Faculty Profiles - UCHIHASHI Takayuki

Structural properties determining low K affinity of the selectivity filter in the TWIK1 K channel

Hisao Tsukamoto, Masahiro Higashi, Hideyoshi Motoki, Hiroki Watanabe, Christian Ganser, Koichi Nakajo, Yoshihiro Kubo, Takayuki Uchihashi, Yuji Furutani

Journal of Biological Chemistry Vol. 293 ( 18 ) page： 6969 - 6984 2018.5

Language：English Publishing type：Research paper (scientific journal) Publisher：American Society for Biochemistry and Molecular Biology Inc.

Canonical K channels are tetrameric and highly K-selective, whereas two-pore– domain K (K2P) channels form dimers, but with a similar pore architecture. A two-pore– domain potassium channel TWIK1 (KCNK1 or K2P1) allows permeation of Na and other monovalent ions, resulting mainly from the presence of Thr-118 in the P1 domain. However, the mechanistic basis for this reduced selectivity is unclear. Using ion-exchange–induced difference IR spectroscopy, we analyzed WT TWIK1 and T118I (highly K-selective) and L228F (substitution in the P2 domain) TWIK1 variants and found that in the presence of K ions, WT and both variants exhibit an amide-I band at 1680 cm1. This band corresponds to interactions of the backbone carbonyls in the selectivity filter with K, a feature very similar to that of the canonical K channel KcsA. Computational analysis indicated that the relatively high frequency for the amide-I band is well explained by impairment of hydrogen bond formation with water molecules. Moreover, concentration-dependent spectral changes indicated that the K affinity of the WT selectivity filter was much lower than those of the variants. Furthermore, only the variants displayed a higher frequency shift of the 1680-cm1 band upon changes from K to Rb or Cs conditions. High-speed atomic force microscopy disclosed that TWIK1’s surface morphology largely does not change in K and Na solutions. Our results reveal the local conformational changes of the TWIK1 selectivity filter and suggest that the amide-I bands may be useful “molecular fingerprints” for assessing the properties of other K channels.

DOI： 10.1074/jbc.RA118.001817

Construction of a Triangle-Shaped Trimer and a Tetrahedron Using an α-Helix-Inserted Circular Permutant of Cytochrome c 555

Akiya Oda, Satoshi Nagao, Masaru Yamanaka, Ikki Ueda, Hiroki Watanabe, Takayuki Uchihashi, Naoki Shibata, Yoshiki Higuchi, Shun Hirota

Chemistry - An Asian Journal Vol. 13 ( 8 ) page： 964 - 967 2018.4

Insight into structural remodeling of the FlhA ring responsible for bacterial flagellar type III protein export

Naoya Terahara, Yumi Inoue, Noriyuki Kodera, Yusuke V. Morimoto, Takayuki Uchihashi, Katsumi Imada, Toshio Ando, Keiichi Namba, Tohru Minamino

Science Advances Vol. 4 ( 4 ) page： eaao7054 2018.4

Language：English Publishing type：Research paper (scientific journal) Publisher：American Association for the Advancement of Science

The bacterial flagellum is a supramolecular motility machine. Flagellar assembly begins with the basal body, followed by the hook and finally the filament. A carboxyl-terminal cytoplasmic domain of FlhA (FlhAC) forms a nonameric ring structure in the flagellar type III protein export apparatus and coordinates flagellar protein export with assembly. However, the mechanism of this process remains unknown. We report that a flexible linker of FlhAC (FlhAL) is required not only for FlhAC ring formation but also for substrate specificity switching of the protein export apparatus from the hook protein to the filament protein upon completion of the hook structure. FlhAL was required for cooperative ring formation of FlhAC. Alanine substitutions of residues involved in FlhAC ring formation interfered with the substrate specificity switching, thereby inhibiting filament assembly at the hook tip. These observations lead us to propose a mechanistic model for export switching involving structural remodeling of FlhAC.

