu

    Biotechnology journal　18　(11)　e2300039　2023/07/17

    DOI： 10.1002/biot.202300039 　

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    Phage display and biopanning are powerful tools for generating binding molecules for a specific target. However, the selection process based only on binding affinity provides no assurance for the antibody's affinity to the target epitope. In this study, we propose a molecular-evolution approach guided by native protein-protein interactions to generate epitope-targeting antibodies. The binding-site sequence in a native protein was grafted into a complementarity-determining region (CDR) in the nanobody, and a nonrelated CDR loop (in the grafted nanobody) was randomized to create a phage display library. In this construction of nanobodies by integrating graft and evolution technology (CAnIGET method), suitable grafting of the functional sequence added functionality to the nanobody, and the molecular-evolution approach enhanced the binding function to inhibit the native protein-protein interactions. To apply for biological tool with growth screening, model nanobodies with an affinity for filamenting temperature-sensitive mutant Z (FtsZ) from Staphylococcus aureus were constructed and completely inhibited the polymerization of FtsZ as a function. Consequently, the expression of these nanobodies drastically decreased the cell division rate. We demonstrate the potential of the CAnIGET method with the use of native protein-protein interactions for steady epitope-specific evolutionary engineering. This article is protected by copyright. All rights reserved.

  19. Simple Method for the Creation of a Bacteria-Sized Unilamellar Liposome with Different Proteins Localized to the Respective Sides of the Membrane. International-journal

    Kosaku Noba, Shogo Yoshimoto, Yoshikazu Tanaka, Takeshi Yokoyama, Tomoaki Matsuura, Katsutoshi Hori

    ACS synthetic biology　12　(5)　1437-1446　2023/05/19

    DOI： 10.1021/acssynbio.2c00564 　

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    Artificial cells are membrane vesicles mimicking cellular functions. To date, giant unilamellar vesicles made from a single lipid membrane with a diameter of 10 μm or more have been used to create artificial cells. However, the creation of artificial cells that mimic the membrane structure and size of bacteria has been limited due to technical restrictions of conventional liposome preparation methods. Here, we created bacteria-sized large unilamellar vesicles (LUVs) with proteins localized asymmetrically to the lipid bilayer. Liposomes containing benzylguanine-modified phospholipids were prepared by combining the conventional water-in-oil emulsion method and the extruder method, and green fluorescent protein fused with SNAP-tag was localized to the inner leaflet of the lipid bilayer. Biotinylated lipid molecules were then inserted externally, and the outer leaflet was modified with streptavidin. The resulting liposomes had a size distribution in the range of 500-2000 nm with a peak at 841 nm (the coefficient of variation was 10.3%), which was similar to that of spherical bacterial cells. Fluorescence microscopy, quantitative evaluation using flow cytometry, and western blotting proved the intended localization of different proteins on the lipid membrane. Cryogenic electron microscopy and quantitative evaluation by α-hemolysin insertion revealed that most of the created liposomes were unilamellar. Our simple method for the preparation of bacteria-sized LUVs with asymmetrically localized proteins will contribute to the creation of artificial bacterial cells for investigating functions and the significance of their surface structure and size.

  20. The difference in the cellular uptake of tocopherol and tocotrienol is influenced by their affinities to albumin. International-journal

    Takashi Nakatomi, Mayuko Itaya-Takahashi, Yosuke Horikoshi, Naoki Shimizu, Isabella Supardi Parida, Mirinthorn Jutanom, Takahiro Eitsuka, Yoshikazu Tanaka, Jean-Marc Zingg, Tatsuya Matsura, Kiyotaka Nakagawa

    Scientific reports　13　(1)　7392-7392　2023/05/06

    DOI： 10.1038/s41598-023-34584-z 　

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    Vitamin E is classified into tocopherol (Toc) and tocotrienol (T3) based on its side chains. T3 generally has higher cellular uptake than Toc, though the responsible mechanism remains unclear. To elucidate this mechanism, we hypothesized and investigated whether serum albumin is a factor that induces such a difference in the cellular uptake of Toc and T3. Adding bovine serum albumin (BSA) to serum-depleted media increased the cellular uptake of T3 and decreased that of Toc, with varying degrees among α-, β-, γ-, and δ-analogs. Such enhanced uptake of α-T3 was not observed when cells were incubated under low temperature (the uptake of α-Toc was also reduced), suggesting that Toc and T3 bind to albumin to form a complex that results in differential cellular uptake of vitamin E. Fluorescence quenching study confirmed that vitamin E certainly bound to BSA, and that T3 showed a higher affinity than Toc. Molecular docking further indicated that the differential binding energy of Toc or T3 to BSA is due to the Van der Waals interactions via their side chain. Overall, these results suggested that the affinity of Toc and T3 to albumin differs due to their side chains, causing the difference in their albumin-mediated cellular uptake. Our results give a better mechanistic insight into the physiological action of vitamin E.

  21. RPL3L-containing ribosomes determine translation elongation dynamics required for cardiac function. International-journal

    Chisa Shiraishi, Akinobu Matsumoto, Kazuya Ichihara, Taishi Yamamoto, Takeshi Yokoyama, Taisuke Mizoo, Atsushi Hatano, Masaki Matsumoto, Yoshikazu Tanaka, Eriko Matsuura-Suzuki, Shintaro Iwasaki, Shouji Matsushima, Hiroyuki Tsutsui, Keiichi I Nakayama

    Nature communications　14　(1)　2131-2131　2023/04/20

    DOI： 10.1038/s41467-023-37838-6 　

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    Although several ribosomal protein paralogs are expressed in a tissue-specific manner, how these proteins affect translation and why they are required only in certain tissues have remained unclear. Here we show that RPL3L, a paralog of RPL3 specifically expressed in heart and skeletal muscle, influences translation elongation dynamics. Deficiency of RPL3L-containing ribosomes in RPL3L knockout male mice resulted in impaired cardiac contractility. Ribosome occupancy at mRNA codons was found to be altered in the RPL3L-deficient heart, and the changes were negatively correlated with those observed in myoblasts overexpressing RPL3L. RPL3L-containing ribosomes were less prone to collisions compared with RPL3-containing canonical ribosomes. Although the loss of RPL3L-containing ribosomes altered translation elongation dynamics for the entire transcriptome, its effects were most pronounced for transcripts related to cardiac muscle contraction and dilated cardiomyopathy, with the abundance of the encoded proteins being correspondingly decreased. Our results provide further insight into the mechanisms and physiological relevance of tissue-specific translational regulation.

  22. Quick and Spontaneous Transformation between [3Fe–4S] and [4Fe–4S] Iron–Sulfur Clusters in the tRNA-Thiolation Enzyme TtuA

    Masato Ishizaka, Minghao Chen, Shun Narai, Yoshikazu Tanaka, Toyoyuki Ose, Masaki Horitani, Min Yao

    International Journal of Molecular Sciences　24　(1)　833-833　2023/01/03

    Publisher: MDPI AG

    DOI： 10.3390/ijms24010833 　

    eISSN： 1422-0067

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    Iron–sulfur (Fe–S) clusters are essential cofactors for enzyme activity. These Fe–S clusters are present in structurally diverse forms, including [4Fe–4S] and [3Fe–4S]. Type-identification of the Fe–S cluster is indispensable in understanding the catalytic mechanism of enzymes. However, identifying [4Fe–4S] and [3Fe–4S] clusters in particular is challenging because of their rapid transformation in response to oxidation–reduction events. In this study, we focused on the relationship between the Fe–S cluster type and the catalytic activity of a tRNA-thiolation enzyme (TtuA). We reconstituted [4Fe–4S]-TtuA, prepared [3Fe–4S]-TtuA by oxidizing [4Fe–4S]-TtuA under strictly anaerobic conditions, and then observed changes in the Fe–S clusters in the samples and the enzymatic activity in the time-course experiments. Electron paramagnetic resonance analysis revealed that [3Fe–4S]-TtuA spontaneously transforms into [4Fe–4S]-TtuA in minutes to one hour without an additional free Fe source in the solution. Although the TtuA immediately after oxidation of [4Fe–4S]-TtuA was inactive [3Fe–4S]-TtuA, its activity recovered to a significant level compared to [4Fe–4S]-TtuA after one hour, corresponding to an increase of [4Fe–4S]-TtuA in the solution. Our findings reveal that [3Fe–4S]-TtuA is highly inactive and unstable. Moreover, time-course analysis of structural changes and activity under strictly anaerobic conditions further unraveled the Fe–S cluster type used by the tRNA-thiolation enzyme.

  23. Structural and molecular insight into antibody recognition of dynamic neoepitopes in membrane tethered MUC1 of pancreatic cancer cells and secreted exosomes International-journal

    Hajime Wakui, Yasuhiro Yokoi, Chieko Horidome, Toyoyuki Ose, Min Yao, Yoshikazu Tanaka, Hiroshi Hinou, Shin-Ichiro Nishimura

    RSC Chemical Biology　4　(8)　564-572　2023

    Publisher: Royal Society of Chemistry (RSC)

    DOI： 10.1039/d3cb00036b 　

    eISSN： 2633-0679

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    We unveil for the first time that pancreatic cancer cells (PANC-1) and secreted exosomes express MUC1 bearing cancer-relevant dynamic epitopes recognized specifically by an anti-MUC1 antibody (SN-131).

  24. A marine sponge-derived lectin reveals hidden pathway for thrombopoietin receptor activation. International-journal

    Hiromi Watari, Hiromu Kageyama, Nami Masubuchi, Hiroya Nakajima, Kako Onodera, Pamela J Focia, Takumi Oshiro, Takashi Matsui, Yoshio Kodera, Tomohisa Ogawa, Takeshi Yokoyama, Makoto Hirayama, Kanji Hori, Douglas M Freymann, Misa Imai, Norio Komatsu, Marito Araki, Yoshikazu Tanaka, Ryuichi Sakai

    Nature communications　13　(1)　7262-7262　2022/11/25

    DOI： 10.1038/s41467-022-34921-2 　

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    N-glycan-mediated activation of the thrombopoietin receptor (MPL) under pathological conditions has been implicated in myeloproliferative neoplasms induced by mutant calreticulin, which forms an endogenous receptor-agonist complex that traffics to the cell surface and constitutively activates the receptor. However, the molecular basis for this mechanism is elusive because oncogenic activation occurs only in the cell-intrinsic complex and is thus cannot be replicated with external agonists. Here, we describe the structure and function of a marine sponge-derived MPL agonist, thrombocorticin (ThC), a homodimerized lectin with calcium-dependent fucose-binding properties. In-depth characterization of lectin-induced activation showed that, similar to oncogenic activation, sugar chain-mediated activation persists due to limited receptor internalization. The strong synergy between ThC and thrombopoietin suggests that ThC catalyzes the formation of receptor dimers on the cell surface. Overall, the existence of sugar-mediated MPL activation, in which the mode of activation is different from the original ligand, suggests that receptor activation is unpredictably diverse in living organisms.

  25. Functional characterization of 12 dihydropyrimidinase allelic variants in Japanese individuals for the prediction of 5-fluorouracil treatment-related toxicity. International-journal

    Eiji Hishinuma, Yoko Narita, Evelyn Marie Gutiérrez Rico, Akiko Ueda, Kai Obuchi, Yoshikazu Tanaka, Sakae Saito, Shu Tadaka, Kengo Kinoshita, Masamitsu Maekawa, Nariyasu Mano, Tomoki Nakayoshi, Akifumi Oda, Noriyasu Hirasawa, Masahiro Hiratsuka

    Drug metabolism and disposition: the biological fate of chemicals　51　(2)　165-173　2022/11/22

    DOI： 10.1124/dmd.122.001045 　

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    The drug 5-fluorouracil (5-FU) is the first-choice chemotherapeutic agent against advanced-stage cancers. However, 10-30% of treated patients experience grade 3-4 toxicity. The deficiency of dihydropyrimidinase (DHPase), which catalyzes the second step of the 5-FU degradation pathway, is correlated with the risk of developing toxicity. Thus, genetic polymorphisms within DPYS, the DHPase-encoding gene, could potentially serve as predictors of severe 5-FU-related toxicity. We identified 12 novel DPYS variants in 3,554 Japanese individuals, but the effects of these mutations on function remain unknown. In the current study, we performed in vitro enzymatic analyses of the 12 newly identified DHPase variants. Dihydrouracil or dihydro-5-FU hydrolytic ring-opening kinetic parameters, Km and Vmax , and intrinsic clearance (CLint = Vmax /Km ) of the wild-type DHPase and eight variants were measured. Five of these variants (R118Q, H295R, T418I, Y448H, and T513A) showed significantly reduced CLint compared with that in the wild-type. The parameters for the remaining four variants (V59F, D81H, T136M, and R490H) could not be determined as dihydrouracil and dihydro-5-FU hydrolytic ring-opening activity was undetectable. We also determined DHPase variant protein stability using cycloheximide and bortezomib. The mechanism underlying the observed changes in the kinetic parameters was clarified using blue-native polyacrylamide gel electrophoresis and three-dimensional structural modeling. The results suggested that the decrease or loss of DHPase enzymatic activity was due to reduced stability and oligomerization of DHPase variant proteins. Our findings support the use of DPYS polymorphisms as novel pharmacogenomic markers for predicting severe 5-FU-related toxicity in the Japanese population. Significance Statement DHPase contributes to the degradation of 5-fluorouracil, and genetic polymorphisms that cause decreased activity of DHPase can cause severe toxicity. In this study, we performed functional analysis of 12 DHPase variants in the Japanese population and identified 9 genetic polymorphisms that cause reduced DHPase function. In addition, we found that the ability to oligomerize and the conformation of the active site are important for the enzymatic activity of DHPase.

  26. An ancestral function of strigolactones as symbiotic rhizosphere signals. International-journal

    Kyoichi Kodama, Mélanie K Rich, Akiyoshi Yoda, Shota Shimazaki, Xiaonan Xie, Kohki Akiyama, Yohei Mizuno, Aino Komatsu, Yi Luo, Hidemasa Suzuki, Hiromu Kameoka, Cyril Libourel, Jean Keller, Keiko Sakakibara, Tomoaki Nishiyama, Tomomi Nakagawa, Kiyoshi Mashiguchi, Kenichi Uchida, Kaori Yoneyama, Yoshikazu Tanaka, Shinjiro Yamaguchi, Masaki Shimamura, Pierre-Marc Delaux, Takahito Nomura, Junko Kyozuka

    Nature communications　13　(1)　3974-3974　2022/07/08

    DOI： 10.1038/s41467-022-31708-3 　

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    In flowering plants, strigolactones (SLs) have dual functions as hormones that regulate growth and development, and as rhizosphere signaling molecules that induce symbiosis with arbuscular mycorrhizal (AM) fungi. Here, we report the identification of bryosymbiol (BSB), an SL from the bryophyte Marchantia paleacea. BSB is also found in vascular plants, indicating its origin in the common ancestor of land plants. BSB synthesis is enhanced at AM symbiosis permissive conditions and BSB deficient mutants are impaired in AM symbiosis. In contrast, the absence of BSB synthesis has little effect on the growth and gene expression. We show that the introduction of the SL receptor of Arabidopsis renders M. paleacea cells BSB-responsive. These results suggest that BSB is not perceived by M. paleacea cells due to the lack of cognate SL receptors. We propose that SLs originated as AM symbiosis-inducing rhizosphere signaling molecules and were later recruited as plant hormone.

  27. The carbohydrate tail of landomycin A is responsible for its interaction with the repressor protein LanK. International-journal

    Atsushi Tsugita, Shiro Uehara, Takashi Matsui, Takeshi Yokoyama, Iryna Ostash, Maksym Deneka, Subbarao Yalamanchili, Clay S Bennett, Yoshikazu Tanaka, Bohdan Ostash

    The FEBS journal　2022/04/16

    DOI： 10.1111/febs.16460 　

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    Landomycin A (LaA) is the largest member of the landomycin group of angucyclic polyketides. Its unusual structure and strong anticancer properties have attracted great interest from chemists and biologists alike. This, in particular, has led to a detailed picture of LaA biosynthesis in Streptomyces cyanogenus S136, the only known LaA producer. LanK is a TetR family repressor protein that limits the export of landomycins from S136 cells until significant amounts of the final product, LaA, have accumulated. Landomycins carrying three or more carbohydrate units in their glycosidic chain are effector molecules for LanK. Yet, the exact mechanism that LanK uses to distinguish the final product, LaA, from intermediate landomycins and sense accumulation of LaA was not known. Here, we report crystal structures of LanK, alone and in complex with LaA, and bioassays of LanK's interaction with synthetic carbohydrate chains of LaA (hexasaccharide) and LaE (trisaccharide). Our data collectively suggest that the carbohydrate moieties are the sole determinants of the interaction of the landomycins with LanK, triggering the latter's dissociation from the lanK-lanJ intergenic region via structure conversion of the helices in the C-terminal ligand-binding domain. Analysis of the available literature suggests that LanK represents an unprecedented type of TetR family repressor that recognises the carbohydrate portion of a natural product, and not an aglycon, as it is the case, for example, with the SimR repressor involved in simocyclinone biosynthesis.

  28. Hidden pathway for cytokine receptor activation: a marine sponge-derived lectin reveals sugar-mediated thrombopoietin receptor activation

    Hiromi Watari, Hiromu Kageyama, Nami Masubuchi, Hiroya Nakajima, Kako Onodera, Pamela Focia, Takumi Oshiro, Takashi Matsui, Yoshio Kodera, Tomohisa Ogawa, Takeshi Yokoyama, Makoto Hirayama, Kanji Hori, Douglas Freymann, Norio Komatsu, Marito Araki, Yoshikazu Tanaka, Ryuichi Sakai

    2022/04/01

    DOI： 10.21203/rs.3.rs-1467606/v1 　

  29. Structural insights reveal the second base catalyst of isomaltose glucohydrolase. International-journal

    Takayoshi Tagami, Minghao Chen, Yuta Furunaga, Asako Kikuchi, Juri Sadahiro, Weeranuch Lang, Masayuki Okuyama, Yoshikazu Tanaka, Tomohito Iwasaki, Min Yao, Atsuo Kimura

    The FEBS journal　289　(4)　1118-1134　2022/02

    DOI： 10.1111/febs.16237 　

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    Glycoside hydrolase family 15 (GH15) inverting enzymes contain two glutamate residues functioning as a general acid catalyst and a general base catalyst, for isomaltose glucohydrolase (IGHase), Glu178 and Glu335, respectively. Generally, a two-catalytic residue-mediated reaction exhibits a typical bell-shaped pH-activity curve. However, IGHase is found to display atypical non-bell-shaped pH-kcat and pH-kcat /Km profiles, theoretically better-fitted to a three-catalytic residue-associated pH-activity curve. We determined the crystal structure of IGHase by the single-wavelength anomalous dispersion method using sulfur atoms and the cocrystal structure of a catalytic base mutant E335A with isomaltose. Although the activity of E335A was undetectable, the electron density observed in its active site pocket did not correspond to an isomaltose but a glycerol and a β-glucose, cryoprotectant, and hydrolysis product. Our structural and biochemical analyses of several mutant enzymes suggest that Tyr48 acts as a second catalytic base catalyst. Y48F mutant displayed almost equivalent specific activity to a catalytic acid mutant E178A. Tyr48, highly conserved in all GH15 members, is fixed by another Tyr residue in many GH15 enzymes; the latter Tyr is replaced by Phe290 in IGHase. The pH profile of F290Y mutant changed to a bell-shaped curve, suggesting that Phe290 is a key residue distinguishing Tyr48 of IGHase from other GH15 members. Furthermore, F290Y is found to accelerate the condensation of isomaltose from glucose by modifying a hydrogen-bonding network between Tyr290-Tyr48-Glu335. The present study indicates that the atypical Phe290 makes Tyr48 of IGHase unique among GH15 enzymes.

