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Affiliation Department |
生命・応用化学科 生命・物質化学分野
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NISHIKINO Tatsuro
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Research Interests
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エネルギー変換
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イオンチャネル
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クライオ電子顕微鏡
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赤外分光法
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細菌べん毛モーター
From School
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Nagoya University Graduate School of Science Graduated
2015.04 - 2020.02
Country：Japan
External Career
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大阪大学 蛋白質研究所 日本学術振興会特別研究員(PD)
2020.04 - 2023.03
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名古屋工業大学大学院工学研究科 特任助教
2023.04 - 2024.03
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Nagoya Institute of Technology Assistant Professor
2024.04
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Nagoya Institute of Technology OptoBioTechnology Research Cemter
2024.04
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名古屋大学 大学院 理学研究科 日本学術振興会特別研究員(PD)
2020.02 - 2020.03
Papers
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Low-temperature FTIR spectroscopy of the L/Q switch of proteorhodopsin. Reviewed International journal
Tatsuro Nishikino, Teppei Sugimoto, Hideki Kandori
Physical chemistry chemical physics : PCCP 26 ( 35 ) 22959 - 22967 2024.09
Authorship：Lead author Language：English Publishing type：Research paper (scientific journal)
Rhodopsins are photoreceptive membrane proteins containing a retinal chromophore, and the color tuning mechanism in rhodopsins is one of the important topics. Color switch is a color-determining residue at the same position, where replacement of red- and blue-shifting amino acids in two wild-type rhodopsins causes spectral blue- and red-shifts, respectively. The first and most famous color switch in microbial rhodopsins is the L/Q switch in proteorhodopsins (PRs). Green- or blue-absorbing PR (GPR or BPR) contains Leu and Gln at position 105 of the C-helix (TM3), respectively, and their replacement converted absorbing colors. The L/Q switch enables bacteria to absorb green or blue light in shallow or deep ocean waters, respectively. Although Gln and Leu are hydrophilic and hydrophobic residues, respectively, a comprehensive mutation study of position 105 in GPR revealed that the λmax correlated with the volume of residues, not the hydropathy index. To gain structural insights into the mechanism, we applied low-temperature FTIR spectroscopy of L105Q GPR, and the obtained spectra were compared with those of GPR and BPR. The difference FTIR spectra of L105Q GPR were similar to those of BPR, not GPR, implying that the L/Q switch converts the GPR structure into a BPR structure in terms of the local environments of the retinal chromophore. It includes retinal skeletal vibration, hydrogen-bonding strength of the protonated Schiff base, amide-A vibration (peptide backbone), and protein-bound water molecules. Consequently color is switched accompanying such structural alterations, and known as the L/Q switch.
DOI： 10.1039/d4cp02248c
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Changes in the hydrophobic network of the FliGMC domain induce rotational switching of the flagellar motor. Reviewed International journal
Tatsuro Nishikino, Atsushi Hijikata, Seiji Kojima, Tsuyoshi Shirai, Masatsune Kainosho, Michio Homma, Yohei Miyanoiri
iScience 26 ( 8 ) 107320 - 107320 2023.08
Language：English Publishing type：Research paper (scientific journal)
The FliG protein plays a pivotal role in switching the rotational direction of the flagellar motor between clockwise and counterclockwise. Although we previously showed that mutations in the Gly-Gly linker of FliG induce a defect in switching rotational direction, the detailed molecular mechanism was not elucidated. Here, we studied the structural changes in the FliG fragment containing the middle and C-terminal regions, named FliGMC, and the switch-defective FliGMC-G215A, using nuclear magnetic resonance (NMR) and molecular dynamics simulations. NMR analysis revealed multiple conformations of FliGMC, and the exchange process between these conformations was suppressed by the G215A residue substitution. Furthermore, changes in the intradomain orientation of FliG were induced by changes in hydrophobic interaction networks throughout FliG. Our finding applies to FliG in a ring complex in the flagellar basal body, and clarifies the switching mechanism of the flagellar motor.
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Site-Specific Isotope Labeling of FliG for Studying Structural Dynamics Using Nuclear Magnetic Resonance Spectroscopy Reviewed
Tatsuro Nishikino, Yohei Miyanoiri
Methods in Molecular Biology 57 - 70 2023.02
Publishing type：Part of collection (book) Publisher：Springer US
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The flagellar motor of Vibrio alginolyticus undergoes major structural remodeling during rotational switching. Reviewed International journal
Brittany L Carroll, Tatsuro Nishikino, Wangbiao Guo, Shiwei Zhu, Seiji Kojima, Michio Homma, Jun Liu
eLife 9 2020.09
Language：English Publishing type：Research paper (scientific journal)
The bacterial flagellar motor switches rotational direction between counterclockwise (CCW) and clockwise (CW) to direct the migration of the cell. The cytoplasmic ring (C-ring) of the motor, which is composed of FliG, FliM, and FliN, is known for controlling the rotational sense of the flagellum. However, the mechanism underlying rotational switching remains elusive. Here, we deployed cryo-electron tomography to visualize the C-ring in two rotational biased mutants in Vibrio alginolyticus. We determined the C-ring molecular architectures, providing novel insights into the mechanism of rotational switching. We report that the C-ring maintained 34-fold symmetry in both rotational senses, and the protein composition remained constant. The two structures show FliG conformational changes elicit a large conformational rearrangement of the rotor complex that coincides with rotational switching of the flagellum. FliM and FliN form a stable spiral-shaped base of the C-ring, likely stabilizing the C-ring during the conformational remodeling.
DOI： 10.7554/eLife.61446
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Characterization of PomA periplasmic loop and sodium ion entering in stator complex of sodium-driven flagellar motor. Reviewed International journal
Tatsuro Nishikino, Hiroto Iwatsuki, Taira Mino, Seiji Kojima, Michio Homma
Journal of biochemistry 167 ( 4 ) 389 - 398 2020.04
Language：English
The bacterial flagellar motor is a rotary nanomachine driven by ion flow. The flagellar stator complex, which is composed of two proteins, PomA and PomB, performs energy transduction in marine Vibrio. PomA is a four transmembrane (TM) protein and the cytoplasmic region between TM2 and TM3 (loop2-3) interacts with the rotor protein FliG to generate torque. The periplasmic regions between TM1 and TM2 (loop1-2) and TM3 and TM4 (loop3-4) are candidates to be at the entrance to the transmembrane ion channel of the stator. In this study, we purified the stator complex with cysteine replacements in the periplasmic loops and assessed the reactivity of the protein with biotin maleimide (BM). BM easily modified Cys residues in loop3-4 but hardly labelled Cys residues in loop1-2. We could not purify the plug deletion stator (ΔL stator) composed of PomBΔ41-120 and WT-PomA but could do the ΔL stator with PomA-D31C of loop1-2 or with PomB-D24N of TM. When the ion channel is closed, PomA and PomB interact strongly. When the ion channel opens, PomA interacts less tightly with PomB. The plug and loop1-2 region regulate this activation of the stator, which depends on the binding of sodium ion to the D24 residue of PomB.
