2021/12/24 更新

写真a

ロビンソン ロバート チャールズ
ROBINSON ROBERT CHAR
ROBINSON ROBERT CHAR
所属
異分野基礎科学研究所 教授(特任)
職名
教授(特任)
外部リンク

学位

  • DPhil ( Oxford University )

研究キーワード

  • Evolution

  • Structural Biology

  • Actin cytoskeleton

  • Asgard archaea

研究分野

  • ライフサイエンス / 進化生物学

学歴

  • Oxford University   Laboratory of Molecular Biophysics   DPhil

    - 1996年

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  • University of British Columbia   Chemistry   MSc

    - 1990年

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  • King’s College, London University   Chemistry   BSc

    - 1987年

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経歴

  • IMCB, Singapore   Research Director

    2011年 - 2018年

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  • IMCB, Singapore   Senior Principal Investigator

    2005年 - 2011年

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  • Uppsala University   IMBIM   Lektor

    2001年 - 2005年

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  • The Salk Institute for Biological Studies   Postdoc

    1996年 - 2001年

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論文

  • Structure of a Minimal α-Carboxysome-Derived Shell and Its Utility in Enzyme Stabilization. 国際誌

    Yong Quan Tan, Samson Ali, Bo Xue, Wei Zhe Teo, Lay Hiang Ling, Maybelle Kho Go, Hong Lv, Robert C Robinson, Akihiro Narita, Wen Shan Yew

    Biomacromolecules   2021年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Bacterial microcompartments are proteinaceous shells that encase specialized metabolic processes in bacteria. Recent advances in simplification of these intricate shells have encouraged bioengineering efforts. Here, we construct minimal shells derived from the Halothiobacillus neapolitanus α-carboxysome, which we term Cso-shell. Using cryogenic electron microscopy, the atomic-level structures of two shell forms were obtained, reinforcing notions of evolutionarily conserved features in bacterial microcompartment shell architecture. Encapsulation peptide sequences that facilitate loading of heterologous protein cargo within the shells were identified. We further provide a first demonstration in utilizing minimal bacterial microcompartment-derived shells for hosting heterologous enzymes. Cso-shells were found to stabilize enzymatic activities against heat shock, presence of methanol co-solvent, consecutive freeze-thawing, and alkaline environments. This study yields insights into α-carboxysome assembly and advances the utility of synthetic bacterial microcompartments as nanoreactors capable of stabilizing enzymes with varied properties and reaction chemistries.

    DOI: 10.1021/acs.biomac.1c00533

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  • A structural model for (GlcNAc)2 translocation via a periplasmic chitooligosaccharide binding protein from marine Vibrio bacteria 国際誌

    Yoshihito Kitaoku, Tamo Fukamizo, Sawitree Kumsaoad, Prakayfun Ubonbal, Robert C. Robinson, Wipa Suginta

    Journal of Biological Chemistry   101071 - 101071   2021年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Elsevier {BV}  

    VhCBP is a periplasmic chitooligo saccharide-specific binding protein mainly responsible for translocation of the chitooligosaccharide (GlcNAc)2 across the double membranes of marine bacteria Vibrio harveyi. However, structural and thermodynamic understanding of the sugar binding/release processes of VhCBP are relatively unknown. VhCBP displayed the greatest affinity towards (GlcNAc)2, with lower affinity for longer-chain chitooligosaccharides [(GlcNAc)3-4]. (GlcNAc)4 partially occupied the closed sugar-binding groove, with two reducing-end GlcNAc units extending beyond the sugar-binding groove and barely characterized by weak electron density. Mutation of three conserved residues (Trp363, Asp365 and Trp513) to Ala resulted in drastic decreases in the binding affinity towards the preferred substrate (GlcNAc)2, indicating their significant contributions to sugar binding. The structure of the W513A-(GlcNAc)2 complex in a 'half-open' conformation unveiled the intermediary step of the (GlcNAc)2 translocation from the soluble CBP in periplasm to the inner membrane-transporting components. Isothermal calorimetry data suggested that VhCBP adopts the high affinity conformation to bind (GlcNAc)2, whilst its low affinity conformation facilitated sugar release. Thus, chitooligosaccharide translocation, conferred by periplasmic VhCBP, is a crucial step in the chitin catabolic pathway, allowing Vibrio bacteria to thrive in oceans where chitin is their major source of nutrients.

    DOI: 10.1016/j.jbc.2021.101071

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  • Functional display of bioactive peptides on the vGFP scaffold. 国際誌

    Sharon Min Qi Chee, Jantana Wongsantichon, Lau Sze Yi, Barindra Sana, Yuri Frosi, Robert C Robinson, Farid J Ghadessy

    Scientific reports   11 ( 1 )   10127 - 10127   2021年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Grafting bioactive peptides into recipient protein scaffolds can often increase their activities by conferring enhanced stability and cellular longevity. Here, we describe use of vGFP as a novel scaffold to display peptides. vGFP comprises GFP fused to a bound high affinity Enhancer nanobody that potentiates its fluorescence. We show that peptides inserted into the linker region between GFP and the Enhancer are correctly displayed for on-target interaction, both in vitro and in live cells by pull-down, measurement of target inhibition and imaging analyses. This is further confirmed by structural studies highlighting the optimal display of a vGFP-displayed peptide bound to Mdm2, the key negative regulator of p53 that is often overexpressed in cancer. We also demonstrate a potential biosensing application of the vGFP scaffold by showing target-dependent modulation of intrinsic fluorescence. vGFP is relatively thermostable, well-expressed and inherently fluorescent. These properties make it a useful scaffold to add to the existing tool box for displaying peptides that can disrupt clinically relevant protein-protein interactions.

    DOI: 10.1038/s41598-021-89421-y

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  • Mythical origins of the actin cytoskeleton. 国際誌

    Caner Akıl, Yoshihito Kitaoku, Linh T Tran, David Liebl, Han Choe, Duangkamon Muengsaen, Wipa Suginta, Albert Schulte, Robert C Robinson

    Current opinion in cell biology   68   55 - 63   2021年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    The origin of the eukaryotic cell is one of the greatest mysteries in modern biology. Eukaryotic-wide specific biological processes arose in the lost ancestors of eukaryotes. These distinctive features, such as the actin cytoskeleton, define what it is to be a eukaryote. Recent sequencing, characterization, and isolation of Asgard archaea have opened an intriguing window into the pre-eukaryotic cell. Firstly, sequencing of anaerobic sediments identified a group of uncultured organisms, Asgard archaea, which contain genes with homology to eukaryotic signature genes. Secondly, characterization of the products of these genes at the protein level demonstrated that Asgard archaea have related biological processes to eukaryotes. Finally, the isolation of an Asgard archaeon has produced a model organism in which the morphological consequences of the eukaryotic-like processes can be studied. Here, we consider the consequences for the Asgard actin cytoskeleton and for the evolution of a regulated actin system in the archaea-to-eukaryotic transition.

    DOI: 10.1016/j.ceb.2020.08.011

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  • The structure of the actin filament uncapping complex mediated by twinfilin 国際誌

    Dennis M. Mwangangi, Edward Manser, Robert C. Robinson

    Science Advances   7 ( 5 )   2021年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:American Association for the Advancement of Science ({AAAS})  

    Uncapping of actin filaments is essential for driving polymerization and depolymerization dynamics from capping protein-associated filaments; however, the mechanisms of uncapping leading to rapid disassembly are unknown. Here, we elucidated the x-ray crystal structure of the actin/twinfilin/capping protein complex to address the mechanisms of twinfilin uncapping of actin filaments. The twinfilin/capping protein complex binds to two G-actin subunits in an orientation that resembles the actin filament barbed end. This suggests an unanticipated mechanism by which twinfilin disrupts the stable capping of actin filaments by inducing a G-actin conformation in the two terminal actin subunits. Furthermore, twinfilin disorders critical actin-capping protein interactions, which will assist in the dissociation of capping protein, and may promote filament uncapping through a second mechanism involving V-1 competition for an actin-binding surface on capping protein. The extensive interactions with capping protein indicate that the evolutionary conserved role of twinfilin is to uncap actin filaments.

    DOI: 10.1126/sciadv.abd5271

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  • Chitoporin from Serratia marcescens: recombinant expression, purification and crystallization

    Rawiporn Amornloetwattana, Robert C. Robinson, Hannadige Sasimali Madusanka Soysa, Bert van den Berg, Wipa Suginta

    Acta Crystallographica Section F Structural Biology Communications   76 ( 11 )   536 - 543   2020年11月

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    掲載種別:研究論文(学術雑誌)   出版者・発行元:International Union of Crystallography ({IUCr})  

    DOI: 10.1107/S2053230X20013874

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  • Insights into the evolution of regulated actin dynamics via characterization of primitive gelsolin/cofilin proteins from Asgard archaea. 国際誌

    Caner Akıl, Linh T Tran, Magali Orhant-Prioux, Yohendran Baskaran, Edward Manser, Laurent Blanchoin, Robert C Robinson

    Proceedings of the National Academy of Sciences of the United States of America   117 ( 33 )   19904 - 19913   2020年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Proceedings of the National Academy of Sciences  

    Asgard archaea genomes contain potential eukaryotic-like genes that provide intriguing insight for the evolution of eukaryotes. The eukaryotic actin polymerization/depolymerization cycle is critical for providing force and structure in many processes, including membrane remodeling. In general, Asgard genomes encode two classes of actin-regulating proteins from sequence analysis, profilins and gelsolins. Asgard profilins were demonstrated to regulate actin filament nucleation. Here, we identify actin filament severing, capping, annealing and bundling, and monomer sequestration activities by gelsolin proteins from Thorarchaeota (Thor), which complete a eukaryotic-like actin depolymerization cycle, and indicate complex actin cytoskeleton regulation in Asgard organisms. Thor gelsolins have homologs in other Asgard archaea and comprise one or two copies of the prototypical gelsolin domain. This appears to be a record of an initial preeukaryotic gene duplication event, since eukaryotic gelsolins are generally comprise three to six domains. X-ray structures of these proteins in complex with mammalian actin revealed similar interactions to the first domain of human gelsolin or cofilin with actin. Asgard two-domain, but not one-domain, gelsolins contain calcium-binding sites, which is manifested in calcium-controlled activities. Expression of two-domain gelsolins in mammalian cells enhanced actin filament disassembly on ionomycin-triggered calcium release. This functional demonstration, at the cellular level, provides evidence for a calcium-controlled Asgard actin cytoskeleton, indicating that the calcium-regulated actin cytoskeleton predates eukaryotes. In eukaryotes, dynamic bundled actin filaments are responsible for shaping filopodia and microvilli. By correlation, we hypothesize that the formation of the protrusions observed from Lokiarchaeota cell bodies may involve the gelsolin-regulated actin structures.

    DOI: 10.1073/pnas.2009167117

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  • Intracellular ion concentrations and cation-dependent remodelling of bacterial MreB assemblies. 国際誌

    Dávid Szatmári, Péter Sárkány, Béla Kocsis, Tamás Nagy, Attila Miseta, Szilvia Barkó, Beáta Longauer, Robert C Robinson, Miklós Nyitrai

    Scientific reports   10 ( 1 )   12002 - 12002   2020年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Here, we measured the concentrations of several ions in cultivated Gram-negative and Gram-positive bacteria, and analyzed their effects on polymer formation by the actin homologue MreB. We measured potassium, sodium, chloride, calcium and magnesium ion concentrations in Leptospira interrogans, Bacillus subtilis and Escherichia coli. Intracellular ionic strength contributed from these ions varied within the 130-273 mM range. The intracellular sodium ion concentration range was between 122 and 296 mM and the potassium ion concentration range was 5 and 38 mM. However, the levels were significantly influenced by extracellular ion levels. L. interrogans, Rickettsia rickettsii and E. coli MreBs were heterologously expressed and purified from E. coli using a novel filtration method to prepare MreB polymers. The structures and stability of Alexa-488 labeled MreB polymers, under varying ionic strength conditions, were investigated by confocal microscopy and MreB polymerization rates were assessed by measuring light scattering. MreB polymerization was fastest in the presence of monovalent cations in the 200-300 mM range. MreB filaments showed high stability in this concentration range and formed large assemblies of tape-like bundles that transformed to extensive sheets at higher ionic strengths. Changing the calcium concentration from 0.2 to 0 mM and then to 2 mM initialized rapid remodelling of MreB polymers.

    DOI: 10.1038/s41598-020-68960-w

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  • Directed Computational Evolution of Quorum-Quenching Lactonases from the Amidohydrolase Superfamily. 国際誌

    Maybelle Kho Go, Li Na Zhao, Bo Xue, Shreyas Supekar, Robert C Robinson, Hao Fan, Wen Shan Yew

    Structure (London, England : 1993)   28 ( 6 )   635 - 642   2020年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    In this work, we present a generalizable directed computational evolution protocol to effectively reduce the sequence space to be explored in rational enzyme design. The protocol involves in silico mutation modeling and substrate docking to rapidly identify mutagenesis hotspots that may enhance an enzyme's substrate binding and overall catalysis. By applying this protocol to a quorum-quenching Geobacillus kaustophilus lactonase, GKL, we generated 1,881 single mutants and docked high-energy intermediates of nine acyl homoserine lactones onto them. We found that Phe28 and Tyr99 were two hotspots that produced most of the predicted top 20 mutants. Of the 180 enzyme-substrate combinations (top 20 mutants × 9 substrates), 51 (28%) exhibited enhanced substrate binding and 22 (12%) had better overall activity when compared with wild-type GKL. X-ray crystallographic studies of Y99C and Y99P provided rationalized explanations for the enhancement in enzyme function and corroborated the utility of the protocol.

    DOI: 10.1016/j.str.2020.03.011

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  • In vivo crystals reveal critical features of the interaction between cystic fibrosis transmembrane conductance regulator (CFTR) and the PDZ2 domain of Na+/H+ exchange cofactor NHERF1 国際誌

    Eleanor R. Martin, Alessandro Barbieri, Robert C. Ford, Robert C. Robinson

    Journal of Biological Chemistry   295 ( 14 )   4464 - 4476   2020年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Elsevier {BV}  

    Crystallization of recombinant proteins has been fundamental to our understanding of protein function, dysfunction, and molecular recognition. However, this information has often been gleaned under extremely nonphysiological protein, salt, and H+ concentrations. Here, we describe the development of a robust Inka1-Box (iBox)-PAK4cat system that spontaneously crystallizes in several mammalian cell types. The semi-quantitative assay described here allows the measurement of in vivo protein-protein interactions using a novel GFP-linked reporter system that produces fluorescent readouts from protein crystals. We combined this assay with in vitro X-ray crystallography and molecular dynamics studies to characterize the molecular determinants of the interaction between the PDZ2 domain of Na+/H+ exchange regulatory cofactor NHE-RF1 (NHERF1) and cystic fibrosis transmembrane conductance regulator (CFTR), a protein complex pertinent to the genetic disease cystic fibrosis. These experiments revealed the crystal structure of the extended PDZ domain of NHERF1 and indicated, contrary to what has been previously reported, that residue selection at positions -1 and -3 of the PDZ-binding motif influences the affinity and specificity of the NHERF1 PDZ2-CFTR interaction. Our results suggest that this system could be utilized to screen additional protein-protein interactions, provided they can be accommodated within the spacious iBox-PAK4cat lattice.

    DOI: 10.1074/jbc.RA119.012015

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  • Tree of motility - A proposed history of motility systems in the tree of life. 国際誌

    Makoto Miyata, Robert C Robinson, Taro Q P Uyeda, Yoshihiro Fukumori, Shun-Ichi Fukushima, Shin Haruta, Michio Homma, Kazuo Inaba, Masahiro Ito, Chikara Kaito, Kentaro Kato, Tsuyoshi Kenri, Yoshiaki Kinosita, Seiji Kojima, Tohru Minamino, Hiroyuki Mori, Shuichi Nakamura, Daisuke Nakane, Koji Nakayama, Masayoshi Nishiyama, Satoshi Shibata, Katsuya Shimabukuro, Masatada Tamakoshi, Azuma Taoka, Yosuke Tashiro, Isil Tulum, Hirofumi Wada, Ken-Ichi Wakabayashi

    Genes to cells : devoted to molecular & cellular mechanisms   25 ( 1 )   6 - 21   2020年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Motility often plays a decisive role in the survival of species. Five systems of motility have been studied in depth: those propelled by bacterial flagella, eukaryotic actin polymerization and the eukaryotic motor proteins myosin, kinesin and dynein. However, many organisms exhibit surprisingly diverse motilities, and advances in genomics, molecular biology and imaging have showed that those motilities have inherently independent mechanisms. This makes defining the breadth of motility nontrivial, because novel motilities may be driven by unknown mechanisms. Here, we classify the known motilities based on the unique classes of movement-producing protein architectures. Based on this criterion, the current total of independent motility systems stands at 18 types. In this perspective, we discuss these modes of motility relative to the latest phylogenetic Tree of Life and propose a history of motility. During the ~4 billion years since the emergence of life, motility arose in Bacteria with flagella and pili, and in Archaea with archaella. Newer modes of motility became possible in Eukarya with changes to the cell envelope. Presence or absence of a peptidoglycan layer, the acquisition of robust membrane dynamics, the enlargement of cells and environmental opportunities likely provided the context for the (co)evolution of novel types of motility.

    DOI: 10.1111/gtc.12737

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  • Author Correction: Coherent diffractive imaging of microtubules using an X-ray laser. 国際誌

    Gisela Brändén, Greger Hammarin, Rajiv Harimoorthy, Alexander Johansson, David Arnlund, Erik Malmerberg, Anton Barty, Stefan Tångefjord, Peter Berntsen, Daniel P DePonte, Carolin Seuring, Thomas A White, Francesco Stellato, Richard Bean, Kenneth R Beyerlein, Leonard M G Chavas, Holger Fleckenstein, Cornelius Gati, Umesh Ghoshdastider, Lars Gumprecht, Dominik Oberthür, David Popp, Marvin Seibert, Thomas Tilp, Marc Messerschmidt, Garth J Williams, N Duane Loh, Henry N Chapman, Peter Zwart, Mengning Liang, Sébastien Boutet, Robert C Robinson, Richard Neutze

    Nature communications   10 ( 1 )   4101 - 4101   2019年9月

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    記述言語:英語  

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

    DOI: 10.1038/s41467-019-12151-3

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  • Glutathione transferase Omega 1-1 (GSTO1-1) modulates Akt and MEK1/2 signaling in human neuroblastoma cell SH-SY5Y. 査読 国際誌

    Saisawang C, Wongsantichon J, Robinson RC, Ketterman AJ

    Proteins   87 ( 7 )   588 - 595   2019年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1002/prot.25683

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  • The role of gelsolin domain 3 in familial amyloidosis (Finnish type). 査読 国際誌

    Zorgati H, Larsson M, Ren W, Sim AYL, Gettemans J, Grimes JM, Li W, Robinson RC

    Proceedings of the National Academy of Sciences of the United States of America   116 ( 28 )   13958 - 13963   2019年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1073/pnas.1902189116

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  • The structure of a 15-stranded actin-like filament from Clostridium botulinum. 査読 国際誌

    Koh F, Narita A, Lee LJ, Tanaka K, Tan YZ, Dandey VP, Popp D, Robinson RC

    Nature communications   10 ( 1 )   2856 - 2856   2019年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1038/s41467-019-10779-9

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  • Coherent diffractive imaging of microtubules using an X-ray laser. 査読 国際誌

    Brändén G, Hammarin G, Harimoorthy R, Johansson A, Arnlund D, Malmerberg E, Barty A, Tångefjord S, Berntsen P, DePonte DP, Seuring C, White TA, Stellato F, Bean R, Beyerlein KR, Chavas LMG, Fleckenstein H, Gati C, Ghoshdastider U, Gumprecht L, Oberthür D, Popp D, Seibert M, Tilp T, Messerschmidt M, Williams GJ, Loh ND, Chapman HN, Zwart P, Liang M, Boutet S, Robinson RC, Neutze R

    Nature communications   10 ( 1 )   2589 - 2589   2019年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1038/s41467-019-10448-x

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  • Development and structural characterization of an engineered multi-copper oxidase reporter of protein-protein interactions. 査読

    Sana B, Chee SMQ, Wongsantichon J, Raghavan S, Robinson RC, Ghadessy FJ

    The Journal of biological chemistry   294 ( 17 )   7002 - 7012   2019年4月

  • Proteomics profiling of arginine methylation defines PRMT5 substrate specificity. 査読

    Musiani D, Bok J, Massignani E, Wu L, Tabaglio T, Ippolito MR, Cuomo A, Ozbek U, Zorgati H, Ghoshdastider U, Robinson RC, Guccione E, Bonaldi T

    Science signaling   12 ( 575 )   2019年4月

  • Structural evidence for the roles of divalent cations in actin polymerization and activation of ATP hydrolysis. 査読 国際誌

    Scipion CPM, Ghoshdastider U, Ferrer FJ, Yuen TY, Wongsantichon J, Robinson RC

    Proceedings of the National Academy of Sciences of the United States of America   115 ( 41 )   10345 - 10350   2018年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1073/pnas.1806394115

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  • Genomes of Asgard archaea encode profilins that regulate actin. 査読 国際誌

    Akıl C, Robinson RC

    Nature   562 ( 7727 )   439 - 443   2018年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1038/s41586-018-0548-6

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  • ATP competes with PIP2 for binding to gelsolin. 査読 国際誌

    Szatmári D, Xue B, Kannan B, Burtnick LD, Bugyi B, Nyitrai M, Robinson RC

    PloS one   13 ( 8 )   e0201826   2018年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1371/journal.pone.0201826

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  • Rapid production of pure recombinant actin isoforms in Pichia pastoris 査読 国際誌

    Tomoyuki Hatano, Salvatore Alioto, Emanuele Roscioli, Saravanan Palani, Scott T. Clarke, Anton Kamnev, Juan Ramon Hernandez-Fernaud, Lavanya Sivashanmugam, Bernardo Chapa-y-Lazo, Alexandra M.E. Jones, Robert C. Robinson, Karuna Sampath, Masanori Mishima, Andrew D. McAinsh, Bruce L. Goode, Mohan K. Balasubramanian

    Journal of Cell Science   131 ( 8 )   2018年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Company of Biologists Ltd  

    Actins are major eukaryotic cytoskeletal proteins, and they are involved in many important cell functions, including cell division, cell polarity, wound healing and muscle contraction. Despite obvious drawbacks, muscle actin, which is easily purified, is used extensively for biochemical studies of the non-muscle actin cytoskeleton. Here, we report a rapid and cost-effective method to purify heterologous actins expressed in the yeast Pichia pastoris. Actin is expressed as a fusion with the actin-binding protein thymosin β4 and purified by means of an affinity tag introduced in the fusion. Following cleavage of thymosin β4 and the affinity tag, highly purified functional full-length actin is liberated. We purify actins from Saccharomyces cerevisiae and Schizosaccharomyces pombe, and the β- and γ-isoforms of human actin. We also report a modification of the method that facilitates expression and purification of arginylated actin, a form of actin thought to regulate dendritic actin networks in mammalian cells. The methods we describe can be performed in all laboratories equipped for molecular biology, and should greatly facilitate biochemical and cell biological studies of the actin cytoskeleton.

    DOI: 10.1242/jcs.213827

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  • Advances in Structural Biology and the Application to Biological Filament Systems 査読 国際誌

    David Popp, Fujiet Koh, Clement P. M. Scipion, Umesh Ghoshdastider, Akihiro Narita, Kenneth C. Holmes, Robert C. Robinson

    BioEssays   40 ( 4 )   e1700213   2018年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:John Wiley and Sons Inc.  

    Structural biology has experienced several transformative technological advances in recent years. These include: development of extremely bright X-ray sources (microfocus synchrotron beamlines and free electron lasers) and the use of electrons to extend protein crystallography to ever decreasing crystal sizes
    and an increase in the resolution attainable by cryo-electron microscopy. Here we discuss the use of these techniques in general terms and highlight their application for biological filament systems, an area that is severely underrepresented in atomic resolution structures. We assemble a model of a capped tropomyosin-actin minifilament to demonstrate the utility of combining structures determined by different techniques. Finally, we survey the methods that attempt to transform high resolution structural biology into more physiological environments, such as the cell. Together these techniques promise a compelling decade for structural biology and, more importantly, they will provide exciting discoveries in understanding the designs and purposes of biological machines.

    DOI: 10.1002/bies.201700213

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  • Tropomyosins Regulate the Severing Activity of Gelsolin in Isoform-Dependent and Independent Manners 査読 国際誌

    Nikolett Kis-Bicskei, Bálint Bécsi, Ferenc Erdődi, Robert C. Robinson, Beáta Bugyi, Tamás Huber, Miklós Nyitrai, Gábor Csaba Talián

    Biophysical Journal   114 ( 4 )   777 - 787   2018年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Biophysical Society  

    The actin cytoskeleton fulfills numerous key cellular functions, which are tightly regulated in activity, localization, and temporal patterning by actin binding proteins. Tropomyosins and gelsolin are two such filament-regulating proteins. Here, we investigate how the effects of tropomyosins are coupled to the binding and activity of gelsolin. We show that the three investigated tropomyosin isoforms (Tpm1.1, Tpm1.12, and Tpm3.1) bind to gelsolin with micromolar or submicromolar affinities. Tropomyosin binding enhances the activity of gelsolin in actin polymerization and depolymerization assays. However, the effects of the three tropomyosin isoforms varied. The tropomyosin isoforms studied also differed in their ability to protect pre-existing actin filaments from severing by gelsolin. Based on the observed specificity of the interactions between tropomyosins, actin filaments, and gelsolin, we propose that tropomyosin isoforms specify which populations of actin filaments should be targeted by, or protected from, gelsolin-mediated depolymerization in living cells.

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  • Structure-activity studies of Mdm2/Mdm4-binding stapled peptides comprising non-natural amino acids 査読

    Sharon Min Qi Chee, Jantana Wongsantichon, Jiawei Siau, Dawn Thean, Fernando Ferrer, Robert C. Robinson, David P. Lane, Christopher J. Brown, Farid J. Ghadessy

    PLOS ONE   12 ( 12 )   e0189379   2017年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PUBLIC LIBRARY SCIENCE  

    As primary p53 antagonists, Mdm2 and the closely related Mdm4 are relevant cancer therapeutic targets. We have previously described a series of cell-permeable stapled peptides that bind to Mdm2 with high affinity, resulting in activation of the p53 tumour suppressor. Within this series, highest affinity was obtained by modification of an obligate tryptophan residue to the non-natural L-6-chlorotryptophan. To understand the structural basis for improved affinity we have solved the crystal structure of this stapled peptide (M011) bound to Mdm2 (residues 6-125) at 1.66 angstrom resolution. Surprisingly, near identity to the structure of a related peptide (M06) without the 6-chloro modification is observed. Further analysis of linear and stapled peptides comprising 6-Me-tryptophan provides mechanistic insight into dual Mdm2/Mdm4 antagonism and confirms L98 of Mdm4 as a mutable steric gate. The results also highlight a possible role of the flexible hinge region in determining Mdm2/Mdm4 plasticity.

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  • Flow-aligned, single-shot fiber diffraction using a femtosecond X-ray free-electron laser 査読 国際誌

    David Popp, N. Duane Loh, Habiba Zorgati, Umesh Ghoshdastider, Lu Ting Liow, Magdalena I. Ivanova, Marten Larsson, Daniel P. DePonte, Richard Bean, Kenneth R. Beyerlein, Cornelius Gati, Dominik Oberthuer, David Arnlund, Gisela Branden, Peter Berntsen, Duilio Cascio, Leonard M. G. Chavas, Joe P. J. Chen, Ke Ding, Holger Fleckenstein, Lars Gumprecht, Rajiv Harimoorthy, Estelle Mossou, Michael R. Sawaya, Aaron S. Brewster, Johan Hattne, Nicholas K. Sauter, Marvin Seibert, Carolin Seuring, Francesco Stellato, Thomas Tilp, David S. Eisenberg, Marc Messerschmidt, Garth J. Williams, Jason E. Koglin, Lee Makowski, Rick P. Millane, Trevor Forsyth, Sebastien Boutet, Thomas A. White, Anton Barty, Henry Chapman, Swaine L. Chen, Mengning Liang, Richard Neutze, Robert C. Robinson

    CYTOSKELETON   74 ( 12 )   472 - 481   2017年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY  

    A major goal for X-ray free-electron laser (XFEL) based science is to elucidate structures of biological molecules without the need for crystals. Filament systems may provide some of the first single macromolecular structures elucidated by XFEL radiation, since they contain one-dimensional translational symmetry and thereby occupy the diffraction intensity region between the extremes of crystals and single molecules. Here, we demonstrate flow alignment of as few as 100 filaments (Escherichia coli pili, F-actin, and amyloid fibrils), which when intersected by femtosecond X-ray pulses result in diffraction patterns similar to those obtained from classical fiber diffraction studies. We also determine that F-actin can be flow-aligned to a disorientation of approximately 5 degrees. Using this XFEL-based technique, we determine that gelsolin amyloids are comprised of stacked -strands running perpendicular to the filament axis, and that a range of order from fibrillar to crystalline is discernable for individual -synuclein amyloids.

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  • Structural basis of small molecule ATPase inhibition of a human mitotic kinesin motor protein 査読 国際誌

    Hee-Won Park, Zhujun Ma, Haizhong Zhu, Shimin Jiang, Robert C. Robinson, Sharyn A. Endow

    SCIENTIFIC REPORTS   7 ( 1 )   15121 - 15121   2017年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATURE PUBLISHING GROUP  

    Kinesin microtubule motor proteins play essential roles in division, including attaching chromosomes to spindles and crosslinking microtubules for spindle assembly. Human kinesin-14 KIFC1 is unique in that cancer cells with amplified centrosomes are dependent on the motor for viable division because of its ability to cluster centrosomes and form bipolar spindles, but it is not required for division in almost all normal cells. Screens for small molecule inhibitors of KIFC1 have yielded several candidates for further development, but obtaining structural data to determine their sites of binding has been difficult. Here we compare a previously unreported KIFC1 crystal structure with new structures of two closely related kinesin-14 proteins, Ncd and KIFC3, to determine the potential binding site of a known KIFC1 ATPase inhibitor, AZ82. We analyze the previously identified kinesin inhibitor binding sites and identify features of AZ82 that favor binding to one of the sites, the alpha 4/alpha 6 site. This selectivity can be explained by unique structural features of the KIFC1 alpha 4/alpha 6 binding site. These features may help improve the drug-like properties of AZ82 and other specific KIFC1 inhibitors.

    DOI: 10.1038/s41598-017-14754-6

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  • Systematic Moiety Variations of Ultrashort Peptides Produce Profound Effects on Self-Assembly, Nanostructure Formation, Hydrogelation, and Phase Transition 査読 国際誌

    Kiat Hwa Chan, Bo Xue, Robert C. Robinson, Charlotte A. E. Hauser

    SCIENTIFIC REPORTS   7 ( 1 )   12897 - 12897   2017年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATURE PUBLISHING GROUP  

    Self-assembly of small biomolecules is a prevalent phenomenon that is increasingly being recognised to hold the key to building complex structures from simple monomeric units. Small peptides, in particular ultrashort peptides containing up to seven amino acids, for which our laboratory has found many biomedical applications, exhibit immense potential in this regard. For next-generation applications, more intricate control is required over the self-assembly processes. We seek to find out how subtle moiety variation of peptides can affect self-assembly and nanostructure formation. To this end, we have selected a library of 54 tripeptides, derived from systematic moiety variations from seven tripeptides. Our study reveals that subtle structural changes in the tripeptides can exert profound effects on self-assembly, nanostructure formation, hydrogelation, and even phase transition of peptide nanostructures. By comparing the X-ray crystal structures of two tripeptides, acetylated leucineleucine-glutamic acid (Ac-LLE) and acetylated tyrosine-leucine-aspartic acid (Ac-YLD), we obtained valuable insights into the structural factors that can influence the formation of supramolecular peptide structures. We believe that our results have major implications on the understanding of the factors that affect peptide self-assembly. In addition, our findings can potentially assist current computational efforts to predict and design self-assembling peptide systems for diverse biomedical applications.

    DOI: 10.1038/s41598-017-12694-9

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  • Direct and convenient measurement of plasmid stability in lab and clinical isolates of E-coli 査読 国際誌

    Siyi Chen, Marten Larsson, Robert C. Robinson, Swaine L. Chen

    SCIENTIFIC REPORTS   7 ( 1 )   4788 - 4788   2017年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATURE PUBLISHING GROUP  

    Plasmids are important mobile elements in bacteria, contributing to evolution, virulence, and antibiotic resistance. Natural plasmids are generally large and maintained at low copy number and thus prone to be lost. Therefore, dedicated plasmid maintenance systems have evolved, leading to plasmid loss rates as low as 1 per 107 divisions. These low rates complicate studies of plasmid loss, as traditional techniques for measuring plasmid loss are laborious and not quantitative. To overcome these limitations, we leveraged a stringent negative selection system to develop a method for performing direct, quantitative measurements of plasmid loss in E. coli. We applied our method to gain mechanistic insights into a heterologously reconstituted segregation system in lab strains and clinical isolates of E. coli. We also performed direct stability studies of a currently circulating resistance plasmid in a clinical isolate, strain EC958, which is a member of the rapidly expanding global ST131 E. coli clone. Our results establish the foundational assays required to screen for small molecules targeting plasmid stability, which could complement current strategies for reducing the spread of antibiotic resistance, complementing other strategies for treating antibiotic resistant bacteria.

    DOI: 10.1038/s41598-017-05219-x

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  • Plastin increases cortical connectivity to facilitate robust polarization and timely cytokinesis 査読

    Wei Yung Ding, Hui Ting Ong, Yusuke Hara, Jantana Wongsantichon, Yusuke Toyama, Robert C. Robinson, Francois Nedelec, Ronen Zaidel-Bar

    JOURNAL OF CELL BIOLOGY   216 ( 5 )   1371 - 1386   2017年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ROCKEFELLER UNIV PRESS  

    The cell cortex is essential to maintain animal cell shape, and contractile forces generated within it by nonmuscle myosin II (NMY-2) drive cellular morphogenetic processes such as cytokinesis. The role of actin cross-linking proteins in cortical dynamics is still incompletely understood. Here, we show that the evolutionarily conserved actin bundling/cross-linking protein plastin is instrumental for the generation of potent cortical actomyosin contractility in the Caenorhabditis elegans zygote. PLST-1 was enriched in contractile structures and was required for effective coalescence of NMY-2 filaments into large contractile foci and for long-range coordinated contractility in the cortex. In the absence of PLST-1, polarization was compromised, cytokinesis was delayed or failed, and 50% of embryos died during development. Moreover, mathematical modeling showed that an optimal amount of bundling agents enhanced the ability of a network to contract. We propose that by increasing the connectivity of the F-actin meshwork, plastin enables the cortex to generate stronger and more coordinated forces to accomplish cellular morphogenesis.

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  • Yersinia effector protein (YopO)-mediated phosphorylation of host gelsolin causes calcium-independent activation leading to disruption of actin dynamics 査読

    Pavithra Singaravelu, Wei Lin Lee, Sheena Wee, Umesh Ghoshdastider, Ke Ding, Jayantha Gunaratne, Jonathan M. Grimes, Kunchithapadam Swaminathan, Robert C. Robinson

    JOURNAL OF BIOLOGICAL CHEMISTRY   292 ( 19 )   8092 - +   2017年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Pathogenic Yersinia bacteria cause a range of human diseases. To modulate and evade host immune systems, these yersiniae inject effector proteins into host macrophages. One such protein, the serine/threonine kinase YopO (YpkA in Yersinia pestis), uses monomeric actin as bait to recruit and phosphorylate host actin polymerization-regulating proteins, including the actin-severing protein gelsolin, to disrupt actin filaments and thus impair phagocytosis. However, the YopO phosphorylation sites on gelsolin and the consequences of YopO-mediated phosphorylation on actin remodeling have yet to be established. Here we determined the effects of YopO-mediated phosphorylation on gelsolin and identified its phosphorylation sites by mass spectrometry. YopO phosphorylated gelsolin in the linker region between gelsolin homology domains G3 and G4, which, in the absence of calcium, are compacted but adopt an open conformation in the presence of calcium, enabling actin binding and severing. Using phosphomimetic and phospho-deletion gelsolin mutants, we found that YopO-mediated phosphorylation partially mimics calcium-dependent activation of gelsolin, potentially contributing to a reduction in filamentous actin and altered actin dynamics in phagocytic cells. In summary, this work represents the first report of the functional outcome of serine/threonine phosphorylation in gelsolin regulation and provides critical insight into how YopO disrupts normal gelsolin function to alter host actin dynamics and thus cripple phagocytosis.

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  • Structural Basis for pH-mediated Regulation of F-actin Severing by Gelsolin Domain 1 査読

    Jing-song Fan, Honzhen Goh, Ke Ding, Bo Xue, Robert C. Robinson, Daiwen Yang

    SCIENTIFIC REPORTS   7   45230   2017年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATURE PUBLISHING GROUP  

    Six-domain gelsolin regulates actin structural dynamics through its abilities to sever, cap and uncap F-actin. These activities are modulated by various cellular parameters like Ca2+ and pH. Until now, only the molecular activation mechanism of gelsolin by Ca2+ has been understood relatively well. The fragment comprising the first domain and six residues from the linker region into the second domain has been shown to be similar to the full-length protein in F-actin severing activity in the absence of Ca2+ at pH 5. To understand how this gelsolin fragment is activated for F-actin severing by lowering pH, we solved its NMR structures at both pH 7.3 and 5 in the absence of Ca2+ and measured the pKa values of acidic amino acid residues and histidine residues. The overall structure and dynamics of the fragment are not affected significantly by pH. Nevertheless, local structural changes caused by protonation of His29 and Asp109 result in the activation on lowering the pH, and protonation of His151 directly effects filament binding since it resides in the gelsolin/actin interface. Mutagenesis studies support that His29, Asp109 and His151 play important roles in the pH-dependent severing activity of the gelsolin fragment.

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  • Mechanisms of Yersinia YopO kinase substrate specificity 査読

    Wei Lin Lee, Pavithra Singaravelu, Sheena Wee, Bo Xue, Khay Chun Ang, Jayantha Gunaratne, Jonathan M. Grimes, Kunchithapadam Swaminathan, Robert C. Robinson

    SCIENTIFIC REPORTS   7   39998   2017年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATURE PUBLISHING GROUP  

    Yersinia bacteria cause a range of human diseases, including yersiniosis, Far East scarlet-like fever and the plague. Yersiniae modulate and evade host immune defences through injection of Yersinia outer proteins (Yops) into phagocytic cells. One of the Yops, YopO (also known as YpkA) obstructs phagocytosis through disrupting actin filament regulation processes - inhibiting polymerization-promoting signaling through sequestration of Rac/Rho family GTPases and by using monomeric actin as bait to recruit and phosphorylate host actin-regulating proteins. Here we set out to identify mechanisms of specificity in protein phosphorylation by YopO that would clarify its effects on cytoskeleton disruption. We report the MgADP structure of Yersinia enterocolitica YopO in complex with actin, which reveals its active site architecture. Using a proteome-wide kinase-interacting substrate screening (KISS) method, we identified that YopO phosphorylates a wide range of actin-modulating proteins and located their phosphorylation sites by mass spectrometry. Using artificial substrates we clarified YopO's substrate length requirements and its phosphorylation consensus sequence. These findings provide fresh insight into the mechanism of the YopO kinase and demonstrate that YopO executes a specific strategy targeting actin-modulating proteins, across multiple functionalities, to compete for control of their native phospho-signaling, thus hampering the cytoskeletal processes required for macrophage phagocytosis.

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  • Directed Evolution of a Fluorinase for Improved Fluorination Efficiency with a Non-native Substrate 査読

    Huihua Sun, Wan Lin Yeo, Yee Hwee Lim, Xinying Chew, Derek John Smith, Bo Xue, Kok Ping Chan, Robert C. Robinson, Edward G. Robins, Huimin Zhao, Ee Lui Ang

    ANGEWANDTE CHEMIE-INTERNATIONAL EDITION   55 ( 46 )   14275 - 14278   2016年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-V C H VERLAG GMBH  

    Fluorinases offer an environmentally friendly alternative for selective fluorination under mild conditions. However, their diversity is limited in nature and they have yet to be engineered through directed evolution. Herein, we report the directed evolution of the fluorinase FlA1 for improved conversion of the non-native substrate 5'-chloro-5'-deoxyadenosine (5'-ClDA) into 5'-fluoro-5'-deoxyadenosine (5'-FDA). The evolved variants, fah2081 (A279Y) and fah2114 (F213Y, A279L), were successfully applied in the radiosynthesis of 5-'[F-18] FDA, with overall radiochemical conversion (RCC) more than 3-fold higher than wild-type FlA1. Kinetic studies of the two-step reaction revealed that the variants show a significantly improved k(cat) value in the conversion of 5'-ClDA into Sadenosyl-l-methionine (SAM) but a reduced k(cat) value in the conversion of SAM into 5'-FDA.

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  • Large-scale purification and in vitro characterization of the assembly of MreB from Leptospira interrogans. 査読

    Barkó S, Szatmári D, Bódis E, Türmer K, Ujfalusi Z, Popp D, Robinson RC, Nyitrai M

    Biochimica et biophysica acta   1860 ( 9 )   1942 - 1952   2016年9月

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    掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/j.bbagen.2016.06.007

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  • Exploring the A22-Bacterial Actin MreB Interaction through Molecular Dynamics Simulations 査読

    Yaw Awuni, Shimin Jiang, Robert C. Robinson, Yuguang Mu

    JOURNAL OF PHYSICAL CHEMISTRY B   120 ( 37 )   9867 - 9874   2016年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER CHEMICAL SOC  

    MreB is an actin-like cytoskeleton protein that plays a vital role in the maintenance of the rod-shaped morphology of many bacteria. S-(3,4-Dichlorobenzyl) isothiourea (A22) is an antibiotic-like small molecule that perturbs the rod cell shape and has been suggested to inhibit MreB by targeting ATP hydrolysis. However, without the elucidation of the structure of the ATP-bound state of MreB in the presence of A22, the mechanism of A22 inhibition is still not clear. Here we apply conventional molecular dynamics simulations to explore the dynamics of the active site of MreB in complex with A22 and different nucleotides. We observe that hydrogen bonding between A22 and the catalytic Glu140 residue is not favored in the ATP-A22-bound state of MreB. Water dynamics analysis in the MreB active site reveals that in the presence of A22 water molecules are able to occupy positions suitable for ATP hydrolysis. Overall, our results are consistent with a mechanism in which A22 affects MreB polymerization/depolymerization dynamics in part through slowing phosphate release rather than by inhibiting ATP hydrolysis. These data can be incorporated in the design/development of the next generation of MreB inhibitors.

    DOI: 10.1021/acs.jpcb.6b05199

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  • Novel actin filaments from Bacillus thuringiensis form nanotubules for plasmid DNA segregation 査読

    Shimin Jiang, Akihiro Narita, David Popp, Umesh Ghoshdastider, Lin Jie Lee, Ramanujam Srinivasan, Mohan K. Balasubramanian, Toshiro Oda, Fujiet Koh, Marten Larsson, Robert C. Robinson

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   113 ( 9 )   E1200 - E1205   2016年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATL ACAD SCIENCES  

    Here we report the discovery of a bacterial DNA-segregating actin-like protein (BtParM) from Bacillus thuringiensis, which forms novel antiparallel, two-stranded, supercoiled, nonpolar helical filaments, as determined by electron microscopy. The BtParM filament features of supercoiling and forming antiparallel double-strands are unique within the actin fold superfamily, and entirely different to the straight, double-stranded, polar helical filaments of all other known ParMs and of eukaryotic F-actin. The BtParM polymers show dynamic assembly and subsequent disassembly in the presence of ATP. BtParR, the DNA-BtParM linking protein, stimulated ATP hydrolysis/phosphate release by BtParM and paired two supercoiled BtParM filaments to form a cylinder, comprised of four strands with inner and outer diameters of 57 angstrom and 145 angstrom, respectively. Thus, in this prokaryote, the actin fold has evolved to produce a filament system with comparable features to the eukaryotic chromosome-segregating microtubule.

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  • Structural complexity of filaments formed from the actin and tubulin folds 査読

    Shimin Jiang, Umesh Ghoshdastider, Akihiro Narita, David Popp, Robert C. Robinson

    Communicative and Integrative Biology   9 ( 6 )   e1242538   2016年

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    記述言語:英語   出版者・発行元:Taylor and Francis Inc.  

    From yeast to man, an evolutionary distance of 1.3 billion years, the F-actin filament structure has been conserved largely in line with the 94% sequence identity. The situation is entirely different in bacteria. In comparison to eukaryotic actins, the bacterial actin-like proteins (ALPs) show medium to low levels of sequence identity. This is extreme in the case of the ParM family of proteins, which often display less than 20% identity. ParMs are plasmid segregation proteins that form the polymerizing motors that propel pairs of plasmids to the extremities of a cell prior to cell division, ensuring faithful inheritance of the plasmid. Recently, exotic ParM filament structures have been elucidated that show ParM filament geometries are not limited to the standard polar pair of strands typified by actin. Four-stranded non-polar ParM filaments existing as open or closed nanotubules are found in Clostridium tetani and Bacillus thuringiensis, respectively. These diverse architectures indicate that the actin fold is capable of forming a large variety of filament morphologies, and that the conception of the "actin" filament has been heavily influenced by its conservation in eukaryotes. Here, we review the history of the structure determination of the eukaryotic actin filament to give a sense of context for the discovery of the new ParM filament structures. We describe the novel ParM geometries and predict that even more complex actin-like filaments may exist in bacteria. Finally, we compare the architectures of filaments arising from the actin and tubulin folds and conclude that the basic units possess similar properties that can each form a range of structures. Thus, the use of the actin fold in microfilaments and the tubulin fold for microtubules likely arose from a wider range of filament possibilities, but became entrenched as those architectures in early eukaryotes.

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  • Actin-Induced Structure in the Beta-Thymosin Family of Intrinsically Disordered Proteins 査読

    B. Xue, R. C. Robinson

    THYMOSINS   102   55 - 71   2016年

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    記述言語:英語   掲載種別:論文集(書籍)内論文   出版者・発行元:ELSEVIER ACADEMIC PRESS INC  

    Thymosin beta 4 (T beta 4) is a 43-amino acid signature motif peptide that defines the betathymosin (beta T) family of proteins. beta Ts are intrinsically unstructured in their free states and undergo disorder-to-order transitions in carrying out their biological functions. This property poses challenges in determining their 3D structures, mainly favoring structural studies on the complexes formed between beta Ts and their interaction partners. One of the beta Ts' primary binding partners is monomeric actin, a major component of the cytoskeleton in eukaryotic cells. T beta 4' s role in this system is to maintain the highly concentrated pool of monomeric actin that can be accessed through profilin by actin filament nucleating machineries. Here, we give an account of the structures of beta Ts that have been illuminated by nuclear magnetic resonance (NMR) and X-ray crystallography. NMR has been the method of choice for probing regions that have intrinsic conformational preference within the largely disordered beta Ts in their native states in solution. X-ray crystallography has demonstrated at atomic detail how beta Ts interact with actin. Detailed analysis of these structures highlights the disorder-to-order transition of T beta 4 in binding to actin and its isoform specificity.

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  • Epsilon glutathione transferases possess a unique class-conserved subunit interface motif that directly interacts with glutathione in the active site 査読

    Jantana Wongsantichon, Robert C. Robinson, Albert J. Ketterman

    BIOSCIENCE REPORTS   35 ( 6 )   2015年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PORTLAND PRESS LTD  

    Epsilon class glutathione transferases (GSTs) have been shown to contribute significantly to insecticide resistance. We report a new Epsilon class protein crystal structure from Drosophila melanogaster for the glutathione transferase DmGSTE6. The structure reveals a novel Epsilon clasp motif that is conserved across hundreds of millions of years of evolution of the insect Diptera order. This histidine-serine motif lies in the subunit interface and appears to contribute to quaternary stability as well as directly connecting the two glutathiones in the active sites of this dimeric enzyme.

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  • Epsilon glutathione transferases possess a unique class-conserved subunit interface motif that directly interacts with glutathione in the active site 査読

    Jantana Wongsantichon, Robert C. Robinson, Albert J. Ketterman

    BIOSCIENCE REPORTS   35   2015年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PORTLAND PRESS LTD  

    Epsilon class glutathione transferases (GSTs) have been shown to contribute significantly to insecticide resistance. We report a new Epsilon class protein crystal structure from Drosophila melanogaster for the glutathione transferase DmGSTE6. The structure reveals a novel Epsilon clasp motif that is conserved across hundreds of millions of years of evolution of the insect Diptera order. This histidine-serine motif lies in the subunit interface and appears to contribute to quaternary stability as well as directly connecting the two glutathiones in the active sites of this dimeric enzyme.

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  • An in cellulo-derived structure of PAK4 in complex with its inhibitor Inka1 査読

    Yohendran Baskaran, Khay C. Ang, Praju V. Anekal, Wee L. Chan, Jonathan M. Grimes, Ed Manser, Robert C. Robinson

    NATURE COMMUNICATIONS   6   8681   2015年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATURE PUBLISHING GROUP  

    PAK4 is a metazoan-specific kinase acting downstream of Cdc42. Here we describe the structure of human PAK4 in complex with Inka1, a potent endogenous kinase inhibitor. Using single mammalian cells containing crystals 50 mm in length, we have determined the in cellulo crystal structure at 2.95 angstrom resolution, which reveals the details of how the PAK4 catalytic domain binds cellular ATP and the Inka1 inhibitor. The crystal lattice consists only of PAK4-PAK4 contacts, which form a hexagonal array with channels of 80 angstrom in diameter that run the length of the crystal. The crystal accommodates a variety of other proteins when fused to the kinase inhibitor. Inka1-GFP was used to monitor the process crystal formation in living cells. Similar derivatives of Inka1 will allow us to study the effects of PAK4 inhibition in cells and model organisms, to allow better validation of therapeutic agents targeting PAK4.

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  • A Single Glycosidase Harnesses Different Pyranoside Ring Transition State Conformations for Hydrolysis of Mannosides and Glucosides 査読

    Anupong Tankrathok, Javier Iglesias-Fernandez, