Faculty Profiles - UCHIHASHI Takayuki

Papers - UCHIHASHI Takayuki

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Single-molecule level dynamic observation of disassembly of the apo-ferritin cage in solution. Reviewed International journal

Basudev Maity, Zhipeng Li, Kento Niwase, Christian Ganser, Tadaomi Furuta, Takayuki Uchihashi, Diannan Lu, Takafumi Ueno

Physical chemistry chemical physics : PCCP Vol. 22 ( 33 ) page： 18562 - 18572 2020.9

Language：English Publishing type：Research paper (scientific journal) Publisher：Royal Society of Chemistry (RSC)

The ferritin cage iron-storage protein assembly has been widely used as a template for preparing nanomaterials. This assembly has a unique pH-induced disassembly/reassembly mechanism that provides a means for encapsulating molecules such as nanoparticles and small enzymes for catalytic and biomaterial applications. Although several researchers have investigated the disassembly process of ferritin, the dynamics involved in the initiation of the process and its intermediate states have not been elucidated due to a lack of suitable methodology to track the process in real-time. We describe the use of high-speed atomic force microscopy (HS-AFM) to image the dynamic event in real-time with single-molecule level resolution. The HS-AFM movies produced in the present work enable direct visualization of the movements of single ferritin cages in solution and formation of a hole prior to disassembly into subunit fragments. Additional support for these observations was confirmed at the atomic level by the results of all-atom molecular dynamics (MD) simulations, which revealed that the initiation process includes the opening of 3-fold symmetric channels. Our findings provide an essential contribution to a fundamental understanding of the dynamics of protein assembly and disassembly, as well as efforts to redesign the apo-ferritin cage for extended applications.

DOI： 10.1039/d0cp02069a

Thermoresponsive structural changes of single poly(N-isopropyl acrylamide) hydrogel microspheres under densely packed conditions on a solid substrate Reviewed

Haruka Minato, Yuichiro Nishizawa, Takayuki Uchihashi, Daisuke Suzuki

Polymer Journal Vol. 52 ( 9 ) page： 1137 - 1141 2020.9

Convergent evolution of processivity in bacterial and fungal cellulases. Reviewed International journal

Taku Uchiyama, Takayuki Uchihashi, Akihiko Nakamura, Hiroki Watanabe, Satoshi Kaneko, Masahiro Samejima, Kiyohiko Igarashi

Proceedings of the National Academy of Sciences of the United States of America Vol. 117 ( 33 ) page： 19896 - 19903 2020.8

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

Cellulose is the most abundant biomass on Earth, and many microorganisms depend on it as a source of energy. It consists mainly of crystalline and amorphous regions, and natural degradation of the crystalline part is highly dependent on the degree of processivity of the degrading enzymes (i.e., the extent of continuous hydrolysis without detachment from the substrate cellulose). Here, we report high-speed atomic force microscopic (HS-AFM) observations of the movement of four types of cellulases derived from the cellulolytic bacteria Cellulomonas fimi on various insoluble cellulose substrates. The HS-AFM images clearly demonstrated that two of them (CfCel6B and CfCel48A) slide on crystalline cellulose. The direction of processive movement of CfCel6B is from the nonreducing to the reducing end of the substrate, which is opposite that of processive cellulase Cel7A of the fungus Trichoderma reesei (TrCel7A), whose movement was first observed by this technique, while CfCel48A moves in the same direction as TrCel7A. When CfCel6B and TrCel7A were mixed on the same substrate, "traffic accidents" were observed, in which the two cellulases blocked each other's progress. The processivity of CfCel6B was similar to those of fungal family 7 cellulases but considerably higher than those of fungal family 6 cellulases. The results indicate that bacteria utilize family 6 cellulases as high-processivity enzymes for efficient degradation of crystalline cellulose, whereas family 7 enzymes have the same function in fungi. This is consistent with the idea of convergent evolution of processive cellulases in fungi and bacteria to achieve similar functionality using different protein foldings.

DOI： 10.1073/pnas.2011366117

Direct visualization of the conformational change of FUS/TLS upon binding to promoter-associated non-coding RNA. Reviewed International journal

Nesreen Hamad, Hiroki Watanabe, Takayuki Uchihashi, Riki Kurokawa, Takashi Nagata, Masato Katahira

Chemical communications (Cambridge, England) Vol. 56 ( 64 ) page： 9134 - 9137 2020.8

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

High-speed AFM revealed the conformational change of fused in sarcoma (FUS) from a compact to an extended structure upon binding of non-coding RNA, which is supposed to allow FUS to bind to CBP/p300 for transcriptional interference. Thus, a mechanistic insight into transcription regulation by FUS and non-coding RNA is provided.

