Organization
School of Life Science and Technology Assistant Professor
Title
Assistant Professor
External link
KANAMARU SHUJI
Degree
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Doctor of Science ( 1999 Tokyo Institute of Technology )
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Master of Pharmaceutical Science ( 1996 Kyushu University )
Research Interests
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Bacteriophage
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Structural biology
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生物物理学
Research Areas
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Life Science / Biophysics
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Life Science / Structural biochemistry
Education
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Tokyo Institute of Technology Bioscience and Biotechnology
1996.4 - 1999.9
Country： Japan
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Kyushu University Graduate School of Pharmaceutical Sciences
1994.4 - 1996.3
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Kyushu University School of Pharmaceutical Sciences
1989.4 - 1994.3
Country： Japan
Research History
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Institute of Science Tokyo Assistant Professor
2004.7
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Tokyo Institute of Technology
2003 - 2004
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Purdue University Researcher
2002 - 2003
Country：United States
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Purdue University
1999 - 2002
Country：United States
Professional Memberships
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日本蛋白質科学会
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日本生化学会
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日本生物物理学会
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The Biophysical Society of Japan
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Protein Science Society of Japan
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The Japanese Biochemical Society
Papers
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Structural and functional insights into the interaction between a PP01 phage gp38 tail fiber tip and an Escherichia coli OmpC receptor. International journal
Haruka Terasaki, Aleksandar Zdravković, Tatsuya Niwa, Ayaka Washizaki, Marina Kawaguchi, Tetsuro Yonesaki, Shuji Kanamaru, Yuichi Otsuka
mBio e0211025 2026.1
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The Swi5-Sfr1 complex regulates Dmc1- and Rad51-driven DNA strand exchange proceeding through two distinct three-stranded intermediates by different mechanisms. International journal
Kentaro Ito, Takahisa Maki, Shuji Kanamaru, Masayuki Takahashi, Hiroshi Iwasaki
Nucleic acids research 52 ( 20 ) 12517 - 12533 2024.11
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Examination of yield, bacteriolytic activity and cold storage of linker deletion mutants based on endolysin S6_ORF93 derived from Staphylococcus giant bacteriophage S6. International journal
Sosuke Munetomo, Jumpei Uchiyama, Iyo Takemura-Uchiyama, Thamonwan Wanganuttara, Yumiko Yamamoto, Toshihiro Tsukui, Hideharu Hagiya, Shuji Kanamaru, Hideyuki Kanda, Osamu Matsushita
PloS one 19 ( 10 ) e0310962 2024
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Rapid and sensitive SARS-CoV-2 detection using a homogeneous fluorescent immunosensor Quenchbody with crowding agents. International journal
Bo Zhu, Nobuyuki Nosaka, Shuji Kanamaru, Jinhua Dong, Yancen Dai, Akihito Inoue, Yinghui Yang, Kaori Kobayashi, Tetsuya Kitaguchi, Hiroshi Iwasaki, Ryuji Koike, Kenji Wakabayashi, Hiroshi Ueda
The Analyst 147 ( 22 ) 4971 - 4979 2022.11
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Cell-free protein crystallization for nanocrystal structure determination. International journal
Satoshi Abe, Junko Tanaka, Mariko Kojima, Shuji Kanamaru, Kunio Hirata, Keitaro Yamashita, Ayako Kobayashi, Takafumi Ueno
Scientific reports 12 ( 1 ) 16031 - 16031 2022.10
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Heterogeneous IgE reactivities to Staphylococcus pseudintermedius strains in dogs with atopic dermatitis, and the identification of DM13-domain-containing protein as a bacterial IgE-reactive molecule. International journal
Iyo Takemura-Uchiyama, Hiroki Tsurui, Hidekatsu Shimakura, Tadahiro Nasukawa, Ichiro Imanishi, Jumpei Uchiyama, Tomoki Fukuyama, Shuji Sakamoto, Keiko Morisawa, Masato Fujimura, Hironobu Murakami, Shuji Kanamaru, Kenji Kurokawa, Keiko Kawamoto, Keita Iyori, Masahiro Sakaguchi
FEMS microbiology letters 369 ( 1 ) 2022.2
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A conserved Ctp1/CtIP C-terminal peptide stimulates Mre11 endonuclease activity. International coauthorship International journal
Aleksandar Zdravković, James M Daley, Arijit Dutta, Tatsuya Niwa, Yasuto Murayama, Shuji Kanamaru, Kentaro Ito, Takahisa Maki, Bilge Argunhan, Masayuki Takahashi, Hideo Tsubouchi, Patrick Sung, Hiroshi Iwasaki
Proceedings of the National Academy of Sciences of the United States of America 118 ( 11 ) e2016287118 - e2016287118 2021.3