  30. Chimeric mutants of staphylococcal hemolysin, which act as both one-component and two-component hemolysin, created by grafting the stem domain. International-journal

    Nouran Ghanem, Natsuki Kanagami, Takashi Matsui, Kein Takeda, Jun Kaneko, Yasuyuki Shiraishi, Christian A Choe, Tomomi Uchikubo-Kamo, Mikako Shirouzu, Tsubasa Hashimoto, Tomohisa Ogawa, Tomoaki Matsuura, Po-Ssu Huang, Takeshi Yokoyama, Yoshikazu Tanaka

    The FEBS journal　2022/01/14

    DOI： 10.1111/febs.16354 　

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    Staphylococcus aureus expresses several hemolytic pore-forming toxins (PFTs), which are all commonly composed of three domains: cap, rim and stem. PFTs are expressed as soluble monomers and assemble to form a transmembrane β-barrel pore in the erythrocyte cell membrane. The stem domain undergoes dramatic conformational changes to form a pore. Staphylococcal PFTs are classified into two groups: one-component α-hemolysin (α-HL) and two-component γ-hemolysin (γ-HL). The α-HL forms a homo-heptamer, whereas γ-HL is an octamer composed of F-component (LukF) and S-component (Hlg2). Because PFTs are used as materials for nanopore-based sensors, knowledge of the functional properties of PFTs is used to develop new, engineered PFTs. However, it remains challenging to design PFTs with a β-barrel pore because their formation as transmembrane protein assemblies requires large conformational changes. In the present study, aiming to investigate the design principles of the β-barrel formed as a consequence of the conformational change, chimeric mutants composed of the cap/rim domains of α-HL and the stem of LukF or Hlg2 were prepared. Biochemical characterization and electron microscopy showed that one of them assembles as a heptameric one-component PFT, whereas another participates as both a heptameric one- and heptameric/octameric two-component PFT. All chimeric mutants intrinsically assemble into SDS-resistant oligomers. Based on these observations, the role of the stem domain of these PFTs is discussed. These findings provide clues for the engineering of staphylococcal PFT β-barrels for use in further promising applications.

  31. Biochemical properties of CumA multicopper oxidase from plant pathogen, Pseudomonas syringae. International-journal

    Konan Ishida, Yuya Tsukamoto, Masaki Horitani, Tomohisa Ogawa, Yoshikazu Tanaka

    Bioscience, biotechnology, and biochemistry　85　(9)　1995-2002　2021/07/09

    DOI： 10.1093/bbb/zbab123 　

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    Multicopper oxidases have a wide range of substrate specificity to be involved in various physiological reactions. Pseudomonas syringae, a plant pathogenic bacterium, has a multicopper oxidase, CumA. Multicopper oxidases have ability to degrade plant cell wall component, lignin. Once P. syringae enter apoplast and colonize, they start to disrupt plant immunity. Therefore, deeper understanding of multicopper oxidases from plant pathogens, help to invent measures to prevent invasion into plant cell, which bring agricultural benefits. Several biochemical studies have reported lower activity of CumA compared with other multicopper oxidase called CotA. However, the mechanisms underlying the difference in activity have not yet been revealed. In order to acquire insight into them, we conducted a biophysical characterization of PsCumA. Our results show that PsCumA has weak type I copper EPR signal, which is essential for oxidation activity. We propose that difference in the coordination of copper ions may decrease reaction frequency.

  32. Anti-EGFR antibody 528 binds to domain III of EGFR at a site shifted from the cetuximab epitope. International-journal

    Koki Makabe, Takeshi Yokoyama, Shiro Uehara, Tomomi Uchikubo-Kamo, Mikako Shirouzu, Kouki Kimura, Kouhei Tsumoto, Ryutaro Asano, Yoshikazu Tanaka, Izumi Kumagai

    Scientific reports　11　(1)　5790-5790　2021/03/11

    DOI： 10.1038/s41598-021-84171-3 　

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    Antibodies have been widely used for cancer therapy owing to their ability to distinguish cancer cells by recognizing cancer-specific antigens. Epidermal growth factor receptor (EGFR) is a promising target for the cancer therapeutics, against which several antibody clones have been developed and brought into therapeutic use. Another antibody clone, 528, is an antagonistic anti-EGFR antibody, which has been the focus of our antibody engineering studies to develop cancer drugs. In this study, we explored the interaction of 528 with the extracellular region of EGFR (sEGFR) via binding analyses and structural studies. Dot blotting experiments with heat treated sEGFR and surface plasmon resonance binding experiments revealed that 528 recognizes the tertiary structure of sEGFR and exhibits competitive binding to sEGFR with EGF and cetuximab. Single particle analysis of the sEGFR-528 Fab complex via electron microscopy clearly showed the binding of 528 to domain III of sEGFR, the domain to which EGF and cetuximab bind, explaining its antagonistic activity. Comparison between the two-dimensional class average and the cetuximab/sEGFR crystal structure revealed that 528 binds to a site that is shifted from, rather than identical to, the cetuximab epitope, and may exclude known drug-resistant EGFR mutations.

  33. Aspergillus oryzae Rutinosidase: Biochemical and Structural Investigation. International-journal

    Koki Makabe, Ruka Hirota, Yoshihito Shiono, Yoshikazu Tanaka, Takuya Koseki

    Applied and environmental microbiology　87　(3)　2021/01/15

    DOI： 10.1128/AEM.02438-20 　

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    The rutinosidase (Rut)-encoding gene Aorut has been expressed in Pichia pastoris with its native signal sequence from Aspergillus oryzae Biochemical and structural investigation of the purified recombinant mature A. oryzae Rut (AoRut), designated rAoRutM, was performed in this study. A 1.7-Å resolution crystal structure of rAoRutM was determined, which is an essential step forward in the utilization of AoRut as a potential catalyst. The crystal structure of rAoRutM was represented by a (β/α)8 TIM barrel fold with structural similarity to that of rutinosidase from Aspergillus niger (AnRut) and an exo-β-(1,3)-glucanase from Candida albicans The crystal structure revealed that the catalytic site was located in a deep cleft, similarly to AnRut, and that internal cavities and water molecules were also present. Purified rAoRutM hydrolyzed not only 7-O-linked and 3-O-linked flavonoid rutinosides but also 7-O-linked and 3-O-linked flavonoid glucosides. rAoRutM displayed high catalytic activity toward quercetin 3-O-linked substrates such as rutin and isoquercitrin, rather than to the 7-O-linked substrate, quercetin-7-O-glucoside. Unexpectedly, purified rAoRutM exhibited increased thermostability after treatment with endo-β-N-acetylglucosaminidase H. Circular dichroism (CD) spectra of purified intact rAoRutM and of the enzyme after N-deglycosylation showed a typical α-helical CD profile; however, the molar ellipticity values of the peaks at 208 nm and 212 nm differed. The Km and k cat values for the substrates modified by rutinose were higher than those for the substrates modified by β-d-glucose.IMPORTANCE Flavonoid glycosides constitute a class of secondary metabolites widely distributed in nature. These compounds are involved in bitter taste or clouding in plant-based foods or beverages, respectively. Flavonoid glycoside degradation can proceed through two alternative enzymatic pathways: one that is mediated by monoglycosidases and another that is catalyzed by a diglycosidase. The present report on the biochemical and structural investigation of A. oryzae rutinosidase provides a potential biocatalyst for industrial applications of flavonoids.

  34. Correction: A straightforward approach to antibodies recognising cancer specific glycopeptidic neoepitopes. International-journal

    Hajime Wakui, Yoshikazu Tanaka, Toyoyuki Ose, Isamu Matsumoto, Koji Kato, Yao Min, Taro Tachibana, Masaharu Sato, Kentaro Naruchi, Fayna Garcia Martin, Hiroshi Hinou, Shin-Ichiro Nishimura

    Chemical science　11　(46)　12588-12589　2020/11/18

    DOI： 10.1039/d0sc90254c 　

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    [This corrects the article DOI: 10.1039/D0SC00317D.].

  35. The N-terminal amino-latch region of Hlg2 component of staphylococcal bi-component γ-haemolysin is dispensable for prestem release to form β-barrel pores. International-journal Peer-reviewed

    Kein Takeda, Yoshikazu Tanaka, Jun Kaneko

    Journal of biochemistry　168　(4)　349-354　2020/10/01

    DOI： 10.1093/jb/mvaa052 　

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    The contribution of N-terminal regions of staphylococcal bi-component γ-haemolysin toxin components to haemolytic activity towards human erythrocyte cells was investigated in this study. A deletion construct of N-terminal amino acids 1-10 of Hlg2 (Hlg2 ΔN10), which is the S-component protein of γ-haemolysin, had little effect on its haemolytic activity, whereas N-terminal 1-11 amino acid deletion (Hlg2 ΔN11) significantly delayed haemolysis. Moreover, a deletion of N-terminal amino acids 1-17 of LukF, which is the F-component protein of γ-haemolysin, increased its haemolytic activity in combination with either the wild-type or Hlg2 ΔN10. Unlike the N-terminal amino-latch region of staphylococcal α-haemolysin, which is a single component β-barrel pore-forming toxin, the N-terminal regions present in γ-haemolysin components are dispensable for the haemolytic activity of the bi-component toxin. These results strengthen our recent proposal that staphylococcal bi-component γ-haemolysin toxin uses an N-terminal amino-latch independent molecular switch for prestem release during the formation of β-barrel pores.

  36. Association behavior and control of the quality of cancer therapeutic bispecific diabodies expressed in Escherichia coli Peer-reviewed

    Hikaru Nakazawa, Tomoko Onodera-Sugano, Aruto Sugiyama, Yoshikazu Tanaka, Takamitsu Hattori, Teppei Niide, Hiromi Ogata, Ryutaro Asano, Izumi Kumagai, Mitsuo Umetsu

    Biochemical Engineering Journal　160　107636-107636　2020/08

    Publisher: Elsevier BV

    DOI： 10.1016/j.bej.2020.107636 　

    ISSN： 1369-703X

    eISSN： 1873-295X

  37. A straightforward approach to antibodies recognising cancer specific glycopeptidic neoepitopes. International-journal Peer-reviewed

    Hajime Wakui, Yoshikazu Tanaka, Toyoyuki Ose, Isamu Matsumoto, Koji Kato, Yao Min, Taro Tachibana, Masaharu Sato, Kentaro Naruchi, Fayna Garcia Martin, Hiroshi Hinou, Shin-Ichiro Nishimura

    Chemical science　11　(19)　4999-5006　2020/04/30

    Publisher: Royal Society of Chemistry (RSC)

    DOI： 10.1039/d0sc00317d 　

    ISSN： 2041-6520

    eISSN： 2041-6539

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    Aberrantly truncated immature O-glycosylation in proteins occurs in essentially all types of epithelial cancer cells, which was demonstrated to be a common feature of most adenocarcinomas and strongly associated with cancer proliferation and metastasis. Although extensive efforts have been made toward the development of anticancer antibodies targeting MUC1, one of the most studied mucins having cancer-relevant immature O-glycans, no anti-MUC1 antibody recognises carbohydrates and the proximal MUC1 peptide region, concurrently. Here we present a general strategy that allows for the creation of antibodies interacting specifically with glycopeptidic neoepitopes by using homogeneous synthetic MUC1 glycopeptides designed for the streamlined process of immunization, antibody screening, three-dimensional structure analysis, epitope mapping and biochemical analysis. The X-ray crystal structure of the anti-MUC1 monoclonal antibody SN-101 complexed with the antigenic glycopeptide provides for the first time evidence that SN-101 recognises specifically the essential epitope by forming multiple hydrogen bonds both with the proximal peptide and GalNAc linked to the threonine residue, concurrently. Remarkably, the structure of the MUC1 glycopeptide in complex with SN-101 is identical to its solution NMR structure, an extended conformation induced by site-specific glycosylation. We demonstrate that this method accelerates dramatically the development of a new class of designated antibodies targeting a variety of "dynamic neoepitopes" elaborated by disease-specific O-glycosylation in the immunodominant mucin domains and mucin-like sequences found in intrinsically disordered regions of many proteins.

  38. The [4Fe-4S] cluster of sulfurtransferase TtuA desulfurizes TtuB during tRNA modification in Thermus thermophilus. International-journal Peer-reviewed

    Minghao Chen, Masato Ishizaka, Shun Narai, Masaki Horitani, Naoki Shigi, Min Yao, Yoshikazu Tanaka

    Communications biology　3　(1)　168-168　2020/04/07

    DOI： 10.1038/s42003-020-0895-3 　

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    TtuA and TtuB are the sulfurtransferase and sulfur donor proteins, respectively, for biosynthesis of 2-thioribothymidine (s2T) at position 54 of transfer RNA (tRNA), which is responsible for adaptation to high temperature environments in Thermus thermophilus. The enzymatic activity of TtuA requires an iron-sulfur (Fe-S) cluster, by which a sulfur atom supplied by TtuB is transferred to the tRNA substrate. Here, we demonstrate that the Fe-S cluster directly receives sulfur from TtuB through its inherent coordination ability. TtuB forms a [4Fe-4S]-TtuB intermediate, but that sulfur is not immediately released from TtuB. Further desulfurization assays and mutation studies demonstrated that the release of sulfur from the thiocarboxylated C-terminus of TtuB is dependent on adenylation of the substrate tRNA, and the essential residue for TtuB desulfurization was identified. Based on these findings, the molecular mechanism of sulfur transfer from TtuB to Fe-S cluster is proposed.

  39. Crystal structure of the catalytic unit of GH 87-type α-1,3-glucanase Agl-KA from Bacillus circulans. International-journal Peer-reviewed

    Shigekazu Yano, Wasana Suyotha, Natsuki Oguro, Takashi Matsui, Shota Shiga, Takafumi Itoh, Takao Hibi, Yoshikazu Tanaka, Mamoru Wakayama, Koki Makabe

    Scientific reports　9　(1)　15295-15295　2019/10/25

    DOI： 10.1038/s41598-019-51822-5 　

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    Glycoside hydrolase (GH) 87-type α-1,3-glucanase hydrolyses the α-1,3-glucoside linkages of α-1,3-glucan, which is found in fungal cell walls and extracellular polysaccharides produced by oral Streptococci. In this study, we report on the molecular structure of the catalytic unit of GH 87-type α-1,3-glucanase, Agl-KA, from Bacillus circulans, as determined by x-ray crystallography at a resolution of 1.82 Å. The catalytic unit constitutes a complex structure of two tandemly connected domains-the N-terminal galactose-binding-like domain and the C-terminal right-handed β-helix domain. While the β-helix domain is widely found among polysaccharide-processing enzymes, complex formation with the galactose-binding-like domain was observed for the first time. Biochemical assays showed that Asp1067, Asp1090 and Asp1091 are important for catalysis, and these residues are indeed located at the putative substrate-binding cleft, which forms a closed end and explains the product specificity.

  40. Protein encapsulation in the hollow space of hemocyanin crystals containing a covalently conjugated ligand. International-journal Peer-reviewed

    Tsubasa Hashimoto, Yuxin Ye, Mihoko Ui, Tomohisa Ogawa, Takashi Matsui, Yoshikazu Tanaka

    Biochemical and biophysical research communications　514　(1)　31-36　2019/06/18

    DOI： 10.1016/j.bbrc.2019.04.062 　

    ISSN： 0006-291X

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    Encapsulation of guest molecules into the vacant space of biomacromolecular crystals has been utilized for various purposes including functioning as a protein container to protect against physical stress and structural determination of the guest. Todarodes pacificus hemocyanin (TpHc) is a hollow cylindrical decameric protein complex with an inner space 110 Å in diameter and 160 Å in height. In the crystal, TpHc forms a straw-like bundle and contains one reactive Cys (Cys3246) in the inner domain of each protomer. Here, we conjugated biotin onto Cys3246 of TpHc followed by incubation with streptavidin. The streptavidin was immobilized into the inner space of TpHc due to its interaction with biotin. Moreover, the complex containing TpHc and streptavidin was crystallized under the same conditions used for unmodified TpHc. In order to expand this methodology for a variety of proteins, we conjugated the ligand nitrilotriacetic acid (NTA) chelated to a Ni2+ ion (Ni2+-NTA) to TpHc. We found that His-tagged green fluorescent protein (GFP) was encapsulated into the Ni2+-NTA-conjugated TpHc via the interaction between the His-tag and the Ni2+-NTA group. X-ray crystallography demonstrated that the crystal packing of the complex containing TpHc and GFP was identical to that of the unmodified TpHc. Our guest immobilization method is distinct from previous approaches that are dependent on diffusion of the guest into the host crystal. Thus, our findings may accelerate the development of proteinaceous crystal engineering.

  41. Cryo-EM reveals the asymmetric assembly of squid hemocyanin. International-journal Peer-reviewed

    Yoshikazu Tanaka, Sanae Kato, Markus Stabrin, Stefan Raunser, Takashi Matsui, Christos Gatsogiannis

    IUCrJ　6　(Pt 3)　426-437　2019/05/01

    DOI： 10.1107/S205225251900321X 　

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    The oxygen transporter of molluscs, hemocyanin, consists of long pearl-necklace-like subunits of several globular domains. The subunits assemble in a complex manner to form cylindrical decamers. Typically, the first six domains of each subunit assemble together to form the cylinder wall, while the C-terminal domains form a collar that fills or caps the cylinder. During evolution, various molluscs have been able to fine-tune their oxygen binding by deleting or adding C-terminal domains and adjusting their inner-collar architecture. However, squids have duplicated one of the wall domains of their subunits instead. Here, using cryo-EM and an optimized refinement protocol implemented in SPHIRE, this work tackled the symmetry-mismatched structure of squid hemocyanin, revealing the precise effect of this duplication on its quaternary structure and providing a potential model for its structural evolution.

  42. SDS-induced oligomerization of Lys49-phospholipase A2 from snake venom. International-journal Peer-reviewed

    Takashi Matsui, Shizuka Kamata, Kentaro Ishii, Takahiro Maruno, Nouran Ghanem, Susumu Uchiyama, Koichi Kato, Atsuo Suzuki, Naoko Oda-Ueda, Tomohisa Ogawa, Yoshikazu Tanaka

    Scientific reports　9　(1)　2330-2330　2019/02/20

    DOI： 10.1038/s41598-019-38861-8 　

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    Phospholipase A2 (PLA2) is one of the representative toxic components of snake venom. PLA2s are categorized into several subgroups according to the amino acid at position 49, which comprises either Asp49, Lys49, Arg49 or Ser49. Previous studies suggested that the Lys49-PLA2 assembles into an extremely stable dimer. Although the behavior on Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions suggested the presence of intermolecular disulfide bonds, these bonds were not observed in the crystal structure of Lys49-PLA2. The reason for this discrepancy between the crystal structure and SDS-PAGE of Lys49-PLA2 remains unknown. In this study, we analyzed a Lys49-PLA2 homologue from Protobothrops flavoviridis (PflLys49-PLA2 BPII), by biophysical analyses including X-ray crystallography, SDS-PAGE, native-mass spectrometry, and analytical ultracentrifugation. The results demonstrated that PflLys49-PLA2 BPII spontaneously oligomerized in the presence of SDS, which is one of the strongest protein denaturants.

  43. Encapsulation of biomacromolecules by soaking and co-crystallization into porous protein crystals of hemocyanin. International-journal Peer-reviewed

    Hashimoto T, Ye Y, Matsuno A, Ohnishi Y, Kitamura A, Kinjo M, Abe S, Ueno T, Yao M, Ogawa T, Matsui T, Tanaka Y

    Biochemical and biophysical research communications　509　(2)　577-584　2019/02

    DOI： 10.1016/j.bbrc.2018.12.096 　

    ISSN： 0006-291X

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    Encapsulation of guest molecules into the hollow spaces of crystals has been applied for a variety of purposes such as structure determination, separation, and catalysis of the guest. Although host-guest studies have been developed mainly in crystals of small molecules, those of biomacromolecules have recently been applied. In those reports, a huge hollow space in the protein crystal is commonly used for encapsulation of the guest. Our previous study revealed that cylindrical hemocyanins stack inside the crystal as a linear hollow structure. The diameter of the linear hollow is approximately 110 Å, which is large enough for most proteins to pass through. In the present study, we evaluated the potential of hemocyanin crystals as a host to encapsulate biomacromolecules. Confocal microscopy revealed that hemocyanin crystals encapsulate proteins of molecular mass up to 250 kDa, i.e., 27 kDa green fluorescence protein, 105 kDa allophycocyanin, 220 kDa C-phycocyanin, and 250 kDa phycoerythrin, and DNAs up to 200-bp long, whereas 440 kDa ferritin not. Further analysis revealed that hemocyanin crystals prefer a negatively charged guest rather than a positive charge to encapsulate. Moreover, a photobleaching experiment showed that the guest does not move once entrapped. This knowledge of the host-guest study using the hollow hemocyanin crystal should be of significance for further application of hollow proteinaceous crystals as a host.