DOI： 10.1093/jb/mvz102
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Molecular architecture of the sheathed polar flagellum in Vibrio alginolyticus. Reviewed International journal
Shiwei Zhu, Tatsuro Nishikino, Bo Hu, Seiji Kojima, Michio Homma, Jun Liu
Proceedings of the National Academy of Sciences of the United States of America 114 ( 41 ) 10966 - 10971 2017.10
Language：English
Vibrio species are Gram-negative rod-shaped bacteria that are ubiquitous and often highly motile in aqueous environments. Vibrio swimming motility is driven by a polar flagellum covered with a membranous sheath, but this sheathed flagellum is not well understood at the molecular level because of limited structural information. Here, we use Vibrio alginolyticus as a model system to study the sheathed flagellum in intact cells by combining cryoelectron tomography (cryo-ET) and subtomogram analysis with a genetic approach. We reveal striking differences between sheathed and unsheathed flagella in V. alginolyticus cells, including a novel ring-like structure at the bottom of the hook that is associated with major remodeling of the outer membrane and sheath formation. Using mutants defective in flagellar motor components, we defined a Vibrio-specific feature (also known as the T ring) as a distinctive periplasmic structure with 13-fold symmetry. The unique architecture of the T ring provides a static platform to recruit the PomA/B complexes, which are required to generate higher torques for rotation of the sheathed flagellum and fast motility of Vibrio cells. Furthermore, the Vibrio flagellar motor exhibits an intrinsic length variation between the inner and the outer membrane bound complexes, suggesting the outer membrane bound complex can shift slightly along the axial rod during flagellar rotation. Together, our detailed analyses of the polar flagella in intact cells provide insights into unique aspects of the sheathed flagellum and the distinct motility of Vibrio species.
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Structural analysis of S-ring composed of FliFG fusion proteins in marine Vibrio polar flagellar motor. Reviewed International journal
Norihiro Takekawa, Tatsuro Nishikino, Jun-Ichi Kishikawa, Mika Hirose, Miki Kinoshita, Seiji Kojima, Tohru Minamino, Takayuki Uchihashi, Takayuki Kato, Katsumi Imada, Michio Homma
mBio e0126124 2024.09
Language：English Publishing type：Research paper (scientific journal)
The marine bacterium Vibrio alginolyticus possesses a polar flagellum driven by a sodium ion flow. The main components of the flagellar motor are the stator and rotor. The C-ring and MS-ring, which are composed of FliG and FliF, respectively, are parts of the rotor. Here, we purified an MS-ring composed of FliF-FliG fusion proteins and solved the near-atomic resolution structure of the S-ring-the upper part of the MS-ring-using cryo-electron microscopy. This is the first report of an S-ring structure from Vibrio, whereas, previously, only those from Salmonella have been reported. The Vibrio S-ring structure reveals novel features compared with that of Salmonella, such as tilt angle differences of the RBM3 domain and the β-collar region, which contribute to the vertical arrangement of the upper part of the β-collar region despite the diversity in the RBM3 domain angles. Additionally, there is a decrease of the inter-subunit interaction between RBM3 domains, which influences the efficiency of the MS-ring formation in different bacterial species. Furthermore, although the inner-surface electrostatic properties of Vibrio and Salmonella S-rings are altered, the residues potentially interacting with other flagellar components, such as FliE and FlgB, are well structurally conserved in the Vibrio S-ring. These comparisons clarified the conserved and non-conserved structural features of the MS-ring across different species.IMPORTANCEUnderstanding the structure and function of the flagellar motor in bacterial species is essential for uncovering the mechanisms underlying bacterial motility and pathogenesis. Our study revealed the structure of the Vibrio S-ring, a part of its polar flagellar motor, and highlighted its unique features compared with the well-studied Salmonella S-ring. The observed differences in the inter-subunit interactions and in the tilt angles between the Vibrio and Salmonella S-rings highlighted the species-specific variations and the mechanism for the optimization of MS-ring formation in the flagellar assembly. By concentrating on the region where the S-ring and the rod proteins interact, we uncovered conserved residues essential for the interaction. Our research contributes to the advancement of bacterial flagellar biology.
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Structural insight into sodium ion pathway in the bacterial flagellar stator from marine<i>Vibrio</i>
Tatsuro Nishikino, Norihiro Takekawa, Jun-ichi Kishikawa, Mika Hirose, Seiji Kojima, Michio Homma, Takayuki Kato, Katsumi Imada
2024.07
Publisher：Cold Spring Harbor Laboratory
Abstract
Many bacteria swim in liquid or swarm on surface using the flagellum rotated by a motor driven by specific ion flow. The motor consists of the rotor and stator, and the stator converts the energy of ion flow to mechanical rotation. However, the ion pathway and the mechanism of stator rotation coupled with specific ion flow are still obscure. Here, we determined the structures of the Na<sup>+</sup>-driven stator ofVibrio, namely PomAB, in the presence and absence of sodium ions and the structure with its specific inhibitor, phenamil, by cryo-electron microscopy. The structures and following functional analysis revealed the sodium ion pathway, the mechanism of ion selectivity, and the inhibition mechanism by phenamil. We propose a model of sodium ion flow coupled with the stator rotation based on the structures. This work provides insights into the molecular mechanisms of ion specificity and conversion of the electrochemical potential into mechanical functions. -
Roles of linker region flanked by transmembrane and peptidoglycan binding region of PomB in energy conversion of the Vibrio flagellar motor. Reviewed International journal
Yusuke Miyamura, Tatsuro Nishikino, Hiroaki Koiwa, Michio Homma, Seiji Kojima
Genes to cells : devoted to molecular & cellular mechanisms 2024.02
Language：English Publishing type：Research paper (scientific journal)
The flagellar components of Vibrio spp., PomA and PomB, form a complex that transduces sodium ion and contributes to rotate flagella. The transmembrane protein PomB is attached to the basal body T-ring by its periplasmic region and has a plug segment following the transmembrane helix to prevent ion flux. Previously we showed that PomB deleted from E41 to R120 (Δ41-120) was functionally comparable to the full-length PomB. In this study, three deletions after the plug region, PomB (Δ61-120), PomB (Δ61-140), and PomB (Δ71-150), were generated. PomB (Δ61-120) conferred motility, whereas the other two mutants showed almost no motility in soft agar plate; however, we observed some swimming cells with speed comparable for the wild-type cells. When the two PomB mutants were introduced into a wild-type strain, the swimming ability was not affected by the mutant PomBs. Then, we purified the mutant PomAB complexes to confirm the stator formation. When plug mutations were introduced into the PomB mutants, the reduced motility by the deletion was rescued, suggesting that the stator was activated. Our results indicate that the deletions prevent the stator activation and the linker and plug regions, from E41 to S150, are not essential for the motor function of PomB but are important for its regulation.
DOI： 10.1111/gtc.13102
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高圧NMR測定による細菌べん毛モータータンパク質FliGの構造変化がモーターの回転方向を決定する分子機構の解明
Tatsuro NISHIKINO, Yohei MIYANOIRI
The Review of High Pressure Science and Technology 33 ( 2 ) 83 - 90 2023.06
Publishing type：Research paper (scientific journal) Publisher：The Japan Society of High Pressure Science and Technology
Misc
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Deciphering the Conformational Change of Bacterial Flagellar Rotor Protein FliG, That Determines the Rotational Direction of the Flagellar Motor, by High-Pressure Solution NMR Analysis Invited Reviewed
錦野達郎, 宮ノ入洋平
高圧力の科学と技術 33 ( 2 ) 2023
Authorship：Lead author, Corresponding author
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Achievements in bacterial flagellar research with focus on Vibrio species Reviewed
Michio Homma, Tatsuro Nishikino, Seiji Kojima
Microbiology and Immunology 2022.01
Publisher：Wiley
Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/1348-0421.12954
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[Flagellar related genes and functions in Vibrio]. Reviewed
Tatsuro Nishikino, Seiji Kojima, Michio Homma
Nihon saikingaku zasshi. Japanese journal of bacteriology 75 ( 3 ) 195 - 214 2020
Language：Japanese
Bacteria can move or swim by flagella. On the other hand, the motile ability is not necessary to live at all. In laboratory, the flagella-deficient strains can grow just like the wild-type strains. The flagellum is assembled from more than 20 structural proteins and there are more than 50 genes including the structural genes to regulate or support the flagellar formation. The cost to construct the flagellum is so expensive. The fact that it evolved as a motor organ means even at such the large cost shows that the flagellum is essential for survival in natural condition. In this review, we would like to focus on the flagella-related researches conducted by the authors and the flagellar research on Vibrio spp.
DOI： 10.3412/jsb.75.195
Presentations
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Clarification of the color turning mechanism between GPR and BPR by FTIR spectroscopy
Tatsuro Nishikino, Teppei Sugimoto, Hideki Kandori
第61回日本生物物理学会年会
Event date： 2023.11
Presentation type：Poster presentation
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Aquifex aeolicus の固定子A サブユニット5量体の単粒子解析
錦野達郎
2021年度べん毛研究交流会 2022.03
Event date： 2022.03
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Structural differences of the C-ring in Vibrio flagellar motor between CW and CCW rotation Invited
Tatsuro Nishikino, Brittany Carroll, Shiwei Zhu, Seiji Kojima, Jun Liu, Michio Homma
Event date： 2020.03
Presentation type：Symposium, workshop panel (public)
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NMR 法とCryo-ET 法を用いたビブリオ菌べん毛モーター回転子FliG のGly-Gly リンカー回転方向変異体の構造解析
錦野達郎, 宮ノ入洋平, Zhu Shiwei, 小嶋誠司, Liu Jun, 本間道夫
日本生体エネルギー研究会第44 回討論会
Event date： 2018.12
Presentation type：Oral presentation (general)
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クライオ電子顕微鏡単粒子解析によるNa+駆動型べん毛モーター固定子のNa+透過経路の解明 Invited
錦野達郎
第17回 MPRCセミナー 2023.09
Presentation type：Oral presentation (invited, special)
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クライオ電子顕微鏡単粒子解析による海洋性ビブリオ菌べん毛モーターPomAPomB固定子阻害剤フェナミルの阻害機構の解明
錦野達郎, 竹川宜宏, 岸川淳一, 廣瀬未果, 小嶋誠司, 本間道夫, 加藤貴之, 今田勝巳
第61回日本細菌学会中部支部総会
Event date： 2024.11
Presentation type：Oral presentation (general)
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低温FTIR解析による2つのプロテオロドプシン GPR と BPR における L/Q スイッチの分子機構の解明
錦野達郎, 杉本哲平, 神取秀樹
文科省・学際領域展開ハブ形成プログラム「マルチスケール量子−古典⽣命インターフェース研究コンソーシアム」第２回合同シンポジウム
Event date： 2024.11
Presentation type：Poster presentation
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内向きプロトンポンプロドプシンNsXeRが持つ機能に最適な光異性化
〇伊藤 侑真, 錦野 達郎, 神取 秀樹, 古谷 祐詞
文科省・学際領域展開ハブ形成プログラム「マルチスケール量子−古典⽣命インターフェース研究コンソーシアム」第２回合同シンポジウム
Event date： 2024.11
Presentation type：Poster presentation
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表⾯増強⾚外分光法による負の⾛光性機能を持つpSRII-pHtrII複合体の光誘起構造変化解析
〇坂本達哉, 唐静一, 加藤壮一郎, 錦野達郎, 古谷祐詞
文科省・学際領域展開ハブ形成プログラム「マルチスケール量子−古典⽣命インターフェース研究コンソーシアム」第２回合同シンポジウム
Event date： 2024.11
Presentation type：Poster presentation
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放線菌由来ヘリオロドプシンの機能解明へ向けた分光解析と遺伝⼦解析
〇山田 航洋, 吉住 玲, 中村 敏規, 古谷 祐詞, 錦野 達郎, 神取 秀樹
文科省・学際領域展開ハブ形成プログラム「マルチスケール量子−古典⽣命インターフェース研究コンソーシアム」第２回合同シンポジウム
Event date： 2024.11
Presentation type：Poster presentation
Awards
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平成31年度名古屋大学学術奨励
2019.06 名古屋大学 海洋性ビブリオ菌べん毛モーター回転子の構造変化の解明
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Taiyo Nippon Sanso award
2021.08 ISMAR-APNMR
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口頭発表優秀賞
2020.12 日本生体エネルギー研究会
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優秀口頭発表賞
2019.12 日本生体エネルギー研究会
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最優秀発表賞
2019.03 日本生物物理学会中部支部会
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Poster award
2018.08 2018 Kuo Symposium on 3D-EM of Macromolecules and Cells/11th K. H. Kuo Summer School of Electron Microscopy & Crystallography
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優秀発表賞
2017.03 日本細菌学会
Scientific Research Funds Acquisition Results
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細菌べん毛モーター固定子に対するアミロライド阻害剤 Phenamil の阻害機構の解明
Grant number：23K14157 2023.04 - 2025.03
日本学術振興会 科学研究費助成事業 若手研究
錦野 達郎
Grant amount：\4550000 （ Direct Cost: \3500000 、 Indirect Cost：\1050000 ）
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べん毛モーター固定子の構造変化により生じるエネルギー変換機構の解明
Grant number：20J00329 2020.04 - 2023.03
日本学術振興会 科学研究費助成事業 特別研究員奨励費 特別研究員奨励費
錦野 達郎
Grant amount：\4810000 （ Direct Cost: \3700000 、 Indirect Cost：\1110000 ）
細菌の運動器官の一つであるべん毛は、回転するモーターをもつ超分子複合体である。モーターは自身が回転する「回転子」と回転子の周りに集合しイオンチャネルとして機能する「固定子」の2種類の複合体から成る。モーターの回転は、固定子への共役イオンの流入とカップルした回転子と固定子の相互作用により、膜内外に形成される電気化学ポテンシャル差が運動エネルギーに変換されることで生じる。固定子は、5分子のAサブユニット(PomA)と2分子の Bサブユニット(PomB) からなる膜貫通タンパク質複合体である。その構造はクライオ条件下での単粒子解析により明らかになっているが、エネルギー変換の際の複合体の構造変化や共役イオンの通り道は未だによくわかっていない。本研究では、これらを明らかにするためにNa+チャネルである海洋性ビブリオ菌の固定子複合体[PomA/PomB]の構造情報をクライオ条件での単粒子解析と溶液核磁気共鳴法(NMR)により取得することを目的としている。
昨年度(研究初年度)は、クライオ条件での単粒子解析法の取得のために高度好熱菌Aquifex aeolicusの固定子Aサブユニット(MotA)複合体の解析とNMR解析技術の習得のために固定子の相互作用相手であり回転子を構成するタンパク質の一つであるFliGの解析を進めた。現在、これら２つの研究成果を論文にまとめ投稿する準備を進めている。
今年度は、クライオ条件でのPomAPomB複合体の単粒子解析を進めた。複合体の密度マップを得ることができたため、モデル構築と精密化の作業を進めている。溶液NMRでの解析では、複合体中のメチオニン残基側鎖のメチル基を13C標識した測定試料を調製し、NMR測定を行った。野生型とNa+イオン透過が阻害されるD24N変異体のスペクトルを取得し比較したところ、スペクトルの変化が見られた。 -
Conformational change of flagellar rotor in marine Vibrio International coauthorship
Grant number：17J11237 2017.04 - 2020.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for JSPS Fellows Grant-in-Aid for JSPS Fellows
Grant amount：\2800000 （ Direct Cost: \2800000 ）
Media Coverage
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Nagoya University achieves clarification of the structure of bacterial flagella using cryo-electron microscopy Newspaper, magazine
The Science News https://sj.jst.go.jp/news/202411/n1112-02k.html 2024.11
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細菌の鞭毛の駆動源 回転モーターの構造解明 Newspaper, magazine
科学新聞 2024.10
Author：Other
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細菌の前進・後退を決めていたタンパク質の構造変化 ―方向制御が可能な極小分子モーターの開発に貢献―
大阪大学、名古屋大学、長浜バイオ大学 2023.08
Author：Other