DOI： 10.1126/sciadv.aao7054

Front Cover: Construction of a Triangle-Shaped Trimer and a Tetrahedron Using an α-Helix-Inserted Circular Permutant of Cytochrome c 555 (Chem. Asian J. 10/2018) Reviewed

Akiya Oda, Satoshi Nagao, Masaru Yamanaka, Ikki Ueda, Hiroki Watanabe, Takayuki Uchihashi, Naoki Shibata, Yoshiki Higuchi, Shun Hirota

Chemistry - An Asian Journal Vol. 13 ( 10 ) page： 1229 2018.4

High-speed AFM reveals detail understanding in adsorption of soft hydrogel microspheres onto solid substrate in aqueous solution

Matsui Shusuke, Uchihashi Takayuki, Suzuki Daisuke

ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY Vol. 255 page： . 2018.3

Real-time adsorption behavior of hydrogel microspheres onto solid/liquid interface

Matsui Shusuke, Uchihashi Takayuki, Suzuki Daisuke

ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY Vol. 255 page： . 2018.3

High-speed AFM reveals detail understanding in adsorption of soft hydrogel microspheres onto solid substrate in aqueous solution

Matsui Shusuke, Uchihashi Takayuki, Suzuki Daisuke

ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY Vol. 255 page： . 2018.3

Real-time adsorption behavior of hydrogel microspheres onto solid/liquid interface

Matsui Shusuke, Uchihashi Takayuki, Suzuki Daisuke

ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY Vol. 255 page： . 2018.3

Negatively Charged Lipids Are Essential for Functional and Structural Switch of Human 2-Cys Peroxiredoxin II Reviewed

Takamitsu Haruyama, Takayuki Uchihashi, Yutaro Yamada, Noriyuki Kodera, Toshio Ando, Hiroki Konno

Journal of Molecular Biology Vol. 430 ( 5 ) page： 602 - 610 2018.3

Language：English Publishing type：Research paper (scientific journal) Publisher：Academic Press

The function of ubiquitous 2-Cys peroxiredoxins (Prxs) can be converted alternatively from peroxidases to molecular chaperones. This conversion has been reported to occur by the formation of high-molecular-weight (HMW) complexes upon overoxidation of or ATP/ADP binding to 2-Cys Prxs, but its mechanism is not well understood. Here, we show that upon binding to phosphatidylserine or phosphatidylglycerol dimeric human 2-Cys PrxII (hPrxII) is assembled to trefoil-shaped small oligomers (possibly hexamers) with full chaperone and null peroxidase activities. Spherical HMW complexes are formed, only when phosphatidylserine or phosphatidylglycerol is bound to overoxidized or ATP/ADP-bound hPrxII. The spherical HMW complexes are lipid vesicles covered with trefoil-shaped oligomers arranged in a hexagonal lattice pattern. Thus, these lipids with a net negative charge, which can be supplied by increased membrane trafficking under oxidative stress, are essential for the structural and functional switch of hPrxII and possibly most 2-Cys Prxs.

DOI： 10.1016/j.jmb.2017.12.020

Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis. International journal

Akihiko Nakamura, Tomoyuki Tasaki, Yasuko Okuni, Chihong Song, Kazuyoshi Murata, Toshiya Kozai, Mayu Hara, Hayuki Sugimoto, Kazushi Suzuki, Takeshi Watanabe, Takayuki Uchihashi, Hiroyuki Noji, Ryota Iino

Physical chemistry chemical physics : PCCP Vol. 20 ( 5 ) page： 3010 - 3018 2018.2

Language：English Publishing type：Research paper (scientific journal)

Serratia marcescens chitinase A is a linear molecular motor that hydrolyses crystalline chitin in a processive manner. Here, we quantitatively determined the rate constants of elementary reaction steps, including binding (kon), translational movement (ktr), and dissociation (koff) with single-molecule fluorescence imaging. The kon for a single chitin microfibril was 2.1 × 109 M-1 μm-1 s-1. The koff showed two components, k (3.2 s-1, 78%) and k (0.38 s-1, 22%), corresponding to bindings to different crystal surfaces. From the kon, k, k and ratio of fast and slow dissociations, dissociation constants for low and high affinity sites were estimated as 2.0 × 10-9 M μm and 8.1 × 10-10 M μm, respectively. The ktr was 52.5 nm s-1, and processivity was estimated as 60.4. The apparent inconsistency between high turnover (52.5 s-1) calculated from ktr and biochemically determined low kcat (2.6 s-1) is explained by a low ratio (4.8%) of productive enzymes on the chitin surface (52.5 s-1 × 0.048 = 2.5 s-1). Our results highlight the importance of single-molecule analysis in understanding the mechanism of enzymes acting on a solid-liquid interface.

DOI： 10.1039/c7cp04606e

Dynamic Observation of Kai Proteins by HS-AFM Reveals a Mechanism of the Robustness in the Cyanobacterial Circadian Oscillator

Correction: Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis. International journal

Physical chemistry chemical physics : PCCP Vol. 20 ( 5 ) page： 3844 - 3844 2018.2

Sugiyama Shogo, Mori Tetsya, Byrne Mark, Uchihashi Takayuki, Johnson Carl H., Ando Toshio

BIOPHYSICAL JOURNAL Vol. 114 ( 3 ) page： 68A - 68A 2018.2

Dynamic Observation of Kai Proteins by HS-AFM Reveals a Mechanism of the Robustness in the Cyanobacterial Circadian Oscillator

Sugiyama Shogo, Mori Tetsya, Byrne Mark, Uchihashi Takayuki, Johnson Carl H, Ando Toshio

BIOPHYSICAL JOURNAL Vol. 114 ( 3 ) page： 68A-68A 2018.2

Applications of high-speed atomic force microscopy to real-time visualization of dynamic biomolecular processes.

Uchihashi T, Scheuring S

Biochimica et biophysica acta Vol. 1862 ( 2 ) page： 229 - 240 2018.2

Dynamic clustering of dynamin-amphiphysin helices regulates membrane constriction and fission coupled with GTP hydrolysis Reviewed

Tetsuya Takeda, Toshiya Kozai, Huiran Yang, Daiki Ishikuro, Kaho Seyama, Yusuke Kumagai, Tadashi Abe, Hiroshi Yamada, Takayuki Uchihashi, Toshio Ando, Kohji Takei

eLife Vol. 7 2018.1

Language：English Publishing type：Research paper (scientific journal) Publisher：eLife Sciences Publications Ltd

Dynamin is a mechanochemical GTPase essential for membrane fission during clathrin-mediated endocytosis. Dynamin forms helical complexes at the neck of clathrin-coated pits and their structural changes coupled with GTP hydrolysis drive membrane fission. Dynamin and its binding protein amphiphysin cooperatively regulate membrane remodeling during the fission, but its precise mechanism remains elusive. In this study, we analyzed structural changes of dynamin-amphiphysin complexes during the membrane fission using electron microscopy (EM) and high-speed atomic force microscopy (HS-AFM). Interestingly, HS-AFM analyses show that the dynamin-amphiphysin helices are rearranged to form clusters upon GTP hydrolysis and membrane constriction occurs at protein-uncoated regions flanking the clusters. We also show a novel function of amphiphysin in size control of the clusters to enhance biogenesis of endocytic vesicles. Our approaches using combination of EM and HS-AFM clearly demonstrate new mechanistic insights into the dynamics of dynamin-amphiphysin complexes during membrane fission.

DOI： 10.7554/eLife.30246

Conversion of functionally undefined homopentameric protein PbaA into a proteasome activator by mutational modification of its C-terminal segment conformation Reviewed International journal

Maho Yagi-Utsumi, Arunima Sikdar, Toshiya Kozai, Rintaro Inoue, Masaaki Sugiyama, Takayuki Uchihashi, Hirokazu Yagi, Tadashi Satoh, Koichi Kato

PROTEIN ENGINEERING DESIGN & SELECTION Vol. 31 ( 1 ) page： 29 - 36 2018.1

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

Recent bioinformatic analyses identified proteasome assembly chaperone-like proteins, PbaA and PbaB, in archaea. PbaB forms a homotetramer and functions as a proteasome activator, whereas PbaA does not interact with the proteasome despite the presence of an apparent C-terminal proteasome activation motif. We revealed that PbaA forms a homopentamer predominantly in the closed conformation with its C-terminal segments packed against the core domains, in contrast to the PbaB homotetramer with projecting C-terminal segments. This prompted us to create a novel proteasome activator based on a well-characterized structural framework. We constructed a panel of chimeric proteins comprising the homopentameric scaffold of PbaA and C-terminal segment of PbaB and subjected them to proteasome-activating assays as well as small-angle X-ray scattering and high-speed atomic force microscopy. The results indicated that the open conformation and consequent proteasome activation activity could be enhanced by replacement of the crystallo-graphically disordered C-terminal segment of PbaA with the corresponding disordered segment of PbaB. Moreover, these effects can be produced just by incorporating two glutamate residues into the disordered C-terminal segment of PbaA, probably due to electrostatic repulsion among the negatively charged segments. Thus, we successfully endowed a functionally undefined protein with proteasome-activating activity by modifying its C-terminal segment.

DOI： 10.1093/protein/gzx066

Visualization of Protein Dynamics using High-Speed Atomic Force Microscopy and Image Analysis

Uchihashi Takayuki

JOURNAL OF COMPUTER CHEMISTRY-JAPAN Vol. 17 ( 1 ) page： 20-30 - 30 2018

Language：English Publishing type：Research paper (scientific journal) Publisher：Society of Computer Chemistry, Japan

<p>Recent progress of high-speed atomic force microscopy (HS-AFM) has enabled us to visualize dynamic conformational change and molecular interaction of proteins in realtime under a physiological condition. Molecular mechanisms through dynamic phenomena that have been deduced from the accumulation of experimental evidence by various analytical techniques are now able to be directly and intuitively understood from AFM movies. While a movie provides a great deal of information compared to a static image, extracting useful information based on objective criteria is essential for the next step. This review describes representative HS-AFM data for three typical dynamic biological phenomena; conformational dynamics of single proteins, linear movement of enzymes and velocity analysis and dynamic molecular interaction together with imaging processing techniques for interpreting AFM data and efficient analysis of successive images.</p>

DOI： 10.2477/jccj.2018-0001

Optimum Substrates for Imaging Biological Molecules with High-Speed Atomic Force Microscopy

CiNii Research

Uchihashi, T; Watanabe, H; Kodera, N

NANOSCALE IMAGING: METHODS AND PROTOCOLS Vol. 1814 page： 159 - 179 2018

Language：English Publisher：Methods in Molecular Biology

Recent progresses in high-speed atomic force microscopy (HS-AFM) have enabled us to directly visualize dynamic processes of various proteins in liquid conditions. One of the key factors leading to successful HS-AFM observations is the selection of an appropriate substrate depending on molecules to be observed. For the HS-AFM imaging, a target molecule must be absorbed on a substrate by controlling its orientation without impairing the dynamics or physiological function of the molecule. In this chapter, we describe protocols for preparation of substrates that have been used for HS-AFM and then introduce observation examples on dynamic processes of biological molecules.

DOI： 10.1007/978-1-4939-8591-3_10

High-speed scanning near-field optical microscopy

Umakoshi T., Fukuda S., Uchihashi T., Verma P., Ando T.

Optics InfoBase Conference Papers Vol. Part F125-JSAP 2018 2018

Language：Japanese Publisher：Optics InfoBase Conference Papers

Scanning near-field optical microscopy (SNOM) has been recognized as a powerful technique for super-resolution optical imaging [1]. It has been even more unique compared with other super-resolution fluorescent imaging techniques because it utilizes near-field light at a metallic tip as a nano-light-source, which realizes super-resolution not only in fluorescence but also in any other optical signals such as Raman scattering, infrared absorption and photoluminescence. Such a strong advantage has been stimulating biological fields as a new class of analytical techniques. However, it has been yet highly challenging to apply SNOM for biological samples. Although there have been several issues for biological applications, one of the major problems is its low imaging speed. Because it requires to physically scan a metallic tip, the imaging rate was typically limited to several minutes per frame, which is too slow to capture dynamic biological samples.