  44. Intermolecular ionic interactions serve as a possible switch for stem release in the staphylococcal bi-component toxin for β-barrel pore assembly. Peer-reviewed

    Yoshikazu Tanaka

    Toxicon : official journal of the International Society on Toxinology　2018/10/10

    DOI： 10.1016/j.toxicon.2018.10.002 　

    ISSN： 0041-0101

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    The β-strand stem release system of staphylococcal β-barrel pore-forming toxin γ-hemolysin was investigated. Mutations at K15 and R16 in the cap domain of Hlg2 decreased hemolytic activity more markedly than their effect on erythrocyte binding. In addition, D122N mutation of LukF prestem lost the activity with Hlg2 R16A, indicating that electrostatic interactions between residues in the Hlg2 cap and prestem of adjacent LukF in the ring-shaped complex might serve as a switch for stem release.

  45. Rim domain loops of staphylococcal β-pore forming bi-component toxin S-components recognize target human erythrocytes in a coordinated manner. Peer-reviewed

    Yoshikazu Tanaka

    Journal of biochemistry　164　(2)　93-102　2018/08/01

    DOI： 10.1093/jb/mvy030 　

    ISSN： 0021-924X

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    Staphylococcus aureus bi-component pore-forming toxins consist of S- and F-components, and form hetero-octameric beta-barrel pores on target blood cell membranes. Among them, γ-haemolysin (Hlg2 and F-component of Luk (LukF)) and LukED (LukE and LukD) possess haemolytic activity, whereas the Panton-Valentine leukocidin (LukS-PV and LukF-PV) does not lyse human erythrocytes. Here, we focussed on four loop structures in the rim domain of S-component, namely loops -1, -2, -3 and -4, and found that replacement of Loop-4 in both Hlg2 and LukE with that of LukS-PV abolished their haemolytic activity. Furthermore, LukS-PV gained haemolytic activity by Loop-4 exchange with Hlg2 or LukE, suggesting that Loop-4 of these S-components determined erythrocyte specificity. LOOP-1 and -2 enhanced the erythrocytes-binding ability of both components. Although Hlg2 and LukE recognize Duffy antigen receptor for chemokines on human erythrocytes, the ability of Loop-4 was not complementary between Hlg2 and LukE. Exchange of Hlg2 with LukE Loop-4 showed weaker activity than intact Hlg2, and LukE mutant with Hlg2 Loop-4 lost its haemolytic activity in combination of LukD. Interestingly, the haemolytic activities of these Loop-4 exchange mutants were affected by F-component, namely LukF enhanced haemolytic activities of these Hlg2 and LukE Loop-4 mutants, and also haemolytic activity of LukS-PV mutant with LukE Loop-4.

  46. Compact Seahorse-Shaped T Cell-Activating Antibody for Cancer Therapy Peer-reviewed

    Fujii, H, Tanaka, Y, Nakazawa, H, Sugiyama, A, Manabe, N, Shinoda, A, Shimizu, N, Hattori, T, Hosokawa, K, Sujino, T, Ito, T, Niide, T, Asano, R, Kumagai, I, Umetsu, M

    Adv. Therapeut.　1　(1700031)　1700031-1700031　2018/06

    Publisher: Wiley

    DOI： 10.1002/adtp.201700031 　

    ISSN： 2366-3987

    eISSN： 2366-3987

  47. Heme Binding to Porphobilinogen Deaminase from Vibrio cholerae Decelerates the Formation of 1-Hydroxymethylbilane Peer-reviewed

    Takeshi Uchida, Takumi Funamizu, Minghao Chen, Yoshikazu Tanaka, Koichiro Ishimori

    ACS Chemical Biology　13　(3)　750-760　2018/03/16

    Publisher: American Chemical Society

    DOI： 10.1021/acschembio.7b00934 　

    ISSN： 1554-8937 1554-8929

  48. pH regulates pore formation of a protease activated Vip3Aa from Bacillus thuringiensis Peer-reviewed

    Thittaya Kunthic, Hirokazu Watanabe, Ryuji Kawano, Yoshikazu Tanaka, Boonhiang Promdonkoy, Min Yao, Panadda Boonserm

    BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES　1859　(11)　2234-2241　2017/11

    DOI： 10.1016/j.bbamem.2017.08.018 　

    ISSN： 0005-2736

    eISSN： 0006-3002

  49. Structural basis for tRNA-dependent cysteine biosynthesis Peer-reviewed

    Meirong Chen, Koji Kato, Yume Kubo, Yoshikazu Tanaka, Yuchen Liu, Feng Long, William B. Whitman, Pascal Lill, Christos Gatsogiannis, Stefan Raunser, Nobutaka Shimizu, Akira Shinoda, Akiyoshi Nakamura, Isao Tanaka, Min Yao

    NATURE COMMUNICATIONS　8　(1)　1521-1521　2017/11

    DOI： 10.1038/s41467-017-01543-y 　

    ISSN： 2041-1723

  50. Characterization of the biosynthesis of 2-thiouridine in transfer RNA catalyzed by an iron-sulfur protein Peer-reviewed

    Chen Minghao, Asai Shin-ichi, Narai Shun, Nambu Shusuke, Omura Naoki, Sakaguchi Yuriko, Suzuki Tsutomu, Ikeda-Saito Masao, Watanabe Kimitsuna, Yao Min, Shigi Naoki, Tanaka Yoshikazu

    JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY　22　S239　2017/07

    ISSN： 0949-8257

  51. Biochemical and structural characterization of oxygen-sensitive 2-thiouridine synthesis catalyzed by an iron-sulfur protein TtuA Peer-reviewed

    Minghao Chen, Shin-ichi Asai, Shun Narai, Shusuke Nambu, Naoki Omura, Yuriko Sakaguchi, Tsutomu Suzuki, Masao Ikeda-Saito, Kimitsuna Watanabe, Min Yao, Naoki Shigi, Yoshikazu Tanaka

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA　114　(19)　4954-4959　2017/05

    DOI： 10.1073/pnas.1615585114 　

    ISSN： 0027-8424

  52. STRUCTURAL PROPERTIES OF PHYCOERYTHRIN FROM DULSE PALMARIA PALMATA Peer-reviewed

    Yoshikatsu Miyabe, Tomoe Furuta, Tomoyuki Takeda, Gaku Kanno, Takeshi Shimizu, Yoshikazu Tanaka, Zuoqi Gai, Hajime Yasui, Hideki Kishimura

    JOURNAL OF FOOD BIOCHEMISTRY　41　(1)　e12301　2017/02

    DOI： 10.1111/jfbc.12301 　

    ISSN： 0145-8884

    eISSN： 1745-4514

  53. Crystallographic study of the 2-thioribothymidine-synthetic complex TtuA-TtuB from Thermus thermophilus Peer-reviewed

    Minghao Chen, Shun Narai, Naoki Omura, Naoki Shigi, Sarin Chimnaronk, Yoshikazu Tanaka, Min Yao

    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS　72　(Pt 10)　777-781　2016/10

    DOI： 10.1107/S2053230X16014242 　

    ISSN： 2053-230X

  54. Structural basis for recognition of a kink-turn motif by an archaeal homologue of human RNase P protein Rpp38 Peer-reviewed

    Kosuke Oshima, Yosuke Kakiuchi, Yoshikazu Tanaka, Toshifumi Ueda, Takashi Nakashima, Makoto Kimura, Min Yao

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS　474　(3)　541-546　2016/06

    DOI： 10.1016/j.bbrc.2016.04.118 　

    ISSN： 0006-291X

    eISSN： 1090-2104

  55. Cytoplasmic Heme-Binding Protein (HutX) from Vibrio cholerae Is an Intracellular Heme Transport Protein for the Heme-Degrading Enzyme, HutZ. International-journal Peer-reviewed

    Yukari Sekine, Takehito Tanzawa, Yoshikazu Tanaka, Koichiro Ishimori, Takeshi Uchida

    Biochemistry　55　(6)　884-93　2016/02/16

    DOI： 10.1021/acs.biochem.5b01273 　

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    HutZ is a cytoplasmic heme-binding protein from Vibrio cholerae. Although we have previously identified HutZ as a heme-degrading enzyme [Uchida, T., et al. (2012) Chem. Commun. 48, 6741-6743], the heme transport protein for HutZ remained unknown. To identify the heme transport protein for HutZ, we focused on the heme utilization operon, hutWXZ. To this end, we constructed an expression system for HutX in Escherichia coli and purified it to homogeneity. An absorption spectral analysis demonstrated that HutX binds heme with a 1:1 stoichiometry and a dissociation constant of 7.4 nM. The crystal structure of HutX displays a fold similar to that of the homologous protein, ChuX, from E. coli O157:H7. A structural comparison of HutX and ChuX, and resonance Raman spectra of heme-HutX, suggest that the axial ligand of the ferric heme is Tyr90. The heme bound to HutX is transferred to HutZ with biphasic dissociation kinetics of 8.3 × 10(-2) and 1.5 × 10(-2) s(-1), values distinctly larger than those for transfer from HutX to apomyoglobin. Surface plasmon resonance experiments confirmed that HutX interacts with HutZ with a dissociation constant of ∼400 μM. These results suggest that heme is transferred from HutX to HutZ via a specific protein-protein interaction. Therefore, we can conclude that HutX is a cytoplasmic heme transport protein for HutZ.

  56. Crystal Structure of the 3.8-MDa Respiratory Supermolecule Hemocyanin at 3.0 angstrom Resolution Peer-reviewed

    Zuoqi Gai, Asuka Matsuno, Koji Kato, Sanae Kato, Md Rafiqul Islam Khan, Takeshi Shimizu, Takeya Yoshioka, Yuki Kato, Hideki Kishimura, Gaku Kanno, Yoshikatsu Miyabe, Tohru Terada, Yoshikazu Tanaka, Min Yao

    STRUCTURE　23　(12)　2204-2212　2015/12

    DOI： 10.1016/j.str.2015.09.008 　

    ISSN： 0969-2126

    eISSN： 1878-4186

  57. Structural basis for pore-forming mechanism of staphylococcal alpha-hemolysin Peer-reviewed

    Takaki Sugawara, Daichi Yamashita, Koji Kato, Zhao Peng, Junki Ueda, Jun Kaneko, Yoshiyuki Kamio, Yoshikazu Tanaka, Min Yao

    TOXICON　108　226-231　2015/12

    DOI： 10.1016/j.toxicon.2015.09.033 　

    ISSN： 0041-0101

  58. A Dye-Decolorizing Peroxidase from Vibrio cholerae Peer-reviewed

    Takeshi Uchida, Miho Sasaki, Yoshikazu Tanaka, Koichiro Ishimorit

    BIOCHEMISTRY　54　(43)　6610-6621　2015/11

    DOI： 10.1021/acs.biochem.5b00952 　

    ISSN： 0006-2960

  59. Zinc Ion-binding Activity of an Anti-ZnO VHH Antibody, 4F2 Peer-reviewed

    Ryosuke Sasaki, Soichiro Kitazawa, Ryo Kitahara, Hikaru Nakazawa, Yoshikazu Tanaka, Izumi Kumagai, Mitsuo Umetsu, Koki Makabe

    CHEMISTRY LETTERS　44　(10)　1309-1311　2015/10

    DOI： 10.1246/cl.150537 　

    ISSN： 0366-7022

    eISSN： 1348-0715

  60. Crystallization and preliminary X-ray crystallographic study of a 3.8-MDa respiratory supermolecule hemocyanin Peer-reviewed

    Asuka Matsuno, Zuoqi Gai, Miyuki Tanaka, Koji Kato, Sanae Kato, Tsuyoshi Katoh, Takeshi Shimizu, Takeya Yoshioka, Hideki Kishimura, Yoshikazu Tanaka, Min Yao

    JOURNAL OF STRUCTURAL BIOLOGY　190　(3)　379-382　2015/06

    DOI： 10.1016/j.jsb.2015.04.015 　

    ISSN： 1047-8477

    eISSN： 1095-8657

  61. Human NAT10 Is an ATP-dependent RNA Acetyltransferase Responsible for N-4-Acetylcytidine Formation in 18 S Ribosomal RNA (rRNA) Peer-reviewed

    Satoshi Ito, Sayuri Horikawa, Tateki Suzuki, Hiroki Kawauchi, Yoshikazu Tanaka, Takeo Suzuki, Tsutomu Suzuki

    JOURNAL OF BIOLOGICAL CHEMISTRY　289　(52)　35724-35730　2014/12

    DOI： 10.1074/jbc.C114.602698 　

    ISSN： 0021-9258

    eISSN： 1083-351X

  62. Anchoring protein crystals to mounting loops with hydrogel using inkjet technology Peer-reviewed

    Akira Shinoda, Yoshikazu Tanaka, Min Yao, Isao Tanaka

    ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY　70　(Pt 11)　2794-2799　2014/11

    DOI： 10.1107/S139900471401476X 　

    ISSN： 1399-0047

  63. Nucleoid compaction by MrgA(Asp56Ala/Glu60Ala) does not contribute to staphylococcal cell survival against oxidative stress and phagocytic killing by macrophages Peer-reviewed

    Yuri Ushijima, Ryosuke L. Ohniwa, Atsushi Maruyama, Shinji Saito, Yoshikazu Tanaka, Kazuya Morikawa

    FEMS MICROBIOLOGY LETTERS　360　(2)　144-151　2014/11

    DOI： 10.1111/1574-6968.12598 　

    ISSN： 0378-1097

    eISSN： 1574-6968

  64. Structural and genomic DNA analysis of the putative TetR transcriptional repressor SCO7518 from Streptomyces coelicolor A3(2) Peer-reviewed

    Takeshi Hayashi, Yoshikazu Tanaka, Naoki Sakai, Ui Okada, Min Yao, Nobuhisa Watanabe, Tomohiro Tamura, Isao Tanaka

    FEBS LETTERS　588　(23)　4311-4318　2014/11

    DOI： 10.1016/j.febslet.2014.09.037 　

    ISSN： 0014-5793

    eISSN： 1873-3468

  65. Molecular basis of transmembrane beta-barrel formation of staphylococcal pore-forming toxins Peer-reviewed

    Daichi Yamashita, Takaki Sugawara, Miyu Takeshita, Jun Kaneko, Yoshiyuki Kamio, Isao Tanaka, Yoshikazu Tanaka, Min Yao

    NATURE COMMUNICATIONS　5　4897-4897　2014/09

    DOI： 10.1038/ncomms5897 　

    ISSN： 2041-1723

  66. Crystallization and preliminary X-ray crystallographic analysis of a bacterial Asn-transamidosome Peer-reviewed

    Tateki Suzuki, Keitaro Yamashita, Yoshikazu Tanaka, Isao Tanaka, Min Yao

    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS　70　(Pt 6)　790-793　2014/06

    DOI： 10.1107/S2053230X14007274 　

    ISSN： 1744-3091

  67. Crystallization and preliminary X-ray crystallographic analysis of a bacterial Asn-transamidosome Peer-reviewed

    Tateki Suzuki, Keitaro Yamashita, Yoshikazu Tanaka, Isao Tanaka, Min Yao

    Acta Crystallographica Section F:Structural Biology Communications　70　(6)　790-793　2014

    Publisher: International Union of Crystallography

    DOI： 10.1107/S2053230X14007274 　

    ISSN： 2053-230X

  68. Direct interaction between EFL1 and SBDS is mediated by an intrinsically disordered insertion domain Peer-reviewed

    Nozomi Asano, Haruka Atsuumi, Akiyoshi Nakamura, Yoshikazu Tanaka, Isao Tanaka, Min Yao

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS　443　(4)　1251-1256　2014/01

    DOI： 10.1016/j.bbrc.2013.12.143 　

    ISSN： 0006-291X

    eISSN： 1090-2104

  69. Crystal structure analysis, overexpression and refolding behaviour of a DING protein with single mutation Invited Peer-reviewed

    Zuoqi Gai, Akiyoshi Nakamura, Yoshikazu Tanaka, Nagisa Hirano, Isao Tanaka, Min Yao

    JOURNAL OF SYNCHROTRON RADIATION　20　(Pt 6)　854-858　2013/11

    DOI： 10.1107/S0909049513020694 　

    ISSN： 0909-0495

    eISSN： 1600-5775

  70. Dynamic elements govern the catalytic activity of CapE, a capsular polysaccharide-synthesizing enzyme from Staphylococcus aureus Peer-reviewed

    Takamitsu Miyafusa, Jose M. M. Caaveiro, Yoshikazu Tanaka, Kouhei Tsumoto

    FEBS LETTERS　587　(23)　3824-3830　2013/11

    DOI： 10.1016/j.febslet.2013.10.009 　

    ISSN： 0014-5793

    eISSN： 1873-3468

  71. SCO4008, a putative TetR transcriptional repressor from Streptomyces coelicolor A3(2), regulates transcription of sco4007 by multidrug recognition Peer-reviewed

    Takeshi Hayashi, Yoshikazu Tanaka, Naoki Sakai, Ui Okada, Min Yao, Nobuhisa Watanabe, Tomohiro Tamura, Isao Tanaka

    Journal of Molecular Biology　425　(18)　3289-3300　2013/09/23

    DOI： 10.1016/j.jmb.2013.06.013 　

    ISSN： 0022-2836 1089-8638

  72. Preliminary X-ray crystallographic study of staphylococcal -haemolysin monomer Peer-reviewed

    Takaki Sugawara, Daichi Yamashita, Yoshikazu Tanaka, Jun Kaneko, Yoshiyuki Kamio, Isao Tanaka, Min Yao

    Acta Crystallographica Section F: Structural Biology and Crystallization Communications　69　(8)　868-870　2013/08

    DOI： 10.1107/S174430911301693X 　

    ISSN： 1744-3091

  73. Structure of dihydrouridine synthase C (DusC) from Escherichia coli Peer-reviewed

    Minghao Chen, Jian Yu, Yoshikazu Tanaka, Miyuki Tanaka, Isao Tanaka, Min Yao

    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS　69　(Pt 8)　834-838　2013/08

    DOI： 10.1107/S1744309113019489 　

    ISSN： 1744-3091

  74. Structural and genomic DNA analysis of a putative transcription factor SCO5550 from Streptomyces coelicolor A3(2): Regulating the expression of gene sco5551 as a transcriptional activator with a novel dimer shape Peer-reviewed

    Takeshi Hayashi, Yoshikazu Tanaka, Naoki Sakai, Nobuhisa Watanabe, Tomohiro Tamura, Isao Tanaka, Min Yao

    Biochemical and Biophysical Research Communications　435　(1)　28-33　2013/05/24

    DOI： 10.1016/j.bbrc.2013.04.017 　

    ISSN： 0006-291X 1090-2104

  75. Crystal structure of a putative methyltransferase SAV1081 from staphylococcus aureus Peer-reviewed

    Shunsuke Kita, Yoshikazu Tanaka, Nagisa Hirano, Satoshi Kimura, Takeo Suzuki, Tsutomu Suzuki, Min Yao, Isao Tanaka

    Protein and Peptide Letters　20　(5)　530-537　2013/05

    DOI： 10.2174/0929866511320050006 　

    ISSN： 0929-8665

  76. Crystal structure of the capsular polysaccharide synthesizing protein CapE of Staphylococcus aureus Peer-reviewed

    Takamitsu Miyafusa, Jose M. M. Caaveiro, Yoshikazu Tanaka, Martin E. Tanner, Kouhei Tsumoto

    BIOSCIENCE REPORTS　33　(3)　463-U109　2013

    DOI： 10.1042/BSR20130017 　

    ISSN： 0144-8463

  77. Directed evolution and structural analysis of nadph-dependent acetoacetyl coenzyme A(acetoacetyl-CoA) reductase from ralstonia eutropha reveals two mutations responsible for enhanced kinetics Peer-reviewed

    Ken'ichiro Matsumoto, Yoshikazu Tanaka, Tsuyoshi Watanabe, Ren Motohashi, Koji Ikeda, Kota Tobitani, Min Yao, Isao Tanaka, Seiichi Taguchi

    Applied and Environmental Microbiology　79　(19)　6134-6139　2013

    DOI： 10.1128/AEM.01768-13 　

    ISSN： 0099-2240 1098-5336

  78. P19 embryonal carcinoma cells exhibit high sensitivity to botulinum type C and D/C mosaic neurotoxins Peer-reviewed

    Kentaro Tsukamoto, Hideyuki Arimitsu, Sadayuki Ochi, Keiji Nakamura, Yoshikazu Tanaka, Nipawan Nuemket, Koki Taniguchi, Shunji Kozaki, Takao Tsuji