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Structure and Function of the T4 Spackle Protein Gp61.3. International journal
Shuji Kanamaru, Kazuya Uchida, Mai Nemoto, Alec Fraser, Fumio Arisaka, Petr G Leiman
Viruses 12 ( 10 ) 2020.9
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Oxidation of aromatic and aliphatic aldehydes to carboxylic acids by Geotrichum candidum aldehyde dehydrogenase
Tomoyasu Hoshino, Emi Yamabe, Muhammad Arisyi Hawari, Mayumi Tamura, Shuji Kanamaru, Keisuke Yoshida, Afifa Ayu Koesoema, Tomoko Matsuda
TETRAHEDRON 76 ( 33 ) 2020.8
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Real-time tracking reveals catalytic roles for the two DNA binding sites of Rad51. International journal
Kentaro Ito, Yasuto Murayama, Yumiko Kurokawa, Shuji Kanamaru, Yuichi Kokabu, Takahisa Maki, Tsutomu Mikawa, Bilge Argunhan, Hideo Tsubouchi, Mitsunori Ikeguchi, Masayuki Takahashi, Hiroshi Iwasaki
Nature communications 11 ( 1 ) 2950 - 2950 2020.6
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Cooperative interactions facilitate stimulation of Rad51 by the Swi5-Sfr1 auxiliary factor complex. International journal
Bilge Argunhan, Masayoshi Sakakura, Negar Afshar, Misato Kurihara, Kentaro Ito, Takahisa Maki, Shuji Kanamaru, Yasuto Murayama, Hideo Tsubouchi, Masayuki Takahashi, Hideo Takahashi, Hiroshi Iwasaki
eLife 9 2020.3
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Rad51 Interaction Analysis Reveals a Functional Interplay Among Recombination Auxiliary Factors
Bilge Argunhan, Masayoshi Sakakura, Negar Afshar, Misato Kurihara, Kentaro Ito, Takahisa Maki, Shuji Kanamaru, Yasuto Murayama, Hideo Tsubouchi, Masayuki Takahashi, Hideo Takahashi, Hiroshi Iwasaki
2019.8
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RecA requires two molecules of Mg2+ ions for its optimal strand exchange activity in vitro Reviewed International journal
Kim Raeyeong, Kanamaru Shuji, Mikawa Tsutomu, Prevost Chantal, Ishii Kentaro, Ito Kentaro, Uchiyama Susumu, Oda Masayuki, Iwasaki Hiroshi, Kim Seog K, Takahashi Masayuki
Nucleic Acids Research 46 ( 5 ) 2548 - 2559 2018.3
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Crystal Structure of the Carboxy-Terminal Region of the Bacteriophage T4 Proximal Long Tail Fiber Protein Gp34. International journal
Meritxell Granell, Mikiyoshi Namura, Sara Alvira, Shuji Kanamaru, Mark J van Raaij
Viruses 9 ( 7 ) 2017.6
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Molecular assembly and structure of the bacteriophage T4 tail. International journal
Fumio Arisaka, Moh Lan Yap, Shuji Kanamaru, Michael G Rossmann
Biophysical reviews 8 ( 4 ) 385 - 396 2016.12
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Purification and characterization of fluorinated ketone reductase from Geotrichum candidum NBRC 5767 (vol 76, pg 13, 2013)
Chen Cao, Takuro Fukae, Takuro Yamamoto, Shuji Kanamaru, Tomoko Matsuda
BIOCHEMICAL ENGINEERING JOURNAL 93 309 - 310 2015.1
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NADH oxidase and alkyl hydroperoxide reductase subunit C (peroxiredoxin) from Amphibacillus xylanus form an oligomeric assembly. International journal
Toshiaki Arai, Shinya Kimata, Daichi Mochizuki, Keita Hara, Tamotsu Zako, Masafumi Odaka, Masafumi Yohda, Fumio Arisaka, Shuji Kanamaru, Takashi Matsumoto, Shunsuke Yajima, Junichi Sato, Shinji Kawasaki, Youichi Niimura
FEBS open bio 5 124 - 31 2015
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Intracellular Protein Delivery System with Protein Needle–GFP Construct Reviewed
Hiroshi Inaba, Nusrat J, M. Sanghamitra, Toshihiro Fukai, Takahiro Matsumoto, Kaname Nishijo, Shuji Kanamaru, Fumio Arisaka, Susumu Kitagawa, Takafumi Ueno
Chemistry Letters 43 ( 9 ) 1505 - 1507 2014.9
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Plasma membrane translocation of a protein needle based on a triple-stranded β-helix motif Reviewed International journal
Nusrat J, M. Sanghamitra, Hiroshi Inaba, Fumio Arisaka, Dan Ohtan Wang, Shuji Kanamaru, Susumu Kitagawa, Takafumi Ueno
Molecular BioSystems 10 ( 10 ) 2677 - 2683 2014.8
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Crystallization of the carboxy-terminal region of the bacteriophage T4 proximal long tail fibre protein gp34. International journal
Meritxell Granell, Mikiyoshi Namura, Sara Alvira, Carmela Garcia-Doval, Abhimanyu K Singh, Irina Gutsche, Mark J van Raaij, Shuji Kanamaru
Acta crystallographica. Section F, Structural biology communications 70 ( Pt 7 ) 970 - 5 2014.7
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Structure and properties of the C-terminal beta-helical domain of VgrG protein from Escherichia coli O157 Reviewed International journal
Kazuya Uchida, Petr G. Leiman, Fumio Arisaka, Shuji Kanamaru
JOURNAL OF BIOCHEMISTRY 155 ( 3 ) 173 - 182 2014.3
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Intermolecular interactions and conformation of antibody dimers present in IgG1 biopharmaceuticals Reviewed International journal
Takafumi Iwura, Jun Fukuda, Katsuyoshi Yamazaki, Shuji Kanamaru, Fumio Arisaka
JOURNAL OF BIOCHEMISTRY 155 ( 1 ) 63 - 71 2014.1
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Acetophenone reductase with extreme stability against a high concentration of organic compounds or an elevated temperature. International journal
Takuro Yamamoto, Yasuo Nakata, Chen Cao, Yosuke Sugiyama, Yoshihisa Asanuma, Shuji Kanamaru, Tomoko Matsuda
Applied microbiology and biotechnology 97 ( 24 ) 10413 - 21 2013.12
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Purification and characterization of fluorinated ketone reductase from Geotrichum candidum NBRC 5767
Chen Cao, Takuro Fukae, Takuro Yamamoto, Shuji Kanamaru, Tomoko Matsuda
BIOCHEMICAL ENGINEERING JOURNAL 76 13 - 16 2013.7
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Protein interactions in the assembly of the tail of bacteriophage T4. International journal
Fumio Arisaka, Shuji Kanamaru
Biophysical reviews 5 ( 2 ) 79 - 84 2013.6
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The Molecular Architecture of the Bacteriophage T4 Neck Reviewed International journal
Andrei Fokine, Zhihong Zhang, Shuji Kanamaru, Valorie D. Bowman, Anastasia A. Aksyuk, Fumio Arisaka, Venigalla B. Rao, Michael G. Rossmann
JOURNAL OF MOLECULAR BIOLOGY 425 ( 10 ) 1731 - 1744 2013.5
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Crystal structure of Cex1p reveals the mechanism of tRNA trafficking between nucleus and cytoplasm. Reviewed International journal
Nozawa K, Ishitani R, Yoshihisa T, Sato M, Arisaka F, Kanamaru S, Dohmae N, Mangroo D, Senger B, Becker HD, Nureki O
Nucleic acids research 41 ( 6 ) 3901 - 3914 2013.4
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Crystallographic Analysis Reveals Octamerization of Viroplasm Matrix Protein P9-1 of Rice Black Streaked Dwarf Virus Reviewed International journal
Akita Fusamichi, Higashiura Akifumi, Shimizu Takumi, Pu Yingying, Suzuki Mamoru, Uehara-Ichiki Tamaki, Sasaya Takahide, Kanamaru Shuji, Arisaka Fumio, Tsukihara Tomitake, Nakagawa Atsushi, Omura Toshihiro
Journal of Virology 86 ( 2 ) 746 - 756 2012.1
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Semi-synthesis of an artificial scandium(iii) enzyme with a β-helical bio-nanotube Reviewed International journal
Hiroshi Inaba, Shuji Kanamaru, Fumio Arisaka, Susumu Kitagawa, Takafumi Ueno
Dalton Transactions 41 ( 37 ) 11424 - 11427 2012
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Screw motion regulates multiple functions of T4 phage protein gene product 5 during cell puncturing. Reviewed International journal
Nishima W, Kanamaru S, Arisaka F, Kitao A
Journal of the American Chemical Society 133 ( 34 ) 13571 - 13576 2011.8
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Dual modification of a triple-stranded β-helix nanotube with Ru and Re metal complexes to promote photocatalytic reduction of CO2 Reviewed International journal
Norihiko Yokoi, Yuki Miura, Chen-Yuang Huang, Nobuyuki Takatani, Hiroshi Inaba, Tomomi Koshiyama, Shuji Kanamaru, Fumio Arisaka, Yoshihito Watanabe, Susumu Kitagawa, Takafumi Ueno
Chemical Communications 47 ( 7 ) 2074 - 2076 2011.1
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Morphogenesis of the T4 tail and tail fibers. International journal
Petr G Leiman, Fumio Arisaka, Mark J van Raaij, Victor A Kostyuchenko, Anastasia A Aksyuk, Shuji Kanamaru, Michael G Rossmann
Virology journal 7 355 - 355 2010.12
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Construction of robust bio-nanotubes using the controlled self-assembly of component proteins of bacteriophage T4 Reviewed International journal
Norihiko Yokoi, Hiroshi Inaba, Makoto Terauchi, Adam Z. Stieg, Nusrat J, M. Sanghamitra, Tomomi Koshiyama, Katsuhide Yutani, Shuji Kanamaru, Fumio Arisaka, Tatsuo Hikage, Atsuo Suzuki, Takashi Yamane, James K. Gimzewski, Yoshihito Watanabe, Susumu Kitagawa, Takafumi Ueno
Small 6 ( 17 ) 1873 - 1879 2010.9
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Zernike phase contrast cryo-electron tomography. International journal
Radostin Danev, Shuji Kanamaru, Michael Marko, Kuniaki Nagayama
Journal of structural biology 171 ( 2 ) 174 - 81 2010.8
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Purification and characterization of acetophenone reductase with excellent enantioselectivity from Geotrichum candidum NBRC 4597. International journal
Yasuo Nakata, Takuro Fukae, Ryoji Kanamori, Shuji Kanamaru, Tomoko Matsuda
Applied microbiology and biotechnology 86 ( 2 ) 625 - 31 2010.3
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The Baseplate Wedges of Bacteriophage T4 Spontaneously Assemble into Hubless Baseplate-Like Structure In Vitro Reviewed International journal
Yap M. L, Mio K, Leiman P. G, Kanamaru S, Arisaka F
Journal of Molecular Biology 395 ( 2 ) 349 - 360 2010
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Sequential Assembly of the Wedge of the Baseplate of Phage T4 in the Presence and Absence of Gp11 as Monitored by Analytical Ultracentrifugation Reviewed International journal
Yap M. L, Mio K, Ali S, Minton A, Kanamaru S, Arisaka F
Macromolecular Bioscience 10 ( 7 ) 808 - 813 2010
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Construction of an energy transfer system in the bio-nanocup space by heteromeric assembly of gp27 and gp5 proteins isolated from bacteriophage T4. Reviewed International journal
Koshiyama T, Ueno T, Kanamaru S, Arisaka F, Watanabe Y