DOI： 10.1039/d0cc03776a

Scanning Probe Microscopy FOREWORD

Nakajima, K; Sumitomo, K; Nakayama, T; Fukui, K; Kageshima, M; Kawai, S; Komeda, T; Takahashi, T; Uchihashi, T

JAPANESE JOURNAL OF APPLIED PHYSICS Vol. 59 2020.8

Assembly mechanism of a supramolecular MS-ring complex to initiate bacterial flagellar biogenesis in Vibrio species. Reviewed International journal

Hiroyuki Terashima, Keiichi Hirano, Yuna Inoue, Takaya Tokano, Akihiro Kawamoto, Takayuki Kato, Erika Yamaguchi, Keiichi Namba, Takayuki Uchihashi, Seiji Kojima, Michio Homma

Journal of bacteriology Vol. 202 ( 16 ) 2020.8

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

The bacterial flagellum is an organelle responsible for motility and has a rotary motor comprising the rotor and the stator. Flagellar biogenesis is initiated by the assembly of the MS-ring, a supramolecular complex embedded in the cytoplasmic membrane. The MS-ring consists of a few dozen copies of the transmembrane FliF protein, and is an essential core structure which is a part of the rotor. The number and location of the flagella are controlled by the FlhF and FlhG proteins in some species. However, there is no clarity on the factors initiating MS-ring assembly, and contribution of FlhF/FlhG to this process. Here, we show that FlhF and a C-ring component FliG facilitate Vibrio MS-ring formation. When Vibrio FliF alone was expressed in Escherichia coli cells, MS-ring formation rarely occurred, indicating the requirement of other factors for MS-ring assembly. Consequently, we investigated if FlhF aided FliF in MS-ring assembly. We found that FlhF allowed GFP-fused FliF to localize at the cell pole in a Vibrio cell, suggesting that it increases local concentration of FliF at the pole. When FliF was co-expressed with FlhF in E. coli cells, the MS-ring was effectively formed, indicating that FlhF somehow contributes to MS-ring formation. The isolated MS-ring structure was similar to the MS-ring formed by Salmonella FliF. Interestingly, FliG facilitates MS-ring formation, suggesting that FliF and FliG assist in each other's assembly into the MS-ring and C-ring. This study aids in understanding the mechanism behind MS-ring assembly using appropriate spatial/temporal regulations.Importance Flagellar formation is initiated by the assembly of the FliF protein into the MS-ring complex, embedded in the cytoplasmic membrane. The appropriate spatial/temporal control of MS-ring formation is important for the morphogenesis of the bacterial flagellum. Here, we focus on the assembly mechanism of Vibrio FliF into the MS-ring. FlhF, a positive regulator of the number and location of flagella, recruits the FliF molecules at the cell pole and facilitates MS-ring formation. FliG also facilitates MS-ring formation. Our study showed that these factors control flagellar biogenesis in Vibrio, by initiating the MS-ring assembly. Furthermore, it also implies that flagellar biogenesis is a sophisticated system linked with the expression of certain genes, protein localization and a supramolecular complex assembly.

DOI： 10.1128/JB.00236-20

ヘリオロドプシンおよびシゾロドプシンの構造から明らかになった微生物型ロドプシンの多様性(Structures of heliorhodopsin and schizorhodopsin elucidate the structural diversity of microbial rhodopsins)

Shihoya Wataru, Inoue Keiichi, Manish Singh, Higuchi Akimitsu, Konno Masae, Yoshizumi Rei, Uchihashi Takayuki, Kandori Hideki, Nureki Osamu

生物物理 Vol. 60 ( Suppl.1-2 ) page： S154 - S154 2020.8

Structural Dynamics of a Protein Domain Relevant to the Water-Oxidizing Complex in Photosystem II as Visualized by High-Speed Atomic Force Microscopy. Reviewed International journal