    MICROBIOLOGY AND IMMUNOLOGY　56　(10)　664-672　2012/10

    DOI： 10.1111/j.1348-0421.2012.00490.x 　

    ISSN： 0385-5600

  79. The binding mechanism of eIF2 beta with its partner proteins, eIF5 and eIF2B epsilon Peer-reviewed

    Zuoqi Gai, Yumie Kitagawa, Yoshikazu Tanaka, Nobutaka Shimizu, Keisuke Komoda, Isao Tanaka, Min Yao

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS　423　(3)　515-519　2012/07

    DOI： 10.1016/j.bbrc.2012.05.155 　

    ISSN： 0006-291X

  80. Crystal structure of the enzyme CapF of Staphylococcus aureus reveals a unique architecture composed of two functional domains Peer-reviewed

    Takamitsu Miyafusa, Jose M. M. Caaveiro, Yoshikazu Tanaka, Kouhei Tsumoto

    BIOCHEMICAL JOURNAL　443　(3)　671-680　2012/05

    DOI： 10.1042/BJ20112049 　

    ISSN： 0264-6021

  81. Amplification of Light-induced Molecular-Shape Change by Supramolecular Machines

    Mihoko Ui, Yoshikazu Tanaka, Kazushi Kinbara

    JOURNAL OF PHOTOPOLYMER SCIENCE AND TECHNOLOGY　25　(5)　655-658　2012

    DOI： 10.2494/photopolymer.25.655 　

    ISSN： 0914-9244

  82. Application of photoactive yellow protein as a photoresponsive module for controlling hemolytic activity of staphylococcal alpha-hemolysin Peer-reviewed

    Mihoko Ui, Yoshikazu Tanaka, Yasuyuki Araki, Takehiko Wada, Toshiaki Takei, Kouhei Tsumoto, Sumire Endo, Kazushi Kinbara

    CHEMICAL COMMUNICATIONS　48　(39)　4737-4739　2012

    DOI： 10.1039/c2cc18118e 　

    ISSN： 1359-7345

    eISSN： 1364-548X

  83. Molecular basis of dihydrouridine formation on tRNA Peer-reviewed

    Futao Yu, Yoshikazu Tanaka, Keitaro Yamashita, Takeo Suzuki, Akiyoshi Nakamura, Nagisa Hirano, Tsutomu Suzuki, Min Yao, Isao Tanaka

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA　108　(49)　19593-19598　2011/12

    DOI： 10.1073/pnas.1112352108 　

    ISSN： 0027-8424

  84. Crystal structure of the octameric pore of staphylococcal gamma-hemolysin reveals the beta-barrel pore formation mechanism by two components Peer-reviewed

    Keitaro Yamashita, Yuka Kawai, Yoshikazu Tanaka, Nagisa Hirano, Jun Kaneko, Noriko Tomita, Makoto Ohta, Yoshiyuki Kamio, Min Yao, Isao Tanaka

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA　108　(42)　17314-17319　2011/10

    DOI： 10.1073/pnas.1110402108 　

    ISSN： 0027-8424

  85. Molecular Basis of Recognition of Antibacterial Porphyrins by Heme-Transporter IsdH-NEAT3 of Staphylococcus aureus Peer-reviewed

    Yoshitaka Moriwaki, Jose M. M. Caaveiro, Yoshikazu Tanaka, Hiroshi Tsutsumi, Itaru Hamachi, Kouhei Tsumoto

    BIOCHEMISTRY　50　(34)　7311-7320　2011/08

    DOI： 10.1021/bi200493h 　

    ISSN： 0006-2960

  86. Structural and mutational analyses of the receptor binding domain of botulinum D/C mosaic neurotoxin: Insight into the ganglioside binding mechanism Peer-reviewed

    Nipawan Nuemket, Yoshikazu Tanaka, Kentaro Tsukamoto, Takao Tsuji, Keiji Nakamura, Shunji Kozaki, Min Yao, Isao Tanaka

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS　411　(2)　433-439　2011/07

    DOI： 10.1016/j.bbrc.2011.06.173 　

    ISSN： 0006-291X

  87. Crystallization and preliminary X-ray crystallographic analysis of dihydrouridine synthase from Thermus thermophilus and its complex with tRNA Peer-reviewed

    Futao Yu, Yoshikazu Tanaka, Shiho Yamamoto, Akiyoshi Nakamura, Shunsuke Kita, Nagisa Hirano, Isao Tanaka, Min Yao

    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS　67　(Pt 6)　685-688　2011/06

    DOI： 10.1107/S1744309111012486 　

    ISSN： 1744-3091

  88. 2-Methyl-2,4-pentanediol induces spontaneous assembly of staphylococcal alpha-hemolysin into heptameric pore structure Peer-reviewed

    Yoshikazu Tanaka, Nagisa Hirano, Jun Kaneko, Yoshiyuki Kamio, Min Yao, Isao Tanaka

    PROTEIN SCIENCE　20　(2)　448-456　2011/02

    DOI： 10.1002/pro.579 　

    ISSN： 0961-8368

  89. Crystal structure of the functional region of Uro-adherence factor A from Staphylococcus saprophyticus reveals participation of the B domain in ligand binding Peer-reviewed

    Eriko Matsuoka, Yoshikazu Tanaka, Makoto Kuroda, Yuko Shouji, Toshiko Ohta, Isao Tanaka, Min Yao

    PROTEIN SCIENCE　20　(2)　406-416　2011/02

    DOI： 10.1002/pro.573 　

    ISSN： 0961-8368

  90. Structural and energetic hot-spots for the interaction between a ladder-like polycyclic ether and the anti-ciguatoxin antibody 10C9Fab Peer-reviewed

    Mihoko Ui, Yoshikazu Tanaka, Takeshi Tsumuraya, Ikuo Fujii, Masayuki Inoue, Masahiro Hirama, Kouhei Tsumoto

    MOLECULAR BIOSYSTEMS　7　(3)　793-798　2011

    DOI： 10.1039/c0mb00162g 　

    ISSN： 1742-206X

    eISSN： 1742-2051

  91. Contribution of the flexible loop region to the function of staphylococcal enterotoxin B Peer-reviewed

    Saeko Yanaka, Motonori Kudou, Yoshikazu Tanaka, Takumi Sasaki, Sumiyo Takemoto, Atsuko Sakata, Yukio Hattori, Tomoyuki Koshi, Shiro Futaki, Kouhei Tsumoto, Toshihiro Nakashima

    PROTEIN ENGINEERING DESIGN & SELECTION　23　(5)　415-421　2010/05

    DOI： 10.1093/protein/gzq006 　

    ISSN： 1741-0126

  92. Preliminary X-ray crystallographic study of the receptor-binding domain of the D/C mosaic neurotoxin from Clostridium botulinum Peer-reviewed

    Nipawan Nuemket, Yoshikazu Tanaka, Kentaro Tsukamoto, Takao Tsuji, Keiji Nakamura, Shunji Kozaki, Min Yao, Isao Tanaka

    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS　66　(Pt 5)　608-610　2010/05

    DOI： 10.1107/S1744309110012182 　

    ISSN： 1744-3091

  93. Temperature and salt concentration alter base-sequence selectivity of a duplex DNA-binding protein Peer-reviewed

    Satoru Nagatoishi, Yoshikazu Tanaka, Motonori Kudou, Kouhei Tsumoto

    MOLECULAR BIOSYSTEMS　6　(1)　98-101　2010

    DOI： 10.1039/b914828k 　

    ISSN： 1742-206X

  94. Deduced RNA binding mechanism of ThiI based on structural and binding analyses of a minimal RNA ligand Peer-reviewed

    Yoshikazu Tanaka, Shiori Yamagata, Yu Kitago, Yoko Yamada, Sarin Chimnaronk, Min Yao, Isao Tanaka

    RNA-A PUBLICATION OF THE RNA SOCIETY　15　(8)　1498-1506　2009/08

    DOI： 10.1261/rna.1614709 　

    ISSN： 1355-8382

  95. Contributions of Interfacial Residues of Human Interleukin15 to the Specificity and Affinity for Its Private alpha-Receptor Peer-reviewed

    Sou Sakamoto, Jose M. M. Caaveiro, Emiko Sano, Yoshikazu Tanaka, Motonori Kudou, Kouhei Tsumoto

    JOURNAL OF MOLECULAR BIOLOGY　389　(5)　880-894　2009/06

    DOI： 10.1016/j.jmb.2009.04.050 　

    ISSN： 0022-2836

  96. Immobilized metal affinity chromatography in the presence of arginine Peer-reviewed

    Ryota Abe, Motonori Kudou, Yoshikazu Tanaka, Tsutomu Arakawa, Kouhei Tsumoto

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS　381　(3)　306-310　2009/04

    DOI： 10.1016/j.bbrc.2009.01.054 　

    ISSN： 0006-291X

  97. The interaction of hyperthermophilic TATA-box binding protein with single-stranded DNA is entropically favorable and exhibits a large negative heat capacity change at high salt concentration Peer-reviewed

    Satoru Nagatoishi, Yoshikazu Tanaka, Motonori Kudou, Kouhei Tsumoto

    MOLECULAR BIOSYSTEMS　5　(9)　957-961　2009

    DOI： 10.1039/b904200h 　

    ISSN： 1742-206X

    eISSN： 1742-2051

  98. Structural and functional characterization of the LldR from Corynebacterium glutamicum: a transcriptional repressor involved in L-lactate and sugar utilization Peer-reviewed

    Yong-Gui Gao, Hiroaki Suzuki, Hiroshi Itou, Yong Zhou, Yoshikazu Tanaka, Masaaki Wachi, Nobuhisa Watanabe, Isao Tanaka, Min Yao

    NUCLEIC ACIDS RESEARCH　36　(22)　7110-7123　2008/12

    DOI： 10.1093/nar/gkn827 　

    ISSN： 0305-1048

  99. Staphylococcus aureus surface protein SasG contributes to intercellular autoaggregation of Staphylococcus aureus Peer-reviewed

    Makoto Kuroda, Ryuta Ito, Yoshikazu Tanaka, Min Yao, Kimio Matoba, Shinji Saito, Isao Tanaka, Toshiko Ohta

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS　377　(4)　1102-1106　2008/12

    DOI： 10.1016/j.bbrc.2008.10.134 　

    ISSN： 0006-291X

  100. Electron microscopy and computational studies of Ebh, a giant cell-wall-associated protein from Staphylococcus aureus Peer-reviewed

    Sou Sakamoto, Yoshikazu Tanaka, Isao Tanaka, Toshiaki Takei, Jian Yu, Makoto Kuroda, Min Yao, Toshiko Ohta, Kouhei Tsumoto

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS　376　(2)　261-266　2008/11

    DOI： 10.1016/j.bbrc.2008.08.117 　

    ISSN： 0006-291X

  101. Structure-based analysis reveals hydration changes induced by arginine hydrochloride Peer-reviewed

    Makoto Nakakido, Yoshikazu Tanaka, Mariko Mitsuhori, Motonori Kudou, Daisuke Ejima, Tsutomu Arakawa, Kouhei Tsumoto

    BIOPHYSICAL CHEMISTRY　137　(2-3)　105-109　2008/10

    DOI： 10.1016/j.bpc.2008.07.009 　

    ISSN： 0301-4622

  102. Structural basis for multimeric heme complexation through a specific protein-heme interaction - The case of the third neat domain of IsdH from Staphylococcus aureus Peer-reviewed

    Masato Watanabe, Yoshikazu Tanaka, Ayuko Suenaga, Makoto Kuroda, Min Yao, Nobuhisa Watanabe, Fumio Arisaka, Toshiko Ohta, Isao Tanaka, Kouhei Tsumoto

    JOURNAL OF BIOLOGICAL CHEMISTRY　283　(42)　28649-28659　2008/10

    DOI： 10.1074/jbc.M803383200 　

    ISSN： 0021-9258

  103. Staphylococcus aureus giant protein Ebh is involved in tolerance to transient hyperosmotic pressure Peer-reviewed

    Makoto Kuroda, Yoshikazu Tanaka, Ryo Aoki, Deng Shu, Kouhei Tsumoto, Toshiko Ohta

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS　374　(2)　237-241　2008/09

    DOI： 10.1016/j.bbrc.2008.07.037 　

    ISSN： 0006-291X

  104. How protein recognizes ladder-like polycyclic ethers - Interactions between ciguatoxin (CTX3C) fragments and its specific antibody 10C9 Peer-reviewed

    Mihoko Ui, Yoshikazu Tanaka, Takeshi Tsumuraya, Ikuo Fujii, Masayuki Inoue, Masahiro Hirama, Kouhei Tsumoto

    JOURNAL OF BIOLOGICAL CHEMISTRY　283　(28)　19440-19447　2008/07

    DOI： 10.1074/jbc.M801282200 　

    ISSN： 0021-9258

    eISSN： 1083-351X

  105. Thermodynamic analysis reveals that GTP binding affects the interaction between the alpha- and gamma-subunits of translation initiation factor 2 Peer-reviewed

    Makoto Nakakido, Yoshikazu Tanaka, Masaaki Sokabe, Kouhei Tsumoto

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS　371　(4)　596-599　2008/07

    DOI： 10.1016/j.bbrc.2008.03.101 　

    ISSN： 0006-291X

  106. Expression, purification, crystallization and preliminary diffraction analysis of CapF, a capsular polysaccharide-synthesis enzyme from Staphylococcus aureus Peer-reviewed

    Takamitsu Miyafusa, Yoshikazu Tanaka, Makoto Kuroda, Toshiko Ohta, Kouhei Tsumoto

    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS　64　(Pt 6)　512-515　2008/06

    DOI： 10.1107/S174430910801213X 　

    ISSN： 2053-230X

  107. Critical contribution of aromatic rings to specific recognition of polyether rings - The case of ciguatoxin CTX3C-ABC and its specific antibody 1C49 Peer-reviewed

    Kouhei Tsumoto, Akiko Yokota, Yoshikazu Tanaka, Mihoko Ui, Takeshi Tsumuraya, Ikuo Fujii, Izumi Kumagai, Yoko Nagumo, Hiroki Oguri, Masayuki Inoue, Masahiro Hirama

    JOURNAL OF BIOLOGICAL CHEMISTRY　283　(18)　12259-12266　2008/05

    DOI： 10.1074/jbc.M710553200 　

    ISSN： 0021-9258

  108. A helical string of alternately connected three-helix bundles for the cell wall-associated adhesion protein Ebh from Staphylococcus aureus Peer-reviewed

    Yoshikazu Tanaka, Sou Sakamoto, Makoto Kuroda, Shuichiro Goda, Yong-Gui Gao, Kouhei Tsumoto, Yuzuru Hiragi, Min Yao, Nobuhisa Watanabe, Toshiko Ohta, Isao Tanaka

    STRUCTURE　16　(3)　488-496　2008/03

    DOI： 10.1016/j.str.2007.12.018 　

    ISSN： 0969-2126

  109. Expression, purification, crystallization and preliminary diffraction analysis of CapF, a capsular polysaccharide-synthesis enzyme from Staphylococcus aureus Peer-reviewed

    Takamitsu Miyafusa, Yoshikazu Tanaka, Makoto Kuroda, Toshiko Ohta, Kouhei Tsumoto

    Acta Crystallographica Section F: Structural Biology and Crystallization Communications　64　(6)　512-515　2008

    DOI： 10.1107/S174430910801213X 　

    ISSN： 1744-3091

  110. Thermodynamic consequences of mutations in Vernier zone residues of a humanized anti-human epidermal growth factor receptor murine antibody-528 Peer-reviewed

    Koki Makabe, Takeshi Nakanishi, Kouhei Tsumoto, Yoshikazu Tanaka, Hidemasa Kondo, Mitsuo Umetsu, Yukiko Sone, Ryutaro Asano, Izumi Kumagai

    JOURNAL OF BIOLOGICAL CHEMISTRY　283　(2)　1156-1166　2008/01

    DOI： 10.1074/jbc.M706190200 　

    ISSN： 0021-9258

    eISSN： 1083-351X

  111. Crystal structure analysis reveals a novel forkhead-associated domain of ESAT-6 secretion system C protein in Staphylocaccus aureus Peer-reviewed

    Yoshikazu Tanaka, Makoto Kuroda, Yoshiaki Yasutake, Min Ao, Kouhei Tsumoto, Nobuhisa Watanabe, Toshiko Ohta, Isao Tanaka

    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS　69　(3)　659-664　2007/11

    DOI： 10.1002/prot.21302 　

    ISSN： 0887-3585

  112. The N-terminal domain of elastin-binding protein of Staphylococcus aureus changes its secondary structure in a membrane-mimetic environment Peer-reviewed

    Makoto Nakakido, Yoshikazu Tanaka, Kouhei Tsumoto

    JOURNAL OF BIOCHEMISTRY　142　(2)　131-134　2007/08

    DOI： 10.1093/jb/mvm131 　

    ISSN： 0021-924X

  113. Molecular properties of two proteins homologous to PduO-Type ATP : cob(I)alamin adenosyltransferase from Sulfolobus tokodaii Peer-reviewed

    Yoshikazu Tanaka, Takashi Sasaki, Izumi Kumagai, Yoshiaki Yasutake, Min Yao, Isao Tanaka, Kouhei Tsumoto

    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS　68　(2)　446-457　2007/08

    DOI： 10.1002/prot.21303 　

    ISSN： 0887-3585

  114. Suppression of protein interactions by arginine: A proposed mechanism of the arginine effects Peer-reviewed

    Tsutomu Arakawa, Daisuke Ejima, Kouhei Tsumoto, Noriyuki Obeyama, Yoshikazu Tanaka, Yoshiko Kita, Serge N. Timasheff

    BIOPHYSICAL CHEMISTRY　127　(1-2)　1-8　2007/04

    DOI： 10.1016/j.bpc.2006.12.007 　

    ISSN： 0301-4622

  115. Corrigendum to "Circular dichroism spectra demonstrate formation of the thrombin-binding DNA aptamer G-quadruplex under stabilizing-cation-deficient conditions" [Biochem. Biophys. Res. Commun. 352 (2007) 812-817] (DOI:10.1016/j.bbrc.2006.11.088) Peer-reviewed

    Satoru Nagatoishi, Yoshikazu Tanaka, Kouhei Tsumoto

    Biochemical and Biophysical Research Communications　354　(3)　837-838　2007/03/16

    DOI： 10.1016/j.bbrc.2007.01.018 　

    ISSN： 0006-291X 1090-2104

  116. Structural consequences of mutations in interfacial Tyr residues of a protein antigen-anti body complex - The case of HyHEL-10-HEL Peer-reviewed

    Mitsunori Shiroishi, Kouhei Tsumoto, Yoshikazu Tanaka, Akiko Yokota, Takeshi Nakanishi, Hidemasa Kondo, Izumi Kumagai

    JOURNAL OF BIOLOGICAL CHEMISTRY　282　(9)　6783-6791　2007/03

    DOI： 10.1074/jbc.M605197200 　

    ISSN： 0021-9258

  117. Structural and mutational analyses of Drp35 from Staphylococcus aureus - A possible mechanism for its lactonase activity Peer-reviewed

    Yoshikazu Tanaka, Kazuya Morikawa, Yu Ohki, Min Yao, Kouhei Tsumoto, Nobuhisa Watanabe, Toshiko Ohta, Isao Tanaka

    JOURNAL OF BIOLOGICAL CHEMISTRY　282　(8)　5770-5780　2007/02

    DOI： 10.1074/jbc.M607340200 　

    ISSN： 0021-9258

  118. Circular dichroism spectra demonstrate formation of the thrombin-binding DNA aptamer G-quadruplex under stabilizing-cation-deficient conditions Peer-reviewed

    Satoru Nagatoishi, Yoshikazu Tanaka, Kouhei Tsumoto

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS　352　(3)　812-817　2007/01

    DOI： 10.1016/j.bbrc.2006.11.088 　

    ISSN： 0006-291X

  119. Crystal structure of the hypothetical protein ST2072 from Sulfolobus tokodaii Peer-reviewed

    Y Tanaka, K Tsumoto, E Tanabe, Y Yasutake, N Sakai, M Yao, Tanaka, I, Kumagai, I

    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS　61　(4)　1127-1131　2005/12

    DOI： 10.1002/prot.20619 　

    ISSN： 0887-3585

  120. Selective detection of the solid-state NMR signals from the bacteriochlorophyll a dimers in a reconstituted light-harvesting 1 complex Peer-reviewed

    M Umetsu, T Kadota, ZY Wang, Y Tanaka, T Adschiri, T Nozawa

    CHEMISTRY LETTERS　34　(7)　940-941　2005/07

    DOI： 10.1246/cl.2005.940 　

    ISSN： 0366-7022

    eISSN： 1348-0715

  121. Structural evidence for guanidine-protein side chain interactions: crystal structure of CutA from Pyrococcus horikoshii in 3 M guanidine hydrochloride Peer-reviewed

    Y Tanaka, K Tsumoto, M Umetsu, T Nakanishi, Y Yasutake, N Sakai, M Yao, Tanaka, I, T Arakawa, Kumagai, I

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS　323　(1)　185-191　2004/10

    DOI： 10.1016/j.bbrc.2004.08.081 　

    ISSN： 0006-291X

  122. How oligomerization contributes to the thermostability of an Archaeon protein - Protein L-isoaspartyl-O-methyltransferase from Sulfolobus tokodaii Peer-reviewed

    Y Tanaka, K Tsumoto, Y Yasutake, M Umetsu, M Yao, H Fukada, Tanaka, I, Kumagai, I

    JOURNAL OF BIOLOGICAL CHEMISTRY　279　(31)　32957-32967　2004/07

    DOI： 10.1074/jbc.M404405200 　

    ISSN： 0021-9258

  123. Crystal structure of conserved protein PH1136 from Pyrococcus horikoshii Peer-reviewed

    Y Tajika, N Sakai, Y Tanaka, M Yao, N Watanabe, Tanaka, I

    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS　55　(1)　210-213　2004/04

    DOI： 10.1002/prot.10644 　

    ISSN： 0887-3585

  124. Structural implications for heavy metal-induced reversible assembly and aggregation of a protein: the case of Pyrococcus horikoshii CutA Peer-reviewed

    Y Tanaka, K Tsumoto, T Nakanishi, Y Yasutake, N Sakai, M Yao, Tanaka, I, Kumagai, I

    FEBS LETTERS　556　(1-3)　167-174　2004/01

    DOI： 10.1016/S0014-5793(03)01402-9 　

    ISSN： 0014-5793

  125. Structural characteristics and refolding of in vivo aggregated hyperthermophilic archaeon proteins Peer-reviewed

    M Umetsu, K Tsumoto, K Ashish, S Nitta, Y Tanaka, T Adschiri, Kumagai, I

    FEBS LETTERS　557　(1-3)　49-56　2004/01

    DOI： 10.1016/S0014-5793(03)01441-8 　

    ISSN： 0014-5793

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    中澤光, 勝木泰司, 松井崇, 田中良和, 梅津光央

    日本蛋白質科学会年会(Web)　25th　2025

  7. ドメイン連結順の変更に伴うがん二重特異性抗体の活性変化の分子機構

    佐藤恭平, 上原史朗, 次田篤史, 石井麻友香, 石山紫衿瑠, 前島敦, 中原維新, 名塚美冴, 松井崇, 松井崇, 横山武司, 熊谷泉, 真壁幸樹, 浅野竜太郎, 田中良和

    日本蛋白質科学会年会(Web)　25th　2025

  8. 新規プレニル基転移酵素PT1の立体構造解析

    大城拓未, 上原秀太, 田中良和, 伊藤卓也, 小寺義男, 小寺義男, 松井崇, 松井崇

    電気泳動(Web)　68　(Suppl)　2024

    ISSN： 2189-2636

  9. 終止コドンリードスルーの分子機構

    田中良和, 大久保雄介, 須藤愛莉咲, 松井崇, 松井崇, 小寺義男, 小寺義男, 横山武司

    日本蛋白質科学会年会(Web)　24th　2024

  10. 海洋放線菌由来新規酵素の基質認識機構の解明

    大城拓未, 上原秀太, 田中良和, 伊藤卓也, 小寺義男, 小寺義男, 松井崇, 松井崇

    日本蛋白質科学会年会(Web)　24th　2024

  11. クライオ電子顕微鏡で明らかにしたドメイン再配列がもたらす二重特異性抗体の高機能化の分子機構

    佐藤恭平, 次田篤史, 松井崇, 横山武司, 熊谷泉, 真壁幸樹, 浅野竜太郎, 田中良和

    日本蛋白質科学会年会(Web)　24th　2024

  12. FtsZのネイティブタンパク質間相互作用から発想するエピトープターゲティング抗体設計

    中澤光, 勝木泰司, 松井崇, 田中良和, 梅津光央

    日本抗体学会学術大会プログラム・抄録集(Web)　2nd　2023

  13. Synthesis of epitope-targeting antibody based on native Protein-Protein interaction

    中澤光, 勝木泰司, 松井崇, 田中良和, 梅津光央

    日本生物工学会大会講演要旨集　75th　2023

  14. Crystal structure analysis of novel enzyme derived from Streptomyces.

    松井崇, 大城拓未, 伊藤卓也, 田中良和, 小寺義男

    KEK Progress Report (Web)　(2023-2)　2023

    ISSN： 1344-6320

  15. Structural analysis of a novel prenyltransferase

    大城拓未, 上原秀太, 田中良和, 伊藤卓也, 小寺義男, 小寺義男, 小寺義男, 松井崇, 松井崇, 松井崇

    量子ビームサイエンスフェスタ(Web)　2022　2023

  16. 新規レクチン様タンパク質の結晶構造解析と糖との結合解析 : 2020年度量子ビームサイエンスフェスタ 学生奨励賞受賞論文—Crystal Structure Analysis and Sugar Binding Analysis of a Novel Lectin Like Protein

    影山 大夢, 小野寺 かこ, 松井 崇, 小川 智久, 横山 武司, 田中 良和

    PF news　39　(4)　11-15　2022/02

    Publisher: つくば : Photon factory news編集委員会

    ISSN： 0916-0604

  17. LC-MS analysis of the protein crystallized by PEG

    大城拓未, 影山大夢, 小野寺かこ, 辺浩美, 中島寛也, 小川智久, 小川智久, 横山武司, 田中良和, 酒井隆一, 松井崇, 松井崇, 小寺義男, 小寺義男

    日本プロテオーム学会大会プログラム・抄録集　2022 (Web)　2022

  18. Structure and Function of Molluscan Hemocyanins

    加藤早苗, 加藤早苗, 松井崇, 田中良和

    人工血液　30　(1)　2022

    ISSN： 1341-1594

  19. FtsZのネイティブタンパク質間相互作用から発想するエピトープターゲティング抗体設計

    中澤光, 勝木泰司, 松井崇, 田中良和, 梅津光央

    化学工学会秋季大会研究発表講演要旨集(CD-ROM)　53rd　2022

  20. ストップコドンリードスルーの分子機構解明

    浅野航佑, 原口大輝, 須藤愛莉咲, 松井崇, 松井崇, 小寺義男, 小寺義男, 小川智久, 横山武司, 田中良和

    日本蛋白質科学会年会プログラム・要旨集　22nd (Web)　2022

  21. Actual Situation of Cryo-Electron Microscopy Single Particle Analysis; From Sample Preparation to Image Analysis

    Tsubasa HASHIMOTO, Takeshi YOKOYAMA, Yoshikazu TANAKA

    Nihon Kessho Gakkaishi　63　(2)　89-96　2021/05/31

    Publisher: The Crystallographic Society of Japan

    DOI： 10.5940/jcrsj.63.89 　

    ISSN： 0369-4585

    eISSN： 1884-5576

  22. 海洋生物に含まれる抗フラビウイルス物質の探索

    中谷晴香, CHIMNARONK Sarin, 田中良和, ROMO Daniel, 酒井隆一

    日本水産学会大会講演要旨集(CD-ROM)　2021　2021

  23. 海洋天然物由来の新規生理活性タンパク質の構造機能解析

    田中良和, 影山大夢, 辺ひろみ, 増渕菜弥, 中島寛也, 小野寺かこ, 大城拓未, 松井崇, 小寺義男, 小川智久, 横山武司, 小松則夫, 荒木真理人, 酒井隆一

    日本生化学会大会(Web)　94th　2021

  24. Marine sponge lectin reveals the mechanism of sugar-chain mediated activation of MPL

    辺浩美, 影山大夢, 中島寛弥, 小野寺かこ, 増渕菜弥, 大城拓未, 松井崇, 小寺義男, 小川智久, 平山真, 堀貫治, 横山武司, 小松則夫, 小松則夫, 荒木真理人, 荒木真理人, 田中良和, 酒井隆一

    日本血液学会学術集会抄録(Web)　83rd　2021

  25. Progress in the understanding of the functional structure of bispecific cancer therapeutic antibodies

    浅野竜太郎, 真壁幸樹, 田中良和, 熊谷泉

    医学のあゆみ　278　(6)　2021

    ISSN： 0039-2359

  26. 新規レクチン様タンパク質の構造と機能の解析

    影山大夢, 小野寺かこ, 松井崇, 松井崇, 小川智久, 小川智久, 横山武司, 田中良和

    日本蛋白質科学会年会プログラム・要旨集　21st　2021

  27. Three-dimensional reconstruction of 3.8 MDa respiratory protein complex Hemocyanin

    92　(1)　113-119　2020/02

    Publisher: 日本生化学会

    ISSN： 0037-1017

  28. そのエピトープは創薬標的となりえるか?:天然タンパク質の相互作用情報を利用したツール抗体設計

    勝木泰司, 中澤光, 松井崇, 田中良和, 梅津光央

    化学工学会秋季大会研究発表講演要旨集(CD-ROM)　51st　2020

  29. Structural analysis of Lys49-phospholipase A2 derived from snake venom.

    鎌田しずか, 松井崇, 小川智久, 田中良和

    KEK Progress Report (Web)　(2020-6)　2020

    ISSN： 1344-6320

  30. 高活性なリガンド創出に向けたユビキチンリガーゼcIAP1-BIR3ドメインの結晶構造解析

    岡部真琴, 佐藤伸一, 佐藤伸一, 横山武司, 友重秀介, 石川稔, 田中良和

    日本分子生物学会年会プログラム・要旨集(Web)　43rd　2020

  31. 放線菌の抗生物質産生に関連する蛋白質LanKの構造解析

    上原史朗, OSTASH Ostash, OSTASH Bohdan, 松井崇, 小川智久, 田中良和

    日本分子生物学会年会プログラム・要旨集(Web)　43rd　2020

  32. Molluscan Hemocyanins. International-journal

    Sanae Kato, Takashi Matsui, Yoshikazu Tanaka

    Sub-cellular biochemistry　94　195-218　2020

    DOI： 10.1007/978-3-030-41769-7_7 　

    More details Close

    Instead of the red blood of vertebrates, most molluscs have blue hemolymph containing hemocyanin, a type-3 copper-containing protein. The hemoglobin of vertebrate blood is replaced in most molluscs with hemocyanin, which plays the role of an  oxygen transporter. Oxygen-binding in hemocyanin changes its hue from colorless deoxygenated hemocyanin into blue oxygenated hemocyanin. Molecules of molluscan hemocyanin are huge, cylindrical multimeric proteins-one of the largest protein molecules in the natural world. Their huge molecular weight (from 3.3 MDa to more than 10 MDa) are the defining characteristic of molluscan hemocyanin, a property that has complicated structural analysis of the molecules for a long time. Recently, the structural analysis of a cephalopod (squid) hemocyanin has succeeded using a hybrid method employing both X-ray crystallography and cryo-EM. In a biochemical breakthrough for molluscan hemocyanin, the first quaternary structure with atomic resolution is on the verge of solving the mystery of molluscan hemocyanin. Here we describe the latest information about the molecular structure, classification and evolution of the molecule, and the physiology of molluscan hemocyanin.

  33. 包括的な物性解析によって見えてきたハブ毒蛋白質のSDSによる多量体化

    松井 崇, 鎌田 しずか, 石井 健太郎, 丸野 孝浩, ガーネム・ノーラン, 内山 進, 加藤 晃一, 鈴木 淳巨, 上田 直子, 小川 智久, 田中 良和

    電気泳動　63　(Suppl.)　263-263　2019/07

    Publisher: 日本電気泳動学会

    ISSN： 2189-2628

    eISSN： 2189-2636

  34. X線結晶構造解析とクライオ電子顕微鏡解析の相関構造解析の有用性―ヘモシアニンを例として―

    田中良和

    日本細胞生物学会大会(Web)　71st　295　2019/06/06

  35. 2成分性βバレル型膜孔形成毒素の膜孔形成に関わるS成分残基の解析

    武田慶胤, 彭昭, 竹下珠由, 田中良和, 金子淳

    日本ゲノム微生物学会年会要旨集　13th　84　2019/03/06

  36. X線結晶構造解析とクライオ電顕の両手法を使って

    田中良和

    KEK Proceedings (Web)　(2018-11)　313‐334 (WEB ONLY)　2019/02

  37. Protein Cyborgization by Main Chain Substitution

    AOKI Yusuke, UI Mihoko, MATSUI Takashi, TANAKA Yoshikazu, MURAOKA Takahiro, SATO Kohei, KINBARA Kazushi

    日本化学会春季年会講演予稿集(CD-ROM)　99th　2019

  38. 蛋白質を包摂可能な新規ホスト分子としての多孔性蛋白質結晶の利用

    橋本翼, 松井崇, 小川智久, 田中良和

    日本生化学会大会(Web)　92nd　2019

  39. Cryo-EM Analysis Reveals the Asymmetric Assembly of the Todarodes pacificus Hemocyanin（TpH）

    MATSUI Takashi, KATO Sanae, TANAKA Yoshikazu

    Nihon Kessho Gakkaishi　61　(4)　211-212　2019

    Publisher: The Crystallographic Society of Japan

    DOI： 10.5940/jcrsj.61.211 　

    ISSN： 0369-4585

  40. 包括的な物性解析によって見えてきたハブ毒蛋白質のSDSによる多量体化

    松井崇, 松井崇, 鎌田しずか, 石井健太郎, 丸野孝浩, GHANEM N, 内山進, 内山進, 加藤晃一, 加藤晃一, 加藤晃一, 鈴木淳巨, 上田直子, 小川智久, 田中良和, 田中良和

    日本プロテオーム学会大会プログラム・抄録集　2019　263　2019

  41. 蛋白質結晶中の空隙へのゲスト蛋白質包摂手法の開発と結晶構造解析への応用

    橋本翼, 松井崇, 小川智久, 田中良和

    生体分子科学討論会講演要旨集　46th　52　2019

  42. tRNA硫黄修飾酵素の活性と鉄硫黄クラスター構造の関連性

    石坂優人, 陳明皓, 奈良井峻, 堀谷正樹, 田中良和, 田中良和, 姚閔

    日本細胞生物学会大会(Web)　71st　ROMBUNNO.1P‐006 (WEB ONLY)　2019

  43. 中空状の蛋白質結晶へのタンパク質ゲストの包摂

    橋本翼, 早河瑞希, 松井崇, 田中良和, 田中良和

    KEK Progress Report (Web)　(2018-7)　ROMBUNNO.238 (WEB ONLY)　2018/12

    ISSN： 1344-6320

  44. 黄色ブドウ球菌二成分性毒素γ‐ヘモリジンのβバレル型膜孔形成機構の解明

    武田慶胤, 田中良和, 阿部直樹, 金子淳

    日本農芸化学会東北支部大会プログラム・講演要旨集　153rd　34　2018/09/22

  45. スルメイカ由来ヘモシアニンのクライオ電顕構造とX線結晶構造の違い

    田中良和, 松井崇, 加藤早苗, GATSOGIANNIS Christos, RAUNSER Stefan

    日本蛋白質科学会年会プログラム・要旨集　18th　124　2018/05/23

  46. 3.8MDaの超巨大酸素運搬タンパク質ヘモシアニン会合体の結晶構造

    加藤早苗, 松井崇, 田中良和

    生化学　90　(2)　238-243　2018/04

    Publisher: 日本生化学会

    DOI： 10.14952/SEIKAGAKU.2018.900238 　

    ISSN： 0037-1017

  47. 黄色ブドウ球菌二成分性毒素γ‐ヘモリジンのβバレル型膜孔形成機構の解明

    武田慶胤, 田中良和, 金子淳

    日本農芸化学会大会講演要旨集(Web)　2018　ROMBUNNO.2A22a05 (WEB ONLY)　2018/03/05

    ISSN： 2186-7976

  48. Molluscan Hemocyanin: Structure, Evolution, and Physiology

    Kato, S, Matsui, T, Gatsogiannis, C, Tanaka, Y

    Biophysical Reviews　2018

  49. GH15 isomaltose glucohydrolaseの基質認識に関わる構造因子の同定

    田上貴祥, 陳明皓, 奥山正幸, 岩崎智仁, 田中良和, 姚閔, 木村淳夫

    応用糖質科学　7　(3)　49　2017/08/20

    ISSN： 2185-6427

  50. 好熱性アーキアにおけるアセチルシチジン修飾はtRNAの耐熱性に寄与する

    大平高之, CHEN Minghao, 栗橋泰子, 山内理恵, 伊藤理, 金子瑛, 折田和泉, 福居俊昭, 鈴木健夫, YAO Min, 田中良和, 鈴木勉

    日本RNA学会年会要旨集　19th　129　2017/07/19

  51. tRNA硫黄修飾塩基の生合成機構

    鴫直樹, 朝井真一, CHEN Minghao, 齋藤正男, 奈良井峻, 大村直樹, 鈴木勉, YAO Min, 田中良和, 田中良和, 田中良和, 渡辺公綱

    日本RNA学会年会要旨集　19th　68　2017/07/19

  52. 鉄硫黄クラスターが関与する新規硫黄転移機構の解明

    陳明皓, 奈良井峻, 大村直樹, 朝井真一, 渡辺公綱, 鴫直樹, 田中良和, 田中良和, 田中良和, 姚閔, 姚閔

    日本蛋白質科学会年会プログラム・要旨集　17th　46　2017/05/22

  53. 黄色ブドウ球菌における二成分性毒素γヘモリジンの膜孔形成機構の解明

    武田慶胤, 田中良和, 金子淳

    日本ブドウ球菌研究会プログラム・講演要旨集　62nd　32　2017

  54. ハブゲノム解読から見出された新規Three‐finger toxinsの発現と機能解析

    土生津光, 齋藤浩唯, 柴田弘紀, 大野素徳, 服部正策, 松井崇, 田中良和, 村本光二, 小川智久

    日本生化学会大会(Web)　90th　ROMBUNNO.3P‐0186 (WEB ONLY)　2017

  55. 溶液フリーマウント法の自動化

    篠田晃, 渡邉信久, 加藤公児, 田中良和, 姚閔, 田中勲

    日本結晶学会年会講演要旨集　2016　115　2016/11/17

  56. 超好熱性古細菌RNaseP構成タンパク質Rpp38のRNA活性化の構造基盤

    大嶋浩介, 高緒柱, 中島崇, 田中良和, 姚閔, 木村誠

    日本結晶学会年会講演要旨集　2016　83　2016/11/17

  57. ラクダ可変領域を利用した自発的多重多価化抗体の提案

    藤井滉人, 田中良和, 浅野竜太郎, 熊谷泉, 梅津光央

    化学工学会秋季大会研究発表講演要旨集(CD-ROM)　48th　ROMBUNNO.LQ221　2016/09/06

  58. 黄色ブドウ球菌のβバレル膜孔形成毒素の膜孔形成及び遺伝子の伝播機構

    金子淳, 田中良和

    トキシンシンポジウム予稿集　63rd　92‐97　2016/06/25

    ISSN： 1344-9346

  59. バクテリアにおける二機能性ユビキチン様タンパク質の機能調節の構造基盤

    陳明皓, 奈良井峻, 大村直樹, 鴫直樹, 田中良和, 田中良和, 姚閔

    日本蛋白質科学会年会プログラム・要旨集　16th　82　2016/05/19

  60. スルメイカヘモシアニン分子のサブユニット間相互作用

    加藤早苗, 吉岡武也, 清水健志, 岸村栄毅, 田中良和

    日本水産学会大会講演要旨集　2016　162　2016/03/26

  61. 超巨大蛋白質会合体ヘモシアニンのX線結晶構造解析

    松野明日香, 田中良和

    日本結晶学会誌　58　(58)　91-95　2016

    DOI： 10.5940/jcrsj.58.91 　

    ISSN： 0369-4585

  62. Crystal Structure Analysis of a Huge Oxygen Transport Supermolecule Hemocyanin

    MATSUNO Asuka, TANAKA Yoshikazu

    Nihon Kessho Gakkaishi　58　(2)　91-95　2016

    Publisher: The Crystallographic Society of Japan

    DOI： 10.5940/jcrsj.58.91 　

    ISSN： 0369-4585

    More details Close

    Many molluscs use hemocyanin freely dissolved in hemolymph for oxygen transportation. Hemocyanin is one of the largest known proteins, of which total molecular mass is larger than 3.3 MDa. Although molluscan hemocyanin is used as immunotherapeutic agents, their detailed structures had not yet been revealed. As its enormous size and the propensity of dissociation hampered crystallization, structural studies have relied mainly on electron microscopy. To understand the oxygen transportation mechanism in detail and promote the biomedical application, high-resolution structure has been desired for a long time. In this study, we successfully determined the X-ray crystal structure of the intact 3.8-MDa hemocyanin from Japanese flying squid at 3.0 Å resolution, which revealed the detailed molecular architecture of the supermolecule.

  63. アミノ酸配列の相同性から考察するDiabody型二重特異性抗体の構造均一性

    小野寺朋子, 梅津光央, 田中良和, 中澤光, 服部峰充, 浅野竜太郎, 熊谷泉

    日本生物工学会大会講演要旨集　67th　188　2015/09/25

  64. 超好熱性アーキアリボヌクレアーゼP構成タンパク質Rpp38によるRNA活性化の作用機構に関する研究

    垣内洋祐, 大嶋浩介, 中島崇, 角田佳充, 田中良和, 姚閔, 木村誠

    日本農芸化学会中四国支部講演会講演要旨集(Web)　43rd　D‐19 (WEB ONLY)　2015/09/17

  65. 黄色ブドウ球菌が産生する膜孔形成毒素の作用機構

    田中良和

    Isotope News　(736)　7-11　2015/08/01

    ISSN： 0285-5518

  66. 超好熱性古細菌RNasePにおける構造安定化に関わるRNA‐タンパク質間相互作用の解析

    大嶋浩介, 中島崇, 角田佳充, 木村誠, 田中良和, YAO Min

    日本RNA学会年会要旨集　17th　140　2015/07/15

  67. 黄色ブドウ球菌由来α‐ヘモリジンの膜孔形成機構

    菅原宇希, 山下大智, 加藤公児, 金子淳, 神尾好是, 田中良和, 姚閔

    日本蛋白質科学会年会プログラム・要旨集　15th　60　2015/05/26

  68. 軟体動物の超巨大酸素運搬蛋白質会合体ヘモシアニンのX線結晶構造解析

    松野明日香, 蓋作啓, 加藤公児, 加藤早苗, 清水健志, 吉岡武哉, 岸村栄毅, 寺田透, 田中良和, 姚閔

    日本蛋白質科学会年会プログラム・要旨集　15th　60　2015/05/26

  69. S‐SAD用溶液フリーマウント法の開発

    篠田晃, 渡邊信久, 姚閔, 田中良和, 加藤公児, 田中勲

    物構研サイエンスフェスタ要旨集　3rd　80　2015

  70. 黄色ブドウ球菌が産生する膜孔形成毒素の作用機構

    田中良和

    Isotope News　(736)　7-11　2015

  71. 黄色ブドウ球菌の2成分性膜孔形成毒素γヘモリジンの膜孔形成メカニズム 失敗から明らかになった毒素の戦略

    田中良和

    化学と生物　53　(53)　136-137　2015

    Publisher: 公益社団法人 日本農芸化学会

    DOI： 10.1271/kagakutoseibutsu.53.136 　

    ISSN： 0453-073X

  72. 2P-056 Influence of amino acid sequence homology on dimerization of diabody-type

    Onodera Tomoko, Umetsu Mitsuo, Tanaka Yoshikazu, Nakazawa Hikaru, Hattori takamitsu, Asano Ryutaro, Kumagai Izumi

    日本生物工学会大会講演要旨集　67　188-188　2015

    Publisher: 日本生物工学会

  73. tRNAジヒドロウリジン合成酵素の分子機構

    田中良和, 陳明皓,姚閔

    生化学　86　(86)　395-399　2014/06

    Publisher: 日本生化学会

    ISSN： 0037-1017

  74. スルメイカヘモシアニンの結晶構造解析

    田中良和, 加藤公児, 蓋作啓, 田中深雪, 田中勲, 姚閔, 加藤佑基, 清水健志, 宮部好克, 菅野岳, 岸村栄毅, 吉岡武也, 加藤早苗

    日本水産学会大会講演要旨集　2014　110　2014/03/27

  75. 一本鎖抗体の自己会合特性を活かした多価・多重特異性抗体設計

    梅津光央, 藤井滉人, 筋野拓馬, 真鍋法義, 中澤光, 田中良和, 浅野竜太郎, 熊谷泉

    化学工学会年会研究発表講演要旨集(CD-ROM)　79th　ROMBUNNO.C305　2014/02/18

  76. Molecular basis of tRNA dihydrouridine synthase

    Yoshikazu Tanaka, Minghao Chen, Min Yao

    Seikagaku　86　(3)　395-399　2014

    Publisher: Japanese Biochemical Society

    ISSN： 2189-0544 0037-1017

  77. 分子量4MDaの巨大酸素運搬蛋白質会合体ヘモシアニンの結晶構造解析

    田中良和, 加藤公児, 蓋作啓, 田中深雪, 加藤早苗, 清水健志, 岸村栄毅, 菅野岳, 宮部好克, 岩崎憲治, 田中勲, 姚閔

    日本結晶学会年会講演要旨集　2013　45　2013/10/12

  78. 動力学的解析および立体構造に基づいた進化型アセトアセチル‐CoAレダクターゼの高活性化メカニズムの解明

    WATANABE TSUYOSHI, MATSUMOTO KEN'ICHIRO, MOTOHASHI REN, IKEDA KOJI, TANAKA YOSHIKAZU, MIN YAO, TANAKA ISAO, TAGUCHI SEIICHI

    日本農芸化学会大会講演要旨集(Web)　2013　4C12A09 (WEB ONLY)　2013/03/05

    ISSN： 2186-7976

  79. 黄色ブドウ球菌の膜孔形成毒素の分子機構

    田中良和

    生物物理　52　(52)　293-295　2012/11/25

    Publisher: 一般社団法人 日本生物物理学会

    DOI： 10.2142/biophys.52.293 　

    ISSN： 0582-4052

  80. 光応答性分子を用いた孔形成毒素alpha‐hemolysinの光制御系の構築

    宇井美穂子, 播磨耕祐, 田中良和, 荒木保幸, 和田健彦, 武井俊朗, 津本浩平, 金原数

    バイオ・高分子シンポジウム講演要旨集　22nd　11-12　2012/06/15

    ISSN： 0917-5202

  81. 光受容蛋白質Phtoactive Yellow proteinを用いたStaphylococcal alpha‐Hemolysinの溶血活性制御

    宇井美穂子, 田中良和, 荒木保幸, 和田健彦, 武井俊朗, 津本浩平, 金原数

    高分子学会予稿集(CD-ROM)　61　(1)　ROMBUNNO.1H31　2012/05/15

  82. 人工分子を用いたStaphylococcus aureus α‐hemolysinの溶血活性の制御

    播磨耕祐, 宇井美穂子, 田中良和, 石井則行, 金原数

    日本化学会講演予稿集　92nd　(3)　776　2012/03/09

    ISSN： 0285-7626

  83. 光応答性蛋白質Phtoactive Yellow protein融合Staphylococcal alpha‐Hemolysinの機能制御

    宇井美穂子, 田中良和, 荒木保幸, 和田健彦, 武井俊朗, 津本浩平, 金原数

    日本化学会講演予稿集　92nd　(3)　816　2012/03/09

    ISSN： 0285-7626

  84. Structural and functional analysis of rRNA methyltransferase from Staphylococcus aureus

    Kita S, Tanaka Y, Yao M, Tanaka I

    XXII International Congress and General Assembly of IUCr　2011/08

  85. 黄色ブドウ球菌由来23S rRNA m5C1962メチル基転移酵素の構造解析

    喜多俊介, 田中良和, YAO Min, 田中勲

    日本蛋白質科学会年会プログラム・要旨集　11th　123　2011/05/27

  86. 黄色ブドウ球菌由来23S rRNA m5C1962メチル基転移酵素ホモログの構造解析

    喜多俊介, 田中良和, 姚閔, 田中勲

    日本結晶学会年会講演要旨集　2011　111　2011

  87. 黄色ブドウ球菌由来rRNAメチル基転移酵素の構造解析

    喜多俊介, 田中良和, 姚閔, 田中勲

    PFシンポジウム要旨集　28th　39　2011

  88. 光応答性タンパク質PYP融合によるStaphylococcal α‐Hemolysinの光制御

    宇井美穂子, 田中良和, 荒木保幸, 和田健彦, 武井俊朗, 津本浩平, 金原数

    東北大学多元物質科学研究所研究発表会講演予稿集　11th　23　2011

  89. Adaptation beyond the Stress Response: Cell Structure Dynamics and Population Heterogeneity in Staphylococcus aureus

    Kazuya Morikawa, Ryosuke L. Ohniwa, Toshiko Ohta, Yoshikazu Tanaka, Kunio Takeyasu, Tarek Msadek

    MICROBES AND ENVIRONMENTS　25　(2)　75-82　2010/05

    DOI： 10.1264/jsme2.ME10116 　

    ISSN： 1342-6311

  90. 黄色ブドウ球菌由来RlmLホモログの構造・機能解析

    喜多俊介, 田中良和, 姚閔, 田中勲

    日本結晶学会年会講演要旨集　2010　109　2010

  91. タンパク質の結晶構造解析を通して得られる予期せぬ発見

    田中 良和

    生物工学会誌 : seibutsu-kogaku kaishi　87　(10)　498-498　2009/10/25

    Publisher: 日本生物工学会

    ISSN： 0919-3758

  92. tRNAチオ化修飾酵素ThiIのRNA認識機構の解析

    田中良和, 山形しおり, 北郷悠, 山田洋子, CHIMNARONK Sarin, YAO Min, 田中勲

    日本RNA学会年会要旨集　11th　117　2009/07/27

  93. tRNAチオ化修飾酵素ThilのtRNA認識機構

    田中良和, 山形しおり, 北郷悠, 山田洋子, チムナロン サリン, 姚閔, 田中勲

    日本蛋白質科学会年会プログラム・要旨集　9th　152　2009/04/24

  94. tRNA修飾酵素ThilのtRNAの認識機構

    田中良和, 山形しおり, 北郷悠, 山田洋子, チムナロンサリン, 姚閔, 田中勲

    PFシンポジウム要旨集　26th　111　2009

  95. 抗体の低分子化の最前線

    田中良和, 津本浩平

    バイオテクノロジージャーナル 7　(6)　646-647　2007

  96. 抗体に代わる新たな抗原結合性蛋白質分子-avimer-

    田中良和, 津本浩平

    バイオテクノロジージャーナル 7　(5)　529-530　2007

  97. 膜タンパク質研究の新たなツール～Nanodisk

    田中良和, 津本浩平

    バイオテクノロジージャーナル 7　(3)　273-274　2007

  98. GFPテクノロジー

    田中良和, 津本浩平

    バイオテクノロジージャーナル 7　(2)　146-147　2007

  99. タンパク質はどこまで熱に対して安定にできるのか？

    田中良和, 津本浩平

    バイオテクノロジージャーナル 7　(1)　13-14　2007

  100. 1P125 Thermodynamic profiles of the interaction between hyperthermostable TATA-box binding protein and a double-stranded DNAat high temperature(Protein engineering, evolutionary engineering, nucleic acid-binding proteins, and nucleic acid,Oral Presentatio

    Nagatoishi Satoru, Tanaka Yoshikazu, Tsumoto Kouhei

    Seibutsu Butsuri　47　(0)　S54　2007

    Publisher: 一般社団法人 日本生物物理学会

    DOI： 10.2142/biophys.47.S54_4 　

    ISSN： 0582-4052

  101. 2P047 The structural and thermodynamic analysis of the interaction between ciguatoxin and its Antibody 10C9 Fab(Proteins-structure and structure-function relationship,Poster Presentations)

    Ui Mihoko, Tanaka Yoshikazu, Tsumuraya Takeshi, Fujii Ikuo, Inoue Masayuki, Hirama Masahiro, Tsumoto Kouhei

    Seibutsu Butsuri　47　(0)　S124　2007

    Publisher: 一般社団法人 日本生物物理学会

    DOI： 10.2142/biophys.47.S124_4 　

    ISSN： 0582-4052

  102. 2P067 Thermodynamic analysis of the assembly of the α-, β-, Y-subunits of archaeral initiation factor 2(Proteins-stability, folding, and other physicochemical properties,Oral Presentations)

    Nakakido Makoto, Tanaka Yoshikazu, Sokabe Masaaki, Sakai Naoki, Yao Min, Tanaka Isao, Tsumoto Kouhei

    Seibutsu Butsuri　47　(0)　S129　2007

    Publisher: 一般社団法人 日本生物物理学会

    DOI： 10.2142/biophys.47.S129_4 　

    ISSN： 0582-4052

  103. 高度好熱古細菌Sulfolobus tokodaii由来protein L-isoaspartyl-O-methyltransferaseの多量体化による耐熱性獲得機構

    田中良和

    生物物理　46　(46)　209-213　2006/07/25

    Publisher: 一般社団法人日本生物物理学会

    DOI： 10.2142/biophys.46.209 　

    ISSN： 0582-4052

    More details Close

    Recent exponential increase of three-dimensional structural information by structural genomics revealed that a lot of proteins from hyper-thermophilic organisms have oligomeric structures. Based on these findings, it has been proposed that oligomerization is one of the strategies for enhancing thermostability of proteins in thermophilic organism. Here I describe how oligomerization contributes to the thermostability in protein <sc><font size = -2>L</font></sc>-isoaspartyl-<i>O</i>-methyltransferase from <i>Sulfolobus tokodaii</i> from its crystal structural and mutational analyses.<br>

  104. The in vivo aggregation of single chain Fv fragment can be replicated in vitro

    Umetsu M, Tsumoto K, Ashish Kumar, Nitta S, Tanaka Y, Adschiri T, Kumagai I

    Seibutsu Butsuri　43　(0)　S62　2003

    Publisher: 一般社団法人 日本生物物理学会

    DOI： 10.2142/biophys.43.S62_1 　

    ISSN： 0582-4052

Show all ︎Show first 5

Books and Other Publications 5

  1. 「クライオ電子顕微鏡ハンドブック」第４章１４節 クライオ電子顕微鏡で見る、リボソームの「かたち」と「動き」

    伴野 詢太, 田中 良和, 横山 武司

    NTS　2023/01

  2. Chapter 7 Molluscan Hemocyanins in Vertebrate and Invertebrate Respiratory Proteins, Lipoproteins and other Body Fluid Proteins edited by Ulrich Hoeger and J. Robin Harris

    Sanae Kato, Takashi Matsui, Yoshikazu Tanaka

    2020

  3. Essentialタンパク質科学

    津本浩平, 植田正, 前仲勝実, 田中良和, 阿部義人, 帯田孝之, 栗栖源嗣, 白石充典, 安達成彦, 千田俊哉, 尾瀬農之, 黒木喜美子, 堀内正隆, 齊藤貴士

    2016/02/25

  4. 環境と微生物の事典

    日本微生物生態学会

    2014

  5. タンパク質結晶の新展開ー新しい育成技術から構造解析・応用研究へー

    高野和文

    2008

Presentations 49

  1. Crystal structure analysis of LanK complexed with landomycinA, a potent antitumor antibiotic

    Atsushi Tsugita, Shiro Uehara, Takashi Matsui, Takeshi Yokoyama, Iryna Ostash, Maksym Deneka, Subbarao Yalamanchili, Clay S. Benne, Bohdan Ostash, YoshikazuTanaka

    e-Asia Joint Symposium on “Marine Biodiversity as a Source of New Chemotypes”　2022/12

  2. クライオ電子顕微鏡単粒子解析による、リボソームを標的とするアミノ配糖体抗菌薬の微細構造差が生み出す阻害活性増強機構の解明

    浅野航佑, 伴野詢太, 鈴木仁人, 横山武司, 田中良和

    第45回日本分子生物学会年会　2022/11

  3. クライオ電子顕微鏡単粒子解析を用いた、非結核性抗酸菌の薬剤耐性因子によるリボソーム解離機構の解明

    千足啄馬, 高田希美, 橋本翼, 深野華子, 山本健太郎, 鈴木仁人, 星野仁彦, 横山武司, 田中良和

    第45回日本分子生物学会年会　2022/11

  4. ヘモリシンの立体構造解析と構造情報を生かしたデザイン Invited

    田中良和

    分子ロボティクス研究会　2022/11

  5. tRNA thiolation by TtuA-TtuB using [4Fe-4S] cluster

    Yoshikazu Tanaka

    he 12th International Conference on the Biology, Chemistry and Therapeutic Applications of Nitric Oxide　2022/10

  6. ヒト由来電位依存性カリウムチャネルKV1.2/KVb2.1複合体のクライオ電子顕微鏡単粒子解析

    関戸菜津子, 飯塚友菜, 相沢智康, 佐々木誠, 不破春彦, 山下-四津まり, 此木敬一, 横山武司, 田中良和

    第60回日本生物物理学会年会　2022/09/28

  7. デングウイルス由来 RNA 依存性 RNA ポリメラーゼと天然物ライブラリーから得られたその阻害剤との複合体の立体構造解析

    細井菜海, 中谷晴香, ラッカナー, ダヴィーポーンクン, 須藤愛莉咲, 坂井直樹, 松浦滉明, 山本雅貴, 松井崇, 小寺義男, サリン チムナロン, 坂井隆一, 横山武司, 田中良和

    第60回日本生物物理学会年会　2022/09/28

  8. タンパク質立体構造解析と質量分析の融合研究 Invited

    田中良和

    日本プロテオーム学会2022年大会　2022/08

  9. ストップコドンリードスルーの分子機構解明

    田中良和

    第22回日本蛋白質科学会年会　2022/06

  10. 海洋生物由来レクチンThCの構造機能相関研究

    影山 大夢, 辺 浩美, 小野寺 かこ, 松井 崇, 小川, 智久, 横山 武司, 酒井 隆一, 田中 良和

    2021年度生物物理学会北海道支部・東北支部合同例会　2022/03/09

  11. 放線菌由来TetR型転写調節蛋白質LanKのX線結晶構造解析

    次田篤史, 上原史朗, 松井崇, 横山武司, Iryna Ostash, Bohdan Ostash, 田中良和

    2021年度生物物理学会北海道支部・東北支部合同例会　2022/03/09

  12. 非結核性抗酸菌Mycobacterium abscessusリボソームにおけるマクロライド耐性機構の解明を目指したクライオ電子顕微鏡構造解析

    橋本翼, 高田希美, 千足啄馬, 深野華子, 山本健太郎, 鈴木仁人, 星野仁彦, 横山武司, 田中良和

    2021年度生物物理学会北海道支部・東北支部合同例会　2022/03/09

  13. クライオ電子顕微鏡単粒子解析法によるウサギ赤血球ライセートに内在する80Sリボソーム構造解析の検討

    千足啄馬, 横山武司, 田中良和

    第44回日本分子生物学会年会

  14. 薬剤耐性獲得機構の解明に向けた、クライオ電子顕微鏡単粒子解析による非結核性抗酸菌Mycobacterium abscessusリボソームへのマクロライド結合様式の解明

    橋本翼, 高田希美, 千足啄馬, 深野華子, 山本健太郎, 鈴木仁人, 星野仁彦, 横山武司, 田中良和

    第44回日本分子生物学会年会　2021/12/01

  15. Elucidation of new action mechanism of aminoglycoside antibiotics on ribosomes using single particle cryo-electron microscopy

    Junta Tomono, Kosuke Asano, Masato Suzuki, Takeshi Yokoyama, Yoshikazu Tanaka

    2021/11/26

  16. Elucidation of the binding mode of a macrolide antibiotic to NTM ribosome for understanding drug resistance mechanism by using cryo-EM

    Tsubasa Hashimoto, Nozomi Takada, Takuma Chiashi, Hanako Fukano, Kentaro Yamamoto, Masato Suzuki, Yoshihiko Hoshino, Takeshi Yokoyama, Yoshikazu Tanaka

    2021/11/25

  17. Design of staphylococcal two-component pore forming toxin to change pore formation property Invited

    2021/11/24

  18. 新規レクチン様タンパク質のX線結晶構造解析

    影山大夢, 小野寺かこ, 松井崇, 小川智久, 横山武司, 田中良和

    令和3年度日本結晶学会年会　2021/11/19

  19. 放線菌由来TetR型転写制御蛋白質LanKのX線結晶構造解析

    次田篤史, 上原史朗, 松井崇, 横山武司, Iryna Ostash, Bordan Ostash, 田中良和

    令和3年度日本結晶学会年会　2021/11/19

  20. 海洋天然物由来の新規生理活性タンパク質の構造機能解析 Invited

    田中良和

    第94回日本生化学会大会　2021/11/03

  21. 巨大タンパク質会合体ヘモシアニンの構造解析 Invited

    田中良和

    農芸化学会　2021/03/19

  22. 新規レクチン様タンパク質の結晶構造解析と糖との結合解析

    影山大夢, 小野寺かこ, 小川智久, 横山武司, 田中良和

    2021/03/10

  23. クライオ電子顕微鏡単粒子解析を用いたアミノ配糖体抗菌薬の新規作用機序の探索

    伴野 詢太, 浅野 航佑, 鈴木 仁人, 横山 武司, 田中 良和

    生物物理学会 北海道支部-東北支部合同年会　2021/03/08

  24. Structural analysis of LanK, a protein associated with the production of antibiotics in actinomycetes

    Shiro Uehara, Iryna Ostash, Bohdan Ostash, Takashi Matsui, Tomohisa Ogawa, Yoshikazu Tanaka

    分子生物学会

  25. Crystal structure analysis of BIR3 domain of ubiquitin ligase in complex with its specific ligand for designing potent ligand

    Makoto Okabe, Shinichi Sato, Takeshi Yokoyama, Shusuke Tomoshige, Minoru Ishikawa, Yoshikazu Tanaka

    分子生物学会

  26. 蛋白質の立体構造解析 -X線結晶構造解析とクライオ電顕単粒子解析- Invited

    田中良和

    酵素工学研究会第８４回講演会　2020/11/20

  27. 巨大タンパク質会合体ヘモシアニンの結晶中の空隙への生体分子の包摂 Invited

    田中良和

    化学工学会第51回秋季大会　2020/09/24

  28. Cryo-electron microscopy single particle analysis of pertussis dermonecrotic toxin

    Atsushi Tsugita, Takashi Matsui, Yasuhiko Horiguchi, Takeshi Yokoyama, Yoshikazu Tanaka

    日本生物物理学会第58回年会　2020/09/16

  29. Cryo-EM study on Saframycin A biosynthesis related protein

    Kiichi Honda, Takashi Matsui, Ryo Tanifuji, Hiroki Oguri, Takeshi Yokoyama, Yoshikazu Tanaka

    日本生物物理学会第58会年会　2020/09/16

  30. 放線菌の抗⽣物質産⽣に関連する蛋⽩質LanKの構造解析

    上原史朗, Iryna Ostash, Bohdan Ostash, 松井崇, 小川智久, 田中良和

    ⽇本⽣物物理学会 東北⽀部会2019　2019/11/08

  31. tRNA thiolation mechanism involving [4Fe-4S] cluster

    Yoshikazu Tanaka

    ISNAC2019　2019/10/29

  32. 蛋白質を包摂可能な新規ホスト分子としての多孔性蛋白質結晶の利用

    橋本翼, 松井崇, 小川智久, 田中良和

    第92回日本生化学会大会　2019/09

  33. X線結晶構造解析とクライオ電子顕微鏡解析の相関構造解析の有用性―ヘモシアニンを例としてー Invited

    田中良和

    日本細胞生物学会大会　2019/06/06

  34. 超巨大タンパク質会合体ヘモシアニンの構造 X線結晶構造解析とクライオ電顕構造解析を併用してわかったこと Invited

    田中良和

    2019年度日本生化学会九州支部例会　2019/06

  35. 蛋白質結晶中の空隙へのゲスト蛋白質包摂手法の開発と結晶構造解析へ の応用

    橋本翼, 松井崇, 小川智久, 田中良和

    第46回生体分子科学討論会　2019/06

  36. Structure aided molecular design of staphylococcal pore-forming toxin

    Nouran Ghanem, Natsuki Kanagami, Kein Takeda, Takashi Matsui, Tomohisa Ogawa, Jun Kaneko, Yoshikazu Tanaka

    2019/06

  37. 蛋白質結晶中の空隙へのゲスト蛋白質包摂手法の開発

    橋本翼, 松井崇, 小川智久, 田中良和

    日本生化学会東北支部第85回例会・シンポジウム　2019/06

  38. ホスホリパーゼA2アナログの立体構造解析とSDSによる多量体化

    松井崇, 鎌田しずか, 石井健太郎, 丸野孝浩, ガーネモノーラン, 内山進, 加藤晃一, 鈴木淳巨, 上田直子, 小川智久, 田中良和

    2018年度量子ビームサイエンスフェスタ（第10回MLFシンポジウム／第36回PFシンポジウム）　2019/03

  39. 巨大蛋白質会合体ヘモシアニンの構造解析

    田中 良和

    日本生物物理学会 東北支部会２０１８　2018/11/09

  40. HIV gp120-inhibition mechanisms of Pteria penguin pearl shell lectins based on their carbohydrate binding properties

    Lakudzala Agness Ethel, Matsui Takashi, Tanaka Yoshikazu, Ogawa Tomohisa

    22nd Research Dissemination Conference　2018/11

  41. X線結晶構造解析とクライオ電顕の両手法を使って Invited

    田中 良和

    PF研究会「X線とクライオ電子顕微鏡で挑む生命の機能とかたち」　2018/09

  42. Encapsulation of protein in a hollow protein crystal Invited

    TANAKA Yoshikazu

    International Conference on Coordination Chemistry　2018/08

  43. HIV gp120-inhibition mechanisms of Pteria penguin pearl shell lectins based on their carbohydrate binding properties

    HIV g, inhibition mechanisms of, Pteria penguin pearl shell lectins based on their carbohydrate binding properties

    The 5th CWRU-Tohoku Joint Workshop　2018/04

  44. Elucidation of the tRNA thiolation mechanism of TtuA involved in Fe-S cluster

    2018

  45. スルメイカ由来ヘモシアニンのクライオ電顕構造とX 線結晶構造の違い

    田中良和, 松井崇, 加藤早苗, Christos Gatsogiannis, Stefan Raunser

    日本蛋白質科学会年会　2018

  46. Investigating a novel tRNA thiolational modification mechanism involving an [4Fe-4S] cluster International-presentation

    Yoshikazu Tanaka, Minghao Chen, Shun Narai, Naoki Omura, Naoki Shigi, Min Yao

    2nd Joint International Symposium of NSRRC and IPR　2017/12/06

  47. 分子量4MDaの巨大蛋白質会合体ヘモシアニンの結晶への蛋白質の包摂

    田中良和

    JST-ACCELプロジェクト「革新的分子構造解析」公開シンポジウム　2017/10/31

  48. 分子量4MDaの巨大蛋白質会合体ヘモシアニンの結晶への蛋白質の包摂

    第17回 リング・チューブ超分子研究会シンポジウム　2017/06/09

  49. Structure analysis of protein using both electron microscopy and X-raycrystallography Invited

    Yoshikazu Tanaka

    ABiS symposium Forefront and Future of Electron Microscopic Imaging　2020/02/15

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Industrial Property Rights 3

  1. ヘモシアニン会合体を用いた包摂体の製造方法

    田中 良和, 松野 明日香, 北村 朗, 金城 政孝, 姚 閔, 上野 隆史, 安部 聡

    Property Type: Patent

  2. 生物学的試料をハイドロゲルで高速固定する方法及びそのための装置

    田中 勲, 篠田 晃, 姚 閔, 田中 良和

    Property Type: Patent

  3. 結晶化促進ポリペプチド

    姚 閔, 薦田 圭介, 于 健, 田中 良和

    Property Type: Patent

Research Projects 39

  1. クライオ電顕と機械学習を利用した終止コドンリードスルーの機構解明

    田中 良和, 横山 武司, 松井 崇, 梅津 光央, 眞壁 幸樹

    Offer Organization: 日本学術振興会

    System: 科学研究費助成事業

    Category: 基盤研究(B)

    Institution: 東北大学

    2025/04/01 - 2028/03/31

  2. Molecular dissection of plant specialized metabolism machineries

    Offer Organization: Japan Society for the Promotion of Science

    System: Grants-in-Aid for Scientific Research

    Category: Grant-in-Aid for Scientific Research (S)

    Institution: Tohoku University

    2023/04/12 - 2028/03/31

  3. Preliminary research on verifying the accuracy of the Codon Table

    Offer Organization: Japan Society for the Promotion of Science

    System: Grants-in-Aid for Scientific Research

    Category: Grant-in-Aid for Challenging Research (Exploratory)

    Institution: Tohoku University

    2025/06/27 - 2027/03/31

  4. クライオ電顕解析による分子配向の最適化に基づく多機能性プロドラッグ抗体の創製

    浅野 竜太郎, 田中 良和, 安永 正浩

    Offer Organization: 日本学術振興会

    System: 科学研究費助成事業

    Category: 基盤研究(B)

    Institution: 東京農工大学

    2023/04/01 - 2026/03/31

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    二重特異性がん治療抗体への機能性分子の融合は、より強力な薬効が期待できるが副作用も懸念される。一方、抗体の結合部位を阻害する配列でマスクしておき、がん組織送達後に外すことができればプロドラッグ抗体となる。本研究は、機能性分子のマスクとしての利用を目指している。二重特異性抗体の結合能と機能性分子の機能を互いに阻害するように融合し、がん組織送達後に両機能が回復するような新規プロドラッグ抗体のデザイン開発を進めている。 本年度は、1) 二重特異性抗体と協働するT細胞活性化因子の探索、と、2) 抗T細胞抗体とT細胞活性化因子との融合検討、を進めた。T細胞は主シグナルであるCD3を介した活性化に引き続く、共刺激分子の作用により持続的に活性化される。共刺激分子として4-1BBとCD28に対するアゴニスト抗体の配列を文献情報から入手し、一本鎖抗体 (scFv) として組換え体を調製した。これらのscFvを二重特異性抗体に添加したところ、T細胞存在下でのがん細胞傷害活性が優位に向上することが明らかになった。また、抗腫瘍性のサイトカインであるIL-2も同様に添加による相加効果が見られた。そこで、これらの分子をマスク配列として利用して、二重特異性抗体への融合検討を行った。興味深いことに融合時のリンカー配列の違いにより、分子の構造安定性に変化が見られた。これらの要因を検証、および考察するためにネガティブ染色電顕での観察へと進めた。今後の精密解析による、融合設計の最適化に期待が持たれる。

  5. 植物特化代謝における超分子的代謝マシナリの形成：多元構造解析による統合的理解

    中山 亨, 和氣 駿之, 田中 良和, 山下 哲, 竹下 浩平, 齋尾 智英

    Offer Organization: 日本学術振興会

    System: 科学研究費助成事業

    Category: 基盤研究(A)

    Institution: 東北大学

    2023/04/01 - 2026/03/31

  6. Visualization of supersulfur molecules based on the structural biology

    Offer Organization: Japan Society for the Promotion of Science

    System: Grants-in-Aid for Scientific Research Grant-in-Aid for Transformative Research Areas (A)

    Category: Grant-in-Aid for Transformative Research Areas (A)

    Institution: University of Miyazaki

    2021/09/10 - 2026/03/31

  7. 終止コドンリードスルーの誘発因子の特定とクライオ電顕による活写

    田中 良和

    Offer Organization: 日本学術振興会

    System: 科学研究費助成事業 学術変革領域研究(A)

    Category: 学術変革領域研究(A)

    Institution: 東北大学

    2023/04/01 - 2025/03/31

  8. 高活性二重特異性抗体の創製に資する至適な抗原配置の先端的構造解析による描像

    熊谷 泉, 田中 良和, 眞壁 幸樹, 浅野 竜太郎

    Offer Organization: 日本学術振興会

    System: 科学研究費助成事業

    Category: 基盤研究(B)

    Institution: 東京農工大学

    2022/04/01 - 2025/03/31

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    異なる2種の抗体の抗原結合ドメインを繋げると、2分子間を架橋可能な二重特異性抗体が作製される。これまでの二重特異性抗体の網羅的な改変体の開発を通して、超高活性のLH型と低活性のHL型が存在することを見出している。本研究は、まずLH型とHL型を含む種々の活性の異なる二重特異性抗体を用いた両標的抗原との三者複合体のクライオ電顕構造解析により、強力ながん細胞傷害活性をもたらし得る両標的抗原の空間配置を解明し、さらに、得られた空間配置を基に高活性の二重特異性抗体の効率的な創製プラットフォームの開発を目指している。 本年度も、クライオ電顕構造解析による二重特異性抗体と両標的抗原の至適な空間配置の解明、を主に目指した。前年度までに、二重特異性抗体と両標的抗原の3者複合体の調製法を確立し、ネガティブ染色電顕での観察、およびクライオ電顕のデータセットの収集を行ったが、原子レベルの構造決定には到らなかった。そこで、引き続き条件検討を行うと共に、二重特異性抗体を構成する各々の抗体の抗原結合部位と対応する標的抗原との複合体の解析も並行して進め、クライオ電顕単粒子解析により得られた像の構造決定の一助とした。また、構造多型分類プログラムを駆使し、ドメインの構造変化も加味した構造情報を取得し、解析を進めた結果、LH型とHL型の構造の違いを明確に示す3者複合体の3次元構造を得ることに成功した。今後さらなる精密解析により、強力ながん細胞傷害活性をもたらし得る両標的抗原の空間配置の解明につながることが期待される。

  9. Exploring Marine Natural Products that Regulate Dynamic Responses of cells

    Offer Organization: Japan Society for the Promotion of Science

    System: Grants-in-Aid for Scientific Research

    Category: Grant-in-Aid for Scientific Research (B)

    Institution: Hokkaido University

    2022/04/01 - 2025/03/31

  10. Structure-based functionalization of novel sugar-binding proteins derived from marine natural products for corona drug discovery

    Offer Organization: Japan Society for the Promotion of Science

    System: Grants-in-Aid for Scientific Research

    Category: Grant-in-Aid for Scientific Research (B)

    Institution: Tohoku University

    2022/04/01 - 2025/03/31

  11. Structure-based functionalization of novel sugar-binding proteins derived from marine natural products for corona drug discovery

    Offer Organization: Japan Society for the Promotion of Science

    System: Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

    Category: Grant-in-Aid for Scientific Research (B)

    Institution: Tohoku University

    2022/04/01 - 2025/03/31

  12. 細胞の動的応答を制御する海洋天然物の探索

    酒井 隆一, 高田 礼人, 田中 良和, 松本 健, 八代田 陽子, 荒木 真理人

    Offer Organization: 日本学術振興会

    System: 科学研究費助成事業 基盤研究(B)

    Category: 基盤研究(B)

    Institution: 北海道大学

    2022/04/01 - 2025/03/31

  13. 高活性二重特異性抗体の創製に資する至適な抗原配置の先端的構造解析による描像

    熊谷 泉, 浅野 竜太郎, 田中 良和, 真壁 幸樹

    Offer Organization: 日本学術振興会

    System: 科学研究費助成事業 基盤研究(B)

    Category: 基盤研究(B)

    Institution: 東京農工大学

    2022/04/01 - 2025/03/31

  14. クライオ電子顕微鏡解析に基づく酵素―電極間直接電子移動制御型センサの開発

    浅野 竜太郎, 田中 良和

    Offer Organization: 日本学術振興会

    System: 科学研究費助成事業 挑戦的研究(開拓)

    Category: 挑戦的研究(開拓)

    Institution: 東京農工大学

    2021/07/09 - 2025/03/31

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    本研究は、汎用的な血中バイオマーカーの連続計測を可能とする酵素―電極間直接電子移動 (Direct Electron Transfer, DET) 制御型センサの開発を目的としている。具体的には、電極へのDET能を有するフラボシトクロムb2 (Fcb2)－シトクロム c (Cyt c) にそれぞれ抗体断片を融合させ、バイオマーカー依存的なDET効率の変化を測定原理とするセンサの開発を目指している。 本年度は、主に1) Fcb2の改変による熱安定性の向上、と2) Fcb2とCyt cのクライオ電子顕微鏡を用いた構造解析、の観点から研究を進めた。 1) Fcb2の改変による熱安定性の向上 ジスルフィド結合の導入による熱安定性の向上に向けて、モデル構造を基に、システインへの変異導入個所を複数選定した。複数の変異体を設計し、それぞれ発現ベクターを作製し、大腸菌を用いた組換え発現を行った。いずれも菌体破砕上清に酵素活性が認められたため、今後安定性評価へと進める予定である。また、精製のためのタグを融合させたFcb2の調製も並行して進めた。結果、興味深いことに酵素活性と安定性の向上が認められた。今後より詳細な評価を行う予定である。 2) Fcb2とCyt cのクライオ電子顕微鏡を用いた構造解析 バイオマーカー依存的なDET効率の変化を測定原理とする新規センサの開発にはFcb2とCyt cの相互作用部位の特定が重要である。それぞれ大腸菌発現系を用いて調製したFcb2とCyt cを量論的に混合させた後、クライオ電子顕微鏡解析を行った。Fcb2とCyt cの相互作用部位の特定には至っていないが、特にFcb2に関して良好な観察像が得られており、今後より詳細な条件検討を行うことで、精密構造が得られる可能性が示された。

  15. Structural basis of the iron-sulfur cluster biogenesis, and the establishment of ex-situ reconstitution system

    Offer Organization: Japan Society for the Promotion of Science

    System: Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

    Category: Grant-in-Aid for Scientific Research (B)

    Institution: University of Miyazaki

    2020/04/01 - 2024/03/31

  16. Snapshot analysis of stop codon read-through by cryo-electron microscopy

    Offer Organization: Japan Society for the Promotion of Science

    System: Grants-in-Aid for Scientific Research Grant-in-Aid for Transformative Research Areas (A)

    Category: Grant-in-Aid for Transformative Research Areas (A)

    Institution: Tohoku University

    2021/09/10 - 2023/03/31

  17. Development of a method to determine RNA-protein complex by cryo-EM

    Offer Organization: Japan Society for the Promotion of Science

    System: Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory)

    Category: Grant-in-Aid for Challenging Research (Exploratory)

    Institution: Tohoku University

    2021/07/09 - 2023/03/31

  18. Design of beta-barrel type transmembrane protein complex by combination of computational and experimental studies

    Offer Organization: Japan Society for the Promotion of Science

    System: Grants-in-Aid for Scientific Research Fund for the Promotion of Joint International Research (Fostering Joint International Research (B))

    Category: Fund for the Promotion of Joint International Research (Fostering Joint International Research (B))

    Institution: Tohoku University

    2019/10/07 - 2023/03/31

  19. Antibodies targeting dynamic epitopes generated by posttranslational protein glycosylation

    Offer Organization: Japan Society for the Promotion of Science

    System: Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)

    Category: Grant-in-Aid for Scientific Research (A)

    Institution: Hokkaido University

    2019/04/01 - 2023/03/31

  20. Development of material for electron microscopy analysis using protein complex with huge internal space

    Tanaka Yoshikazu

    Offer Organization: Japan Society for the Promotion of Science

    System: Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

    Category: Grant-in-Aid for Scientific Research (B)

    Institution: Tohoku University

    2019/04/01 - 2022/03/31

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    Method to encapsulate the guest protein inside the hemocyanin via various linkers was developed. The effect of guest binding on the decameric structure of the host differed depending on the linker. This was suggested to be due to the strength of the binding of the guest inside the host. Electron microscopic observation also showed that by improving the conditions, it was possible to maintain a stable decameric structure with the guest encapsulated inside. Evaluation using gold nanoparticles suggested the importance of improving the encapsulation ratio.

  21. 合成単分子シャペロンの開発

    金原 数, 田中 良和

    Offer Organization: 日本学術振興会

    System: 科学研究費助成事業 基盤研究(B)

    Category: 基盤研究(B)

    Institution: 東京工業大学

    2019/04/01 - 2022/03/31

  22. 細胞のバリアを攻略する海洋天然物の探索

    酒井 隆一, 田中 良和, 及川 雅人

    Offer Organization: 日本学術振興会

    System: 科学研究費助成事業 基盤研究(B)

    Category: 基盤研究(B)

    Institution: 北海道大学

    2019/04/01 - 2022/03/31

  23. 次世代抗体設計に資するマルチスケール解析による二重特異性抗体の超活性化機構の理解

    熊谷 泉, 田中 良和, 真壁 幸樹, 浅野 竜太郎

    Offer Organization: 日本学術振興会

    System: 科学研究費助成事業 基盤研究(B)

    Category: 基盤研究(B)

    Institution: 東京農工大学

    2019/04/01 - 2022/03/31

  24. Elucidation of a tRNA conversion mechanism of transsulfursome by the structural analysis of quaternary complex

    Yao Min

    Offer Organization: Japan Society for the Promotion of Science

    System: Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

    Category: Grant-in-Aid for Scientific Research (B)

    Institution: Hokkaido University

    2017/04/01 - 2020/03/31

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    To translate the protein according to the genetic code on the ribosome, the aminoacyl-tRNA (aa-tRNA) must be synthesized correctly. Generally, the aminoacyl-tRNA synthetase (aaRS) directly catalyzes this synthesis. However, in methanogenic archaea, a ternary complex called transsulfursome (SepRS, SepCys, SepCysS) synthesizes Cys-tRNA(Cys) in an indirect pathway. In this study, we analyzed the relationship of structure and function of transsulfursome-tRNA complex by using X-ray crystallography, small-angle X-ray scattering, electron microscopic observation, and biochemical methods. Taken results together, we understood the dynamic molecular mechanism for the Cys-tRNA(Cys) synthesis of the transsulfursome.

  25. 構造情報に基づいた機能変換による膜孔形成毒素の会合機構の解明

    田中 良和

    Offer Organization: 日本学術振興会

    System: 科学研究費助成事業 新学術領域研究(研究領域提案型)

    Category: 新学術領域研究(研究領域提案型)

    2016/04/01 - 2018/03/31

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    病原性微生物がホスト細胞に障害を与えるために産生する膜孔形成毒素は，可溶性の単量体として分泌されるが，ターゲット細胞に接すると膜上でpreporeと呼ばれる円状の会合体を形成した後，劇的な構造変化を起こして膜孔を形成する．黄色ブドウ球菌は，3種類の非常に類似した膜孔形成毒素（Hla，LukF，Hlg2）を分泌するが，Hlaはホモ7量体の膜孔を形成するのに対し，LukF，Hlg2は交互に会合したヘテロ8量体の膜孔を形成する．毒素同士がどのようにして特定のパートナーとのみ特異的に会合して膜孔を形成するのか，また，7量体・8量体という分子の会合数がどのようにして決定されるのかについては，明らかにできていない． 本研究では，構造解析から提唱された機構を基に変異体蛋白質を合理設計し，毒素の会合特性を改変することを通して，これらの毒素の自己組織化機構を解明することを目指した．立体構造情報をもとに，Hlg2とLukFの接触界面を移植したキメラ変異体を2種類作成した．また，コンピュータシミュレーションにより単量体として機能できるように，界面の残基の変異体を設計した．4種類の変異体を作成した．これらに加え，2成分性毒素の各成分に対し，ステム領域をHlaのもので置換した変異体と，さらにその周辺の重要残基に変異を加えたキメラ変異体を合計16種類作成した．これらのキメラ変異体の溶血活性を種々の条件下で評価した結果，LukFのステム領域はHlaのステム領域で置換可能であることがわかった．また，Hlaのステムで置換したHlg2の種々の変異体のCDスペクトルから，ステム領域の入れ替えにより，単量体の構造に変化が起きていることが示された．また，一連の実験の過程で， Hlg2，LukFのリフォールディングの手法が確立された．

  26. Development of antibodies for crystallization based on anti-peptide antibody to elucidate the functional structure of small bispecific antibodies

    ASANO Ryutaro

    Offer Organization: Japan Society for the Promotion of Science

    System: Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research

    Category: Grant-in-Aid for Challenging Exploratory Research

    Institution: Tokyo University of Agriculture and Technology

    2015/04/01 - 2018/03/31

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    We tried to develop a novel method for preparation of antibodies for crystallization to elucidate the functional structure of small bispecific antibodies. We selected the pair of anti-peptide antibodies and its antigen peptide from Protein Data Bank, an archive-information about the 3D shapes of proteins, to use anti-peptide antibodies as antibodies for crystallization after integrating the antigen peptide into small bispecific antibodies. Although further investigation is needed to obtain soluble small bispecific antibody integrated with the antigen peptide, it was shown that the antigen peptide can integrate into the linker region in small bispecific antibody.

  27. Structural study of a huge respiratory supermolecule hemocyanin (international collaboration work)(Fostering Joint International Research)

    Tanaka Yoshikazu, Raunser Stefan

    Offer Organization: Japan Society for the Promotion of Science

    System: Grants-in-Aid for Scientific Research Fund for the Promotion of Joint International Research (Fostering Joint International Research)

    Category: Fund for the Promotion of Joint International Research (Fostering Joint International Research)

    2015 - 2018

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    Hemocyanin is an oxygen transporter of mollusks. Squid hemocyanin is a huge cylindrical protein complex with a molecular mass approximately 4 MDa. In a previous study, we determined crystal structure of squid hemocyanin at 3.0 angstrom resolution. However, due to the crystal packing and symmetry of the outer wall region, we could not determine precise structure of the inside domains. Therefore, in the present study, we used cryo-electrom microscopy single particle analysis method to determine the structure. The revealed strcutre showed that the inner domains are arranged asymmetrically. As well, insight into molecular evolution of hemocyanin was revealed.

  28. A direct structure determination using lectin-carbohydrate complex

    Sakai Ryuichi, TANAKA Yoshikazu

    Offer Organization: Japan Society for the Promotion of Science

    System: Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research

    Category: Grant-in-Aid for Challenging Exploratory Research

    Institution: Hokkaido University

    2015/04/01 - 2017/03/31

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    Lectins, carbohydrate binding proteins, bind to sugars. We have isolated a galactose binding lectin CchG from the marine sponge Cinachyrella sp. that forms a rigid tetramer in the crystalline state. If the lectin-carbohydrate complex can be crystallized and its structure solved, the structure of the carbohydrate bind to the protein will be visualized. We extended this idea to determine the structure of glycosylated small molecules; that is, if a small molecule was glycosylated with the sugar that can bind to the lectin, the structure of the glycoside would be visualized directly by X-ray crystallography at the atomic level. We thus tested our concept by synthesizing 1-fluoro-2,4-dinitrobenzene-5-(1-aminolacoside) (LacDNFB) which can readily couple with amino acids. In crystallization of CchG-lactose, CchG-LacDNFB, and CchG-LacDNFB-alanine complexes, and crystal structure for CchG-lactose showing that +1-end of the sugar had a space to hold molecule to be visualized, was solved.

  29. Structural study of a huge respiratory supermolecule hemocyanin

    Tanaka Yoshikazu

    Offer Organization: Japan Society for the Promotion of Science

    System: Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

    Category: Grant-in-Aid for Scientific Research (B)

    Institution: Hokkaido University

    2014/04/01 - 2017/03/31

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    In this study, we have determined the X-ray crystal structure of intact hemocyanin from Japanese flying squid at 3.0 angstrom resolution. The structure urevealed the architecture of hemocyanin composed of a cylindrical wall region and five collar regions, in which the dimers of functional units (FUs) hierarchically associated to form the entire decamer. Furthermore, the roles of carbohydrates in assembly, and conserved sulfur-containing residues in copper incorporation, were revealed.

  30. 膜孔形成毒素の動的な作用機構の解明

    田中 良和

    Offer Organization: 日本学術振興会

    System: 科学研究費助成事業 新学術領域研究(研究領域提案型)

    Category: 新学術領域研究(研究領域提案型)

    Institution: 北海道大学

    2014/07/10 - 2016/03/31

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    院内感染の原因菌として知られる黄色ブドウ球菌は，様々な膜孔形成毒素を分泌し，宿主の血球細胞を破壊する．αヘモリジン（αHL）は膜孔形成毒素の一つであり，βバレル型の膜孔を形成して赤血球を破壊する．αHLは可溶性の単量体として分泌されるが，赤血球の膜上で会合してpreporeと呼ばれる中間体構造を形成した後，大きく構造変化して膜孔を形成する．膜孔形成機構の理解に向け，様々な膜孔形成毒素において，各段階の結晶構造解析が行われてきたが，αHLは膜孔へと会合しやすく，これまで，膜孔の構造のみしか決定されていなかった．そこで本研究では，過去の報告をもとに，安定な単量体およびpreporeを形成する変異体を取得し（H35A変異体およびW179A/R200A二重変異体），これら結晶構造解析によりαHLの一連の動的な構造変化を明らかにした．また，結晶構造解析の結果，Asp45-Tyr118間の水素結合，およびN末端アミノラッチ領域が機能発現に重要な役割を担う事が明らかになったため，これらの部位の変異体蛋白質を調製し，その膜孔形成活性を評価した．これらの結果に基づき，αHLの動的な膜孔形成機構を解明した．得られた成果を，Toxicon誌に報告したほか，第15回日本蛋白質科学会年会，生理学研究所 研究会「膜システムの機能的・構造的統合」，The 3rd International Life-Science Symposium，Hokkaido University / Pusan National University Joint Symposium 2016 in Busan，2015年度量子ビームサイエンスフェスタ・第7回MLFシンポジウム・第33回PFシンポジウムにて発表した．

  31. Bio-design for interface molecules in nano scale

    KUMAGAI IZUMI, UMETSU MITSUO, TSUMOTO KOUHEI, TANAKA YOSHIKAZU, MAKABE KOKI, ASANO RYUTARO

    Offer Organization: Japan Society for the Promotion of Science

    System: Grants-in-Aid for Scientific Research Grant-in-Aid for Specially Promoted Research

    Category: Grant-in-Aid for Specially Promoted Research

    Institution: Tohoku University

    2012 - 2016

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    An attainment of molecular and nano-scale manipulations are expected to open the way for making an effective linkage among biomolecules and cells, and for fabricating bottom-up structures from nanomaterials. Recently, we focus on antibodies which have high-binding affinity for a specific antigen in immune system, and we have identified the antibodies with high affinity for low organic molecules, receptors on cells, and inorganic surfaces by using molecular evolution techniques. In this study, we constructed a new method which enables to generate the antibodies against various materials in organic, inorganic, and biological fields, and the interface molecules were designed from the antibodies to spontaneously assemble cells or nanomaterials for application of cancer therapy and bottom-up fabrication of nano-devices.

  32. development of preparation of modified tRNA using protein

    TANAKA Yoshikazu

    Offer Organization: Japan Society for the Promotion of Science

    System: Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research

    Category: Grant-in-Aid for Challenging Exploratory Research

    Institution: Hokkaido University

    2012/04/01 - 2014/03/31

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    In this study, I tried to establish conventional method to prepare highly pure modified tRNA from E. coli cell using tRNA-protein complex spontaneously formed in the cells. First, I established preparation system for tRNA(Met) using E. coli tRNA(Met) acetyltransferase. For this purpose, I selected K205A mutant, which has high affinity for tRNA. Moreover, crystals of the purified tRNA(Met) were obtained. The crystals diffracted about 6-angstrom resolution at synchrotron radiation facility. Furthermore, co-expression system of selenium introducing enzyme with tRNA(Gln), tRNA(Glu), and tRNA(Lys) in E. coli were constructed, and pure tRNAs were prepared from the tRNA-protein complex spontaneously formed in the E. coli cell.

  33. ナノ世界のインターフェイス構築へのタンパク質工学的デザイン学

    熊谷 泉, 梅津 光央, 浅野 竜太郎, 田中 良和, 真壁 幸樹

    Offer Organization: 日本学術振興会

    System: 科学研究費助成事業 基盤研究(S)

    Category: 基盤研究(S)

    Institution: 東北大学

    2010 - 2012

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    本研究は、研究代表者がこれまで研究してきた機能抗体断片を構成要素として接合対象素材によらず目的素材間をナノレベルで正確に接合・アセンブリ制御できるバイオインターフェイス分子を創出するプラットフォーム技術を開発し、様々な生体分子・細胞・工学ナノ材料に応じたインターフェイス分子の迅速な適応・進化を可能にする接合分子デザイン工学を提言することを目的とする。そのために、本年度は、下記の研究を行った。 1.タンパク質フォーマットを利用した組換えバイオインターフェイス分子の構築 異なった機能を有する複数の免疫グロブリンフォールドを機能・構造単位と捕らえ、抗体分子骨格を母体として高次多重特異性を持つ組換えバイオインターフェイス分子を、X線結晶構造解析情報を基に作製するため、抗体の抗原結合ドメインのみをペプチドリンカーで融合した二重特異性抗体を大腸菌発現系で調製し、リンカーの設計によってタンパク質の発現量が変化することが分かった。 2.組換えバイオインターフェイス分子の構造・機能解析 1.より作製したバイオインターフェイス分子の構造解析をX線結晶解析などによって行う検討を始めた。細胞架橋用バイオインターフェイス分子については、単独結晶化による構造解析へ向けたサンプル調製の最適化のプロセスを設計した。また、ナノ工学材料特異的抗体断片に関しては、電子顕微鏡によるバイオインターフェイス分子による観察を行うために最適なサンプル調製を見出した。

  34. Structural and functional analysis of Uro-adherence factor A(UafA) from Staphylococcus saprophyticus

    TANAKA Yoshikazu

    Offer Organization: Japan Society for the Promotion of Science

    System: Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)

    Category: Grant-in-Aid for Young Scientists (B)

    Institution: Hokkaido University

    2009 - 2011

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    Pathogenic bacteria express several cell wall-anchored proteins to adhere to the surface of host cell, whereas Staphylococcus saprophyticus, a uropathogen involved in urinary tract infection of young females, possesses a unique cell wall-anchored protein, UafA. In this study, we investigated the adhesion mechanism of UafA by structure analysis and biochemical assay.

  35. ボツリヌス神経毒素の結晶構造解析と受容体結合様式の特殊性の解明

    塚本 健太郎, 小崎 俊司, 田中 良和

    Offer Organization: 日本学術振興会

    System: 科学研究費助成事業 特定領域研究

    Category: 特定領域研究

    Institution: 藤田保健衛生大学

    2009 - 2010

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    C型およびD型に分類される4種の毒素受容体結合ドメイン(Hc)について結晶構造と結合特異性についての解析を試みた。4種のHcのうち、DCモザイク毒素由来のOFD05Hcの立体構造をSe-SAD法にて2.8Aの分解能で決定した。しかし、N末端ドメインのフレキシビリティが高く、良好なモデルを構築することが出来なかった。そこで、N,C末端の各ドメインを別々に調製し、それぞれの構造を分子置換法により決定した上、これらの構造を上記のHcの構造に重ね合わせることにより、全体構造を構築した。明らかになった構造を機知のボツリヌス毒素のHcと比較したところ,本Hcには特徴的な長いループ領域の存在が確認された。このループには硫酸イオンが結合しており、機能発現における重要性が示唆された。また、他のボツリヌス毒素と同様にC末端領域にはポケット構造が存在していた。次に、立体構造をもとに31種類のAla残基置換体とループ領域の欠失変異体を作製し、その受容体結合活性を表面プラズモン共鳴と培養細胞への結合アッセイにより評価した。その結果、ループ領域とポケット周辺への変異導入により、結合活性が著しく減少することが明らかになった。更に、ガングリオシド中に含まれるSialyllactoseとの複合体の結晶構造を3.4Aの分解能で決定した。明らかになった構造において、単糖分子の電子密度が上記のポケット中に確認された。この部位は、他のボツリヌス毒素においてもガングリオシド結合ポケットとして機能することが報告されており、OFD05Hcにおいても同様の機構によりガングリオシドを認識することが示唆された。このことは、変異体解析の結果からも強く裏付けられる。一方、長いループ周辺には有意な電子密度は確認されず、このループが直接ガングリオシドを認識しているかどうか明らかにするためには、より詳細な解析が必要と考えられた。

  36. Replication and pathogenesis of plus strand RNA viruses and double strand RNA viruses

    NOMOTO Akio, TANIGUCHI Kouki, OHKA Seii, MUNAKATA Tsubasa, TANAKA Yoshikazu

    Offer Organization: Japan Society for the Promotion of Science

    System: Grants-in-Aid for Scientific Research

    Category: Grant-in-Aid for Scientific Research on Priority Areas

    2006 - 2010

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    Reverse genetics systems for poliovirus, Aichivirus, and hepatitis C virus were established. For these viruses, interaction between viral and host molecules were investigated to give an insight into molecular mechanisms of viral replication and pathogenesis. As for rotavirus whose reverse genetics is not completely established, mainly antigenic structure and cleavage region of viral VP4 were studied. At the same time, we made an effort to establish better reverse genetics system for rotavirus.

  37. Characterization of expression and adherent property of staphylococcal surface proteins associated with staphylococcus infectious diseases.

    KURODA Makoto

    Offer Organization: Japan Society for the Promotion of Science

    System: Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

    Category: Grant-in-Aid for Scientific Research (C)

    Institution: National Institute of Infectious Diseases

    2007 - 2008

  38. Structural and functional analysis of iron uptake related cell-wall anchored protein Isd from Staphylococcus aureus

    TANAKA Yoshikazu

    Offer Organization: Japan Society for the Promotion of Science

    System: Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)

    Category: Grant-in-Aid for Young Scientists (B)

    Institution: Hokkaido University

    2007 - 2008

  39. 黄色ブドウ球菌の莢膜合成関連蛋白質群の構造解析

    田中 良和

    Offer Organization: 日本学術振興会

    System: 科学研究費助成事業 若手研究(B)

    Category: 若手研究(B)

    2005 - 2006

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    本研究では,黄色ブドウ球菌由来の4種類の莢膜合成蛋白質,すなわち,CapE, CapF, CapG, CapLの結晶構造解析を目指したが,その中で,CapFの立体構造をSe-MAD法で2.6Åの分解能で決定することに成功した.明らかになった構造は,結晶学的に関連付けられた2量体構造であった.これはゲルろ過の結果とよく一致するものであるとともに,他の結晶系でも同様の2量体を形成していたことから,溶液中での本蛋白質の立体構造を反映するものと考えられる.単量体のCapFの構造は2つのドメイン(Nドメイン,Cドメイン)から構成されており,各ドメインの構造と類似した構造の蛋白質を検索したところ,NドメインはRm1D, CドメインはRm1Cという,いずれも莢膜を合成する蛋白質と類似していることが明らかになった.Rm1C, Rm1Dは一連の莢膜合成反応の中で,連続する2つの反応を触媒する酵素である.活性点について,CapFとRm1C, Rm1Dとを比較したところ,CapFにおいてもRm1C,Dと同様な基質結合ポケットが存在することが明らかになった.しかしながら,Rm1C, Rm1Dの触媒残基は保存されていなかったことから,同様な活性部位は有するが,そのメカニズムは異なると考えられる.さらに,His残基が3つ集中するHisクラスターが存在することが明らかになり,これらの残基の中心には金属イオンと思われる電子密度マップが確認された.蛋白質調製中には金属イオンは添加していないため,培地中の金属イオンを取り込んだものと推察される.今後,活性評価を行うことにより,これらの構造学的特徴が機能に及ぼす影響について考察する予定である.

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    2022/11/25 -

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    仙台市立吉成小学校で「東北大学出前授業」を行いました。「結晶をつくるとなにがわかるの？」をテーマに、実際にマイクロピペッターを用いて結晶を作成しました。

  8. 仙台市理科特別授業（小学５年生対象）「 塩以外にも結晶になるの だろうか」

    2022/11/11 -

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    仙台市立馬場小学校で「仙台市理科特別授業」を行いました。5年生向け特別授業「塩以外にも結晶になるのだろうか」で、自分たちで作成したタンパク質の結晶に子供達は見入っていました。

  9. 出前授業

    2017/11/30 -

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    聖ウルスラ学院英智高等学校 3年生を対象に，タンパク質科学に関する体験型の授業を行った．

  10. 自然科学カフェの集い

    2017/05/27 -

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    「分子の形を見るとわかること」という演題で，タンパク質科学研究に関する市民講演を行った．

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