Organic & biomolecular chemistry 7 ( 12 ) 2649 - 2654 2009.6
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Structural similarity of tailed phages and pathogenic bacterial secretion systems
Shuji Kanamaru
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 106 ( 11 ) 4067 - 4068 2009.3
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ORF334 in Vibrio phage KVP40 plays the role of gp27 in T4 phage to form a heterohexameric complex
Mai Nemoto, Kazuhiro Mio, Shuji Kanamaru, Fumio Arisaka
JOURNAL OF BACTERIOLOGY 190 ( 10 ) 3606 - 3612 2008.5
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Molecular design of heteroprotein assemblies providing a bionanocup as a chemical reactor
Tomomi Koshiyama, Norihiko Yokoi, Takafumi Ueno, Shuji Kanamaru, Shingo Nagano, Yoshitsugit Shiro, Fumio Arisaka, Yoshihito Watanabe
SMALL 4 ( 1 ) 50 - 54 2008.1
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Domain interaction analyses of gp7, gp10 and gp11 of bacteriophage T4 for crystallization
Shuji Kanamaru, Tomoko Nakao, Tatsuya Nagao, Fumio Arisaka
ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES 64 C339 - C339 2008
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From structure of the complex to understanding of the biology
Michael G. Rossmann, Fumio Arisaka, Anthony J. Battisti, Valorie D. Bowman, Paul R. Chipman, Andrei Fokine, Susan Hafenstein, Shuji Kanamaru, Victor A. Kostyuchenko, Vadim V. Mesyanzhinov, Mikhail M. Shneider, Marc C. Morais, Petr G. Leiman, Laura M. Palermo, Colin R. Parrish, Chuan Xiao
ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY 63 9 - 16 2007.1
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The neck of bacteriophage T4 is a ring-like formed by a hetero-oligomer of gp13 and structure gp14 Reviewed International journal
Akhter T, Zhao L, Kohda A, Mio K, Kanamaru S, Arisaka F
Biochimica Et Biophysica Acta-Proteins and Proteomics 1774 ( 8 ) 1036 - 1043 2007
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Association and dissociation of the cell puncturing complex of bacteriophage T4 is controlled by both pH and temperature. International journal
Subodh Kumar Sarkar, Yoko Takeda, Shuji Kanamaru, Fumio Arisaka
Biochimica et biophysica acta 1764 ( 9 ) 1487 - 92 2006.9
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Association and dissociation of the cell puncturing complex of bacteriophage T4 is controlled by both pH and temperature
Subodh Kumar Sarkar, Yoko Takeda, Shuji Kanamaru, Fumio Arisaka
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 1764 ( 9 ) 1487 - 1492 2006.9
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Bionanotube tetrapod assembly by in situ synthesis of a gold nanocluster with (Gp5-His6)3 from bacteriophage T4
Takafumi Ueno, Tomonn Koshiyama, Toshimitsu Tsuruga, Toshiaki Goto, Shuji Kanamaru, Fumio Arisaka, Yoshihito Watanabe
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 45 ( 27 ) 4508 - 4512 2006
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3D Rearrangement of Proteins in the Tail of Bacteriophage T4 on Infection of its Host
Michael G. Rossmann, Petr G. Leiman, Paul R. Chipman, Victor A. Kostyuchenko, Shuji Kanamaru, Fumio Arisaka, Vadim V. Mesyanzhinov
ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES 61 C75 - C76 2005
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Conformational switching by the scaffolding protein D directs the assembly of bacteriophage phiX174. International journal
Marc C Morais, Megan Fisher, Shuji Kanamaru, Laralynne Przybyla, John Burgner, Bentley A Fane, Michael G Rossmann
Molecular cell 15 ( 6 ) 991 - 7 2004.9
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The functional domains of bacteriophage t4 terminase. International journal
Shuji Kanamaru, Kiran Kondabagil, Michael G Rossmann, Venigalla B Rao
The Journal of biological chemistry 279 ( 39 ) 40795 - 801 2004.9
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Three-dimensional structure of bacteriophage T4 baseplate. International journal
Victor A Kostyuchenko, Petr G Leiman, Paul R Chipman, Shuji Kanamaru, Mark J van Raaij, Fumio Arisaka, Vadim V Mesyanzhinov, Michael G Rossmann
Nature structural biology 10 ( 9 ) 688 - 93 2003.9
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Bacteriophage phi29 scaffolding protein gp7 before and after prohead assembly. International journal
Marc C Morais, Shuji Kanamaru, Mohammed O Badasso, Jaya S Koti, Barbara A L Owen, Cynthia T McMurray, Dwight L Anderson, Michael G Rossmann
Nature structural biology 10 ( 7 ) 572 - 6 2003.7
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P15 and P3, the tail completion proteins of bacteriophage T4, both form hexameric rings. International journal
Li Zhao, Shuji Kanamaru, Chatree'chalerm Chaidirek, Fumio Arisaka
Journal of bacteriology 185 ( 5 ) 1693 - 700 2003.3
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The tail lysozyme complex of bacteriophage T4. International journal
Fumio Arisaka, Shuji Kanamaru, Petr Leiman, Michael G Rossmann
The international journal of biochemistry & cell biology 35 ( 1 ) 16 - 21 2003.1
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[The structural biology and infection mechanism of bacteriophage T4].
Shuji Kanamaru, Fumio Arisaka
Seikagaku. The Journal of Japanese Biochemical Society 74 ( 2 ) 131 - 5 2002.2
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Structure of the cell-puncturing device of bacteriophage T4. International journal
Shuji Kanamaru, Petr G Leiman, Victor A Kostyuchenko, Paul R Chipman, Vadim V Mesyanzhinov, Fumio Arisaka, Michael G Rossmann
Nature 415 ( 6871 ) 553 - 7 2002.1
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The C-terminal fragment of the precursor tail lysozyme of bacteriophage T4 stays as a structural component of the baseplate after cleavage. International journal
S Kanamaru, N C Gassner, N Ye, S Takeda, F Arisaka
Journal of bacteriology 181 ( 9 ) 2739 - 44 1999.5
MISC
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バクテリオファージの構造と感染機構
金丸周司, 有坂文雄
蛋白質 核酸 酵素 50 ( 10 ) 1341 - 1348 2005
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Structural Analysis of the Baseplate of Bacteriophage T4 and Elucidation of the Infection Mechanism
KANAMARU Shuji, LEIMAN Petr, ARISAKA Fumio
X-RAYS 45 ( 1 ) 48 - 53 2003
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The Syringe of T4 Tail Has Needle
KANAMARU Shuji, ARISAKA Fumio
Seibutsu Butsuri 43 ( 1 ) 21 - 24 2003
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Ｔ４ファージの構造生物学と感染のメカニズム
金丸周司, 有坂文雄
生化学 74 ( 2 ) 131 - 135 2002
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processing of T4 tall lysozyme and its domein structure.
Kanamaru S., Arisaka F.
Biophysics 38 ( 2 ) S44 1998.9
Presentations
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Shuji Kanamaru, Kazuya Uchida, Takahiro Momiyama, Kaname Nishijo, Fumio Arisaka
Seibutsu Butsuri 2014 The Biophysical Society of Japan General Incorporated Association
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Iura Takafumi, Fukuda Jun, Yamazaki Katsuyoshi, Kanamaru Shuji, Arisaka Fumio
Seibutsu Butsuri 2013 The Biophysical Society of Japan General Incorporated Association
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Uchida Kazuya, Kanamaru Shuji, Leiman Petr, Arisaka Fumio
Seibutsu Butsuri 2013 The Biophysical Society of Japan General Incorporated Association
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Kanamaru Shuji, Namura Mikiyoshi, Arisaka Fumio
Seibutsu Butsuri 2013 The Biophysical Society of Japan General Incorporated Association
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Nishima Wataru, Kanamaru Shuji, Arisaka Fumio, Kitao Akio
Biophysics 2007.11 The Biophysical Society of Japan
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Protein-protein Interactions in the assembly of the Baseplate of Bacteriophage T4
Takeda Y, Kanamaru S, Arisaka F
Seibutsu Butsuri 2000 The Biophysical Society of Japan General Incorporated Association
Awards
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手島研究論文賞
2003
Research Projects
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改変型人工抗菌酵素による薬剤耐性グラム陰性桿菌感染症の治療基盤の確立
Grant number：25K02692 2025.4 - 2028.3
日本学術振興会 科学研究費助成事業 基盤研究(B)
内山 淳平, 西藤 公司, 高井 まどか, 金丸 周司
Grant amount：\18200000 （ Direct Cost: \14000000 、 Indirect Cost：\4200000 ）
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Structural biology of phage long tail fiber protein and host recognition
Grant number：25440066 2013.4 - 2017.3
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
Kanamaru Shuji
Grant amount：\4810000 （ Direct Cost: \3700000 、 Indirect Cost：\1110000 ）
We over-expressed, purified and crystallized a C-terminal 564 residues of gp34, proximal long tail fiber, of bacteriophage T4. A determined crystal structure of gp34C774-1289 is trimeric fibrous protein which is 18nm long. In detail, there are three-stranded beta-helix and beta-prism domains which are found in other phage tail protein. Thus we conclude that the phage tail proteins are evolved with utilizing the “tail parts domains” to obtain their ability for their infection.
We also purified gp38 as a complex with C-terminal portion of gp37 by co-expression system. -
ファージの重複感染シグナルがもたらす溶菌阻止現象の分子メカニズムの解明
Grant number：23121538 2011.4 - 2013.3
日本学術振興会 科学研究費助成事業 新学術領域研究(研究領域提案型)
金丸 周司
Grant amount：\1430000 （ Direct Cost: \1100000 、 Indirect Cost：\330000 ）
1.gpT-62CTDの発現・精製
gpTのC末端側のペリプラズムドメイン(CTD)のN末端にpelBのシグナル配列を付加したgpT-CTDは精製過程で62残基目のリジンで一部切断が起きていることが確認された。そこで、gpTの残基目のリジンからC末端側のペリプラズムドメイン(62CTD)のN末端にpelBのシグナル配列を付加したgpT-62CTDの大腸菌における大量発現系を構築した。gpT-CTDと同様に不溶性各分として回収されたgpT-62CTDは、pH9.5, 1.2Mアルギニンの条件で可溶化することに成功した。
2. gpT-62CTD, gpRI-CTD複合体の結晶化
pH9.5, 1.2Mアルギニンの条件で可溶化したgpT-62CTDに精製したgpRI-CTDを添加しその後アルギニンを透析により徐々に除くことにより、gpT-62CTD_gpRI-CTD複合体を得ることに成功した。超遠心分析によりこの精製標品は溶液中で2：2の複合体とその重合体が平衡して存在することが示された。また、pHを9.5からpH7.5にすると、2：2の複合体が3量化しているもののみが存在することが分かった。さらに、この複合体について結晶化を試み、gpT-CTD_gpRI-CTD複合体とは異なった条件で結晶化に成功した。
3. gpT-62CTD, gpRI-CTD複合体の結晶構造解析
gpT-62CTD_gpRI-CTD複合体の結晶構造解析に成功し、2.4Å分解能の複合体の結晶構造を得ることができた。その結果、溶液中の基本単位の2：2の複合体と思われるユニットを結晶中に見出すことができた。また、gpRIはgpTの周囲の3か所に結合しgpTの重合を阻害していることが示唆された。 -
蛋白質ケージを用いたウイルス様粒子作成技術基盤の確立－SARS-Cov2モデル－
Grant number：21K05268 2021.4 - 2024.3
日本学術振興会 科学研究費助成事業 基盤研究(C)
金丸 周司
Grant amount：\4160000 （ Direct Cost: \3200000 、 Indirect Cost：\960000 ）
本研究では、ウイルスの検査試薬・ウイルスの構造解析・ワクチン開発等に利用するために高い熱安定性・pH安定性を有する蛋白質ケージ（Scaffold）を用いてウイルス様粒子（VLP）を作成することを目的としている。
現在、モデル分子を用いて大腸菌での発現系の構築と精製法の確立のために必要な技術基盤開発を行っている。
現在までのところ、モデル分子のフラグメント化や大腸菌発現条件の検討、分子シャペロン等の共発現などを行っているが期待する可溶性のVLPは得られていない。
今後は、引き続き大腸菌での発現条件の検討を行うとともに、他の生物種での発現を検討している。そのための、学内での遺伝子組換え実験の変更を行っている。 -
バクテリオファージに学ぶ発動分子システムの創成
Grant number：18H05421 2018.6 - 2023.3
日本学術振興会 科学研究費助成事業 新学術領域研究(研究領域提案型)
上野 隆史, 金丸 周司
Grant amount：\72930000 （ Direct Cost: \56100000 、 Indirect Cost：\16830000 ）
生命環境では、様々なタンパク質が特異な集合構造を形成し、自発的に機動する分子機関を構築されている。本研究では、代表者が開発した、T4ファージ由来の全長15nmのタンパク質針を基盤とし、新しい分子機関の合成をめざした。具体的には、分子機関としてのタンパク質針精密設計として、タンパク質針の結晶構造やHSAFMの実験結果と分子動力学計算を元に、分子機関としての機能を精密に制御したタンパク質針を構築し、タンパク質針をユニットとしてタンパク質針を階層的に集積し、アクティブマターとしての新しい機能の発現を目指した。高速AFMの実験結果を数理モデルを用いた解析により理解することによって、理論計算のモデルを構築し新たな集合体形成のメカニズム解明へとつなげることができた。さらに、これらの結果を踏まえ、タンパク質針の集団運動の制御に向けた精密分子設計の指針ができた。
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フェリチンを用いた新規「蛋白質ケージ構造解析法」の技術基盤の確立
Grant number：18K05314 2018.4 - 2023.3
日本学術振興会 科学研究費助成事業 基盤研究(C)
金丸 周司
Grant amount：\4420000 （ Direct Cost: \3400000 、 Indirect Cost：\1020000 ）
本研究では、高い熱安定性・pH安定性を有する蛋白質ケージ（Scaffold）のもつ直径約8nmの内部空洞に標的蛋白質を閉じ込め、粒子画像抽出が容易なScaffoldごとクライオ電顕単粒子解析法によって立体構造を決定すること、そのために必要な技術基盤を構築することを目的としている。
現在までに、モデル分子を用いて発現ベクターの構築から、精製、電顕観察、画像解析までを繰り返し、必要な技術基盤開発を行っている
Scaffold蛋白質と標的蛋白質の発現量を調節し効率よくScaffold-標的蛋白質複合体を形成させることは可能となった。また、適切な精製タグを用いることでScaffold-標的蛋白質複合体を高純度で精製することに成功した。
しかし、構造解析を行うとScaffoldの構造は比較的高分解能で決まるものの、標的蛋白質はScaffold内部で様々な方向を向き本研究のめざす分解能の構造が得られていない。そこで、Scaffoldと標的蛋白質を繋ぐリンカーを種々のαヘリックス形成配列に置換し、標的蛋白質を含まないケージを作成し結晶構造解析にて評価した。ところが、ケージの高い対称性と異なる対称要素を持つため、もしくは期待した硬い構造を形成できないために、いずれの場合もリンカー部分の電子密度を観察できなかった。そこで、Scaffold外側に画像平均化のための目印となる蛋白質を付加することで、Scaffold-標的蛋白質複合体の構造解析を容易にするような方法で解決しようとしている。
また、内部空洞の小さい蛋白質ケージを用いた標的蛋白質の固定化も継続して行っている。 -
Structure and assembly of the central hub of the baseplate of bacteriophage T4
Grant number：23570190 2011 - 2013
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
ARISAKA Fumio, KANAMARU Shuji
Grant amount：\5460000 （ Direct Cost: \4200000 、 Indirect Cost：\1260000 ）
1. Tube initiator protein, gp48, which were fused with SlyD at the N-terminus was expressed in a soluble fraction and crystallized. 2. The C-terminal domain of gp37 from phage PP01 which constitutes the tail fiber together with gp38 were co-expressed and the complex with 3:1 stoichiometry was isolated and crystallized. It was shown that the C-terminal 13 kDa polypeptide was self-cleaved. 3. The complex of the soluble domain of the holin and the rapid lysis protein gp rI was crystallized and the structure has been successfully determined, which is expected to contribute to understanding the mechanisma of lysis inhibition.
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Structural analysis of novel type 6 secretion apparatus proteins
Grant number：21770164 2009 - 2011
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)
KANAMARU Shuji
Grant amount：\3380000 （ Direct Cost: \2600000 、 Indirect Cost：\780000 ）
To determine the crystal structure of VgrG protein of Type 6 secresion apparatus from E. coli O157 and E. coli CFT073, over-expression vectors for both full-length and C-terminal domain of VgrG were constructed. We succeeded in establish the purification methods for VgrG from E. coli O157. Purified protein was treated with trypsin to get stable VgrG1^M467-his. This VgrG1^M467-his was examined thousands of crystallization condition. One of the conditions gave nice crystal formation for structure determination. Now we are trying get isomorphous heavy atom derivatives for phase determination. Moreover, we are going to establish other VgrG purification strategy.
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Structure Formation of the Neck which Links the Head and the Tail of Bacteriophage
Grant number：18570147 2006 - 2007
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
ARISAKA Fumio, KANAMARU Shuji
Grant amount：\4110000 （ Direct Cost: \3600000 、 Indirect Cost：\510000 ）
During the assembly of phage T4, the neck is formed to block the DNA leakage and to prepare for the tail association after DNA packaging into the head is completed. The neck is composed of gp13 (gp: gene product) and gp14 each consists of 309 and 256 amino acid residues with the molecular weight of 41,226 and 32,868, respectively. In order to elucidate the structure and the subunit arrangement of the neck, genes coding for the two proteins were cloned by PCR method, an overexpression system was constructed and the genes were induced by IPTG. The two proteins were purified by ammonium sulfate fractionation, ion exchange chromatography, gen filtration as a single band in SDS-PAGE. Far-ultraviolet circular dichroism spectra of the two proteins indicated that gp13 is rich in α-helix, whereas gp13 is rich in β-sheet. On the other hand, sedimentation velocity indicated that both proteins are monomers with the sedimentation coefficient of 2.80S and 2.00S, respectively and that the frictional ratios were 1.22 and 1.73, respectively, which indicated that gp14 is more elongated than gp13. The two proteins did not interact each other under normal physiological conditions, but it was found that the two proteins formed a specific complex with the sedimentation coefficient of 16.6S. The complex had the molecular weight of 497,000 which together with the molar ratio of 2:1 based on SDS-PAGE indicated that the complex is a hetero 15-mer, (gp 13)10(gp 14)5. This complex is considered to be identical with the complex which was observed when the two proteins were co-expressed. Electron microscopic observations revealed that the complex formed a ring with the diameter of 156 Å which coincided with the electron density of the three-dimensional image of the neck reconstructed from electron micrograph.
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原子レベルでの構造解析に基づくウィルスのDNAパッケージングモーターの動作基盤
Grant number：17687014 2005 - 2007
日本学術振興会 科学研究費助成事業 若手研究(A)
金丸 周司
Grant amount：\19370000 （ Direct Cost: \14900000 、 Indirect Cost：\4470000 ）
本研究は、T4ファージを対象として、DNAがウィルス頭殻に詰め込まれる現象を原子レベルで明らかにしようとするものである。本年度の成果は下記の通りである:
1.ネック蛋白質の発現・精製
ネック蛋白質(gp13,gp14)について、発現・精製を行った。gp13,gp14はそれぞれ、単独で精製することに成功し、それぞれが単量体で存在することがわかった。また、0.4M以上濃度の硫酸アンモニウム存在下で二つの蛋白質を混合すると、(gp13)_<10>-(gp14)_5からなる複合体が形成することがわかった。
2.ネック蛋白質の物理化学的解析
上記(gp13)_<10>-(gp14)_5の電子顕微鏡観察、超遠心分析を行いこの複合体が溶液中で均一のリング状の構造を持ち、ファージ粒子中に存在する複合体であることを示した。
3.結晶化、構造解析
gp13,(gp13)_<10>-(gp14)_5について、結晶化に適した精製試料を用いて結晶化を試みている。具体的には、市販のグローバルな結晶化条件スクリーニングキットを用いて、蒸気拡散法により結晶化条件の初期検索を行っている。現在、構造解析に向けて充分な質と大きさを持った結晶を得るための条件を探索中である。 -
Assembly and Mechanism of Infection of Bacteriophage
Grant number：16087204 2004 - 2009
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Priority Areas
ARISAKA Fumio, KANAMARU Shuji, TAKEDA Shigeki, MOH LAN Yap, NEMOTO Mai, TAHMINA Akhter, SUBODH Sarkar, ABURAI Kohei, MONZAKI Yasunori, OKUDA Mako, UCHIDA Kazuya, NAMURA Mikiyoshi, TERAUCHI Makoto, ITO Masako, HORIKOSHI Yusuke, FUJITA Daigo, SUGAI Shinnji, NAGAO Tatsuya, DONNY Sunanda, NAKAO Tomoko
Grant amount：\91800000 （ Direct Cost: \91800000 ）
All the subunit proteins were localized in the baseplate before and after infection by a combination of X-ray crystallography and three-dimensional image econstruction from electron-micrographs. As a result, the gross structural change of the baseplate upon infection can be interpreted by the change of the relative positions of the subunits. Also, a new method was developed, which indicated that the stringent sequential assembly of the baseplate wedge is based on the induced-fit of the subunits upon binding.
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T4ファージの粒子形成に関わる分子間相互作用の解明
Grant number：16041213 2004 - 2005
日本学術振興会 科学研究費助成事業 特定領域研究
有坂 文雄, 金丸 周司
Grant amount：\6000000 （ Direct Cost: \6000000 ）
本研究は、蛋白質複合体のモデル系としてバクテリオファージに注目し、分子集合の過程における水溶液中の分子間相互作用を明らかにしようとするものである。本年度は特に基盤蛋白質gp13,gp14,gp16,gp17,gp48,gp54に焦点を合わせて実験を行い、以下の結果を得た。
1.gp13及びgp14の単離・精製と性状解析
gp13とgp14はネックを構成する蛋白質で頭部形成の最終段階で結合し、尻尾との結合部に存在すると考えられる。両蛋白質は超遠心分析の結果、単独では単量体として存在すること、また両者を混合するとgp13:gp14=1:2の比率で結合することが分かった。
2.DNAパッケージング蛋白質gp16とgp17の単離・精製と性状解析
2本鎖DNAファージには共通して大小2種のパッケージング蛋白質が必要である。T4ファージでは大サブユニットがgp17、小サブユニットがgp16で、後者にはATPaseはないが、前者のATPaseを50倍以上促進する。両蛋白質を単離精製し、超遠心分析を行ったところ、gp17が単量体であるのに対して、gp16は20量体と思われる分子種が主要成分で、少量の10量体と思われる分子種と平衡にあることが分かった。一方、gp16とgp17の間には弱い相互作用しか見られなかった。
3.基盤形成の最終ステップに結合するgp48とgp54の単離・精製と性状解析
gp48とgp54は基盤形成の最終段階で結合する蛋白質で、gp54は尾管イニシエーターを呼ばれる。両蛋白質遺伝子をクローニングし、単離精製することを試みたが、良く発現はするものの、不溶性画分に入り、発現温度、IPTG濃度など発現条件を検討したほか、コールドショックプロモーターを試みたが、可溶性画分として精製することができなかった。しかし、gp48については発現温度22℃、15時間で半分以上を可溶性画分に回収することができた。現在、結晶化の準備を進めている。 -
Structure and Assembly of the Contractile Tail of Bacteriophage
Grant number：15370065 2003 - 2005
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
ARISAKA Fumio, KANAMARU SHUJI, TAKEDA SHIGEKI
Grant amount：\14900000 （ Direct Cost: \14900000 ）
The purpose of the present study is to elucidate the mechanism of assembly and function of bacteriophage as a model system in terms of its molecular and atomic level. The major results we obtained were the following :
The most important progress in elucidating the mechanism of the structural transformation of the contractile tail of phage T4 came from the collaboration with Michael Rossmann's group at Purdue. Leiman in Rossmann's group succeeded in determining the low resolution structure of the contracted tail by 3D image reconstruction from cryo-EM. The fact that the same high resolution structure of the subunits can be fitted into the electron density of the contracted conformation indicates that the subunits themselves do not change their conformation significantly, but change the relative positions in the baseplate during conformational change of the baseplate. Using the extended and contracted images of the tail and interpolation, we were able obtain a reasonably detailed model of how the subunit rearrangement takes place. Gp11 strongly binds to the domain III and the N-terminus of gp12 which is the short tail fiber binds to domain IV of gp10 before structural change. The protein which plays a key role during the "hexagon" to "star" conformational change of the baseplate is gp10. Upon infection, as gp10 rotates around the longitudinal axis, gp11 which had been bound to gp12 now binds to gp34, the proximal long tail fiber, and the short tail fiber extends out from the bottom of the baseplate.
Secondly, we have succeeded in elucidating the tail lysozyme (gp5) structure, where the amino acid at 351^<st> position was replaced with seven different amino acids. One of them, S351L, of which the cleavage was completely suppressed, was expressed, purified and crystallized for X-ray diffraction. The structure of the S351L mutant showed that the cleavage site was highly exposed, as would be required for efficient digestion during phage maturation. Since the structure of gp5 does not resemble any known protease, the cleavage is most likely not autocatalytic, but the result of an E.coli protease. The mutant structure further demonstrates that the inhibition peptide from the neighboring subunit is already at the substrate recognition site prior to cleavage.