Takaya Tokano, Yuki Kato, Shogo Sugiyama, Takayuki Uchihashi, Takumi Noguchi

The journal of physical chemistry. B Vol. 124 ( 28 ) page： 5847 - 5857 2020.7

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

Photosystem II (PSII) is a multiprotein complex that has a function of light-driven water oxidation. The catalytic site of water oxidation is the Mn4CaO5 cluster, which is bound to the lumenal side of PSII through amino acid residues from the D1 and CP43 proteins and is further surrounded by the extrinsic proteins. In this study, we have for the first time visualized the structural dynamics of the lumenal region of a PSII core complex using high-speed atomic force microscopy (HS-AFM). The HS-AFM images of a PSII membrane fragment showed stepwise dissociation of the PsbP and PsbO extrinsic proteins. Upon subsequent destruction of the Mn4CaO5 cluster, the lumenal domain of CP43 was found to undergo a conformational fluctuation. The observed structural flexibility and conformational fluctuation of the CP43 lumenal domain are suggested to play important roles in the biogenesis of PSII and the photoassembly of the Mn4CaO5 cluster.

DOI： 10.1021/acs.jpcb.0c03892

Dynamic behavior of an artificial protein needle contacting a membrane observed by high-speed atomic force microscopy. Reviewed International journal

Takafumi Ueno, Kento Niwase, Daisho Tsubokawa, Kosuke Kikuchi, Natsumi Takai, Tadaomi Furuta, Ryuji Kawano, Takayuki Uchihashi

Nanoscale Vol. 12 ( 15 ) page： 8166 - 8173 2020.4

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

Bacteriophage T4 and other bacteriophages have a protein component known as a molecular needle which is used for the transmembrane reaction in the infection process. In this paper, the transmembrane reaction mechanisms of artificial protein needles (PNs) constructed by protein engineering of the component protein of bacteriophage T4 are elucidated by observation of single-molecules by high-speed atomic force microscopy (HS-AFM) and molecular dynamics (MD) simulations. The HS-AFM images indicate that the tip of the needle structure stabilizes the interaction of the needle with the membrane surface and is involved in controlling the contact angle and angular velocity with respect to the membrane. The MD simulations indicate that the dynamic behavior of PN is governed by hydrogen bonds between the membrane phosphate fragments and the tip. Moreover, quartz crystal microbalance (QCM) and electrophysiological experiments indicate that the tip structure of PN affects its kinetic behavior and membrane potential. These results demonstrate that protein assemblies derived from natural biosupramolecules can be used to create nanomaterials with rationally-designed functionality.

DOI： 10.1039/d0nr01121e

Dynamics of oligomer and amyloid fibril formation by yeast prion Sup35 observed by high-speed atomic force microscopy. Reviewed International journal

Hiroki Konno, Takahiro Watanabe-Nakayama, Takayuki Uchihashi, Momoko Okuda, Liwen Zhu, Noriyuki Kodera, Yousuke Kikuchi, Toshio Ando, Hideki Taguchi

Proceedings of the National Academy of Sciences of the United States of America Vol. 117 ( 14 ) page： 7831 - 7836 2020.4

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

The yeast prion protein Sup35, which contains intrinsically disordered regions, forms amyloid fibrils responsible for a prion phenotype [PSI+]. Using high-speed atomic force microscopy (HS-AFM), we directly visualized the prion determinant domain (Sup35NM) and the formation of its oligomers and fibrils at subsecond and submolecular resolutions. Monomers with freely moving tail-like regions initially appeared in the images, and subsequently oligomers with distinct sizes of ∼1.7 and 3 to 4 nm progressively accumulated. Nevertheless, these oligomers did not form fibrils, even after an incubation for 2 h in the presence of monomers. Fibrils appeared after much longer monomer incubation. The fibril elongation occurred smoothly without discrete steps, suggesting gradual conversions of the incorporated monomers into cross-β structures. The individual oligomers were separated from each other and also from the fibrils by respective, identical lengths on the mica surface, probably due to repulsion caused by the freely moving disordered regions. Based on these HS-AFM observations, we propose that the freely moving tails of the monomers are incorporated into the fibril ends, and then the structural conversions to cross-β structures gradually occur.

DOI： 10.1073/pnas.1916452117

Schizorhodopsins: A family of rhodopsins from Asgard archaea that function as light-driven inward H+ pumps. Reviewed International journal

Keiichi Inoue, Satoshi P Tsunoda, Manish Singh, Sahoko Tomida, Shoko Hososhima, Masae Konno, Ryoko Nakamura, Hiroki Watanabe, Paul-Adrian Bulzu, Horia L Banciu, Adrian-Ştefan Andrei, Takayuki Uchihashi, Rohit Ghai, Oded Béjà, Hideki Kandori

Science advances Vol. 6 ( 15 ) page： eaaz2441 2020.4

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

Schizorhodopsins (SzRs), a rhodopsin family first identified in Asgard archaea, the archaeal group closest to eukaryotes, are present at a phylogenetically intermediate position between typical microbial rhodopsins and heliorhodopsins. However, the biological function and molecular properties of SzRs have not been reported. Here, SzRs from Asgardarchaeota and from a yet unknown microorganism are expressed in Escherichia coli and mammalian cells, and ion transport assays and patch clamp analyses are used to demonstrate SzR as a novel type of light-driven inward H+ pump. The mutation of a cytoplasmic glutamate inhibited inward H+ transport, suggesting that it functions as a cytoplasmic H+ acceptor. The function, trimeric structure, and H+ transport mechanism of SzR are similar to that of xenorhodopsin (XeR), a light-driven inward H+ pumping microbial rhodopsins, implying that they evolved convergently. The inward H+ pump function of SzR provides new insight into the photobiological life cycle of the Asgardarchaeota.

DOI： 10.1126/sciadv.aaz2441

Recent advances in bioimaging with high-speed atomic force microscopy. Reviewed International journal

Takayuki Uchihashi, Christian Ganser

Biophysical reviews Vol. 12 ( 2 ) page： 363 - 369 2020.4

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

Among various microscopic techniques for characterizing protein structures and functions, high-speed atomic force microscopy (HS-AFM) is a unique technique in that it allows direct visualization of structural changes and molecular interactions of proteins without any labeling in a liquid environment. Since the development of the HS-AFM was first reported in 2001, it has been applied to analyze the dynamics of various types of proteins, including motor proteins, membrane proteins, DNA-binding proteins, amyloid proteins, and artificial proteins. This method has now become a versatile tool indispensable for biophysical research. This short review summarizes some bioimaging applications of HS-AFM reported in the last few years and novel applications of HS-AFM utilizing the unique ability of AFM to gain mechanical properties of samples in addition to structural information.

DOI： 10.1007/s12551-020-00670-z

Single-molecule imaging analysis reveals the mechanism of a high-catalytic-activity mutant of chitinase A from Serratia marcescens Reviewed International journal

Akasit Visootsat, Akihiko Nakamura, Paul Vignon, Hiroki Watanabe, Takayuki Uchihashi, Ryota Iino

JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 295 ( 7 ) page： 1915 - 1925 2020.2

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Chitin degradation is important for biomass conversion and has potential applications for agriculture, biotechnology, and the pharmaceutical industry. Chitinase A from the Gram-negative bacterium Serratia marcescens (SmChiA) is a processive enzyme that hydrolyzes crystalline chitin as it moves linearly along the substrate surface. In a previous study, the catalytic activity of SmChiA against crystalline chitin was found to increase after the tryptophan substitution of two phenylalanine residues (F232W and F396W), located at the entrance and exit of the substrate binding cleft of the catalytic domain, respectively. However, the mechanism underlying this high catalytic activity remains elusive. In this study, single-molecule fluorescence imaging and high-speed atomic force microscopy were applied to understand the mechanism of this high-catalytic-activity mutant. A reaction scheme including processive catalysis was used to reproduce the properties of SmChiA WT and F232W/F396W, in which all of the kinetic parameters were experimentally determined. High activity of F232W/F396W mutant was caused by a high processivity and a low dissociation rate constant after productive binding. The turnover numbers for both WT and F232W/F396W, determined by the biochemical analysis, were well-replicated using the kinetic parameters obtained from single-molecule imaging analysis, indicating the validity of the reaction scheme. Furthermore, alignment of amino acid sequences of 258 SmChiA-like proteins revealed that tryptophan, not phenylalanine, is the predominant amino acid at the corresponding positions (Phe-232 and Phe-396 for SmChiA). Our study will be helpful for understanding the kinetic mechanisms and further improvement of crystalline chitin hydrolytic activity of SmChiA mutants.

DOI： 10.1074/jbc.RA119.012078

Induced-Fit Pathway Accelerated Binding of Agitoxin-2 to A K<SUP>+</SUP> Channel Imaged by HS-AFM

Sumino, A; Sumikama, T; Uchihashi, T; Oiki, S

BIOPHYSICAL JOURNAL Vol. 118 ( 3 ) page： 236A - 236A 2020.2

Construction of a Hexameric Hemoprotein Sheet and Direct Observation of Dynamic Process of its Formation Reviewed

Koji Oohora, Shota Hirayama, Takayuki Uchihashi, Takashi Hayashi

Chemistry Letters Vol. 49 ( 2 ) page： 186 - 190 2020.2

Language：English Publishing type：Research paper (scientific journal) Publisher：The Chemical Society of Japan

<p>A two-dimensional sheet assembly of a hexameric hemoprotein was constructed. A single cysteine residue was introduced onto each subunit surface in the hexameric hemoprotein and modified with a maleimide-tethering tripeptide FGG tag to provide a host–guest interaction between one cucurbit[8]uril molecule (CB8) and two FGG tags. The addition of CB8 into the engineered protein forms the assembly as a sheet-like precipitate. High-speed atomic force microscopic measurements directly reveal the detailed structure and the dynamic process of formation.</p>

DOI： 10.1246/cl.190855

High-speed near-field fluorescence microscopy combined with high-speed atomic force microscopy for biological studies Reviewed International journal

Takayuki Umakoshi, Shingo Fukuda, Ryota Iino, Takayuki Uchihashi, Toshio Ando

BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS Vol. 1864 ( 2 ) page： 129325 2020.2

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

Background: High-speed atomic force microscopy (HS-AFM) has successfully visualized a variety of protein molecules during their functional activity. However, it cannot visualize small molecules interacting with proteins and even protein molecules when they are encapsulated. Thus, it has been desired to achieve techniques enabling simultaneous optical/AFM imaging at high spatiotemporal resolution with high correlation accuracy.Methods: Scanning near-field optical microscopy (SNOM) is a candidate for the combination with HS-AFM. However, the imaging rate of SNOM has been far below that of HS-AFM. We here developed HS-SNOM and metal tip-enhanced total internal reflection fluorescence microscopy (TIRFM) by exploiting tip-scan HS-AFM and exploring methods to fabricate a metallic tip on a tiny HS-AFM cantilever.Results: In tip-enhanced TIRFM/HS-AFM, simultaneous video recording of the two modalities of images was demonstrated in the presence of fluorescent molecules in the bulk solution at relatively high concentration. By using fabricated metal-tip cantilevers together with our tip-scan HS-AFM setup equipped with SNOM optics, we could perform simultaneous HS-SNOM/HS-AFM imaging, with correlation analysis between the two overlaid images being facilitated.Conclusions: This study materialized simultaneous tip-enhanced TIRFM/HS-AFM and HS-SNOM/HS-AFM imaging at high spatiotemporal resolution. Although some issues remain to be solved in the future, these correlative microscopy methods have a potential to increase the versatility of HS-AFM in biological research.General significance: We achieved an imaging rate of similar to 3 s/frame for SNOM imaging, more than 100-times higher than the typical SNOM imaging rate. We also demonstrated similar to 39 nm resolution in HS-SNOM imaging of fluorescently labeled DNA in solution.

DOI： 10.1016/j.bbagen.2019.03.011

Supramolecular tholos-like architecture constituted by archaeal proteins without functional annotation Reviewed International journal

Maho Yagi-Utsumi, Arunima Sikdar, Chihong Song, Jimin Park, Rintaro Inoue, Hiroki Watanabe, Raymond N. Burton-Smith, Toshiya Kozai, Tatsuya Suzuki, Atsuji Kodama, Kentaro Ishii, Hirokazu Yagi, Tadashi Satoh, Susumu Uchiyama, Takayuki Uchihashi, Keehyoung Joo, Jooyoung Lee, Masaaki Sugiyama, Kazuyoshi Murata, Koichi Kato

SCIENTIFIC REPORTS Vol. 10 ( 1 ) page： 1540 - 1540 2020.1

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

Euryarchaeal genomes encode proteasome-assembling chaperone homologs, PbaA and PbaB, although archaeal proteasome formation is a chaperone-independent process. Homotetrameric PbaB functions as a proteasome activator, while PbaA forms a homopentamer that does not interact with the proteasome. Notably, PbaA forms a complex with PF0014, an archaeal protein without functional annotation. In this study, based on our previous research on PbaA crystal structure, we performed an integrative analysis of the supramolecular structure of the PbaA/PF0014 complex using native mass spectrometry, solution scattering, high-speed atomic force microscopy, and electron microscopy. The results indicated that this highly thermostable complex constitutes ten PbaA and ten PF0014 molecules, which are assembled into a dumbbell-shaped structure. Two PbaA homopentameric rings correspond to the dumbbell plates, with their N-termini located outside of the plates and C-terminal segments left mobile. Furthermore, mutant PbaA lacking the mobile C-terminal segment retained the ability to form a complex with PF0014, allowing 3D modeling of the complex. The complex shows a five-column tholos-like architecture, in which each column comprises homodimeric PF0014, harboring a central cavity, which can potentially accommodate biomacromolecules including proteins. Our findings provide insight into the functional roles of Pba family proteins, offering a novel framework for designing functional protein cages.

DOI： 10.1038/s41598-020-58371-2

Thermoresponsive Micellar Assembly Constructed from a Hexameric Hemoprotein Modified with Poly(N-isopropylacrylamide) toward an Artificial Light-Harvesting System. Reviewed International journal

Shota Hirayama, Koji Oohora, Takayuki Uchihashi, Takashi Hayashi

Journal of the American Chemical Society Vol. 142 ( 4 ) page： 1822 - 1831 2020.1

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

Artificial protein assemblies inspired by nature have significant potential in development of emergent functional materials. In order to construct an artificial protein assembly, we employed a mutant of a thermostable hemoprotein, hexameric tyrosine-coordinated heme protein (HTHP), as a building block. The HTHP mutant which has cysteine residues introduced on the bottom surface of its columnar structure was reacted with maleimide-tethering thermoresponsive poly(N-isopropylacrylamide), PNIPAAm, to generate the protein assembly upon heating. The site-specific modification of the cysteine residues with PNIPAAm on the protein surface was confirmed by SDS-PAGE and analytical size exclusion chromatography (SEC). The PNIPAAm-modified HTHP (PNIPAAm-HTHP) is found to provide a 43 nm spherical structure at 60 °C, and the structural changes observed between the assembled and the disassembled forms were duplicated at least five times. High-speed atomic force microscopic measurements of the micellar assembly supported by cross-linkage with glutaraldehyde indicate that the protein matrices are located on the surface of the sphere and cover the inner PNIPAAm core. Furthermore, substitution of heme with a photosensitizer, Zn protoporphyrin IX (ZnPP), in the micellar assembly provides an artificial light-harvesting system. Photochemical measurements of the ZnPP-substituted micellar assembly demonstrate that energy migration among the arrayed ZnPP molecules occurs within the range of several tens of picoseconds. Our present work represents the first example of an artificial light-harvesting system based on an assembled hemoprotein oligomer structure to replicate natural light-harvesting systems.

DOI： 10.1021/jacs.9b10080

Corrigendum to "Dynamics of Inter-Molecular Interactions Between Single Aβ42 Oligomeric and Aggregate Species by High-Speed Atomic Force Microscopy". Reviewed International journal

Lei Feng, Hiroki Watanabe, Paul Molino, Gordon G Wallace, Son L Phung, Takayuki Uchihashi, Michael J Higgins

Journal of molecular biology Vol. 432 ( 2 ) page： 633 - 633 2020.1

Rad50 zinc hook functions as a constitutive dimerization module interchangeable with SMC hinge. Reviewed International journal

Hisashi Tatebe, Chew Theng Lim, Hiroki Konno, Kazuhiro Shiozaki, Akira Shinohara, Takayuki Uchihashi, Asako Furukohri

Nature communications Vol. 11 ( 1 ) page： 370 - 370 2020.1

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

The human Mre11/Rad50 complex is one of the key factors in genome maintenance pathways. Previous nanoscale imaging by atomic force microscopy (AFM) showed that the ring-like structure of the human Mre11/Rad50 complex transiently opens at the zinc hook of Rad50. However, imaging of the human Mre11/Rad50 complex by high-speed AFM shows that the Rad50 coiled-coil arms are consistently bridged by the dimerized hooks while the Mre11/Rad50 ring opens by disconnecting the head domains; resembling other SMC proteins such as cohesin or condensin. These architectural features are conserved in the yeast and bacterial Mre11/Rad50 complexes. Yeast strains harboring the chimeric Mre11/Rad50 complex containing the SMC hinge of bacterial condensin MukB instead of the RAD50 hook properly functions in DNA repair. We propose that the basic role of the Rad50 hook is similar to that of the SMC hinge, which serves as rather stable dimerization interface.

DOI： 10.1038/s41467-019-14025-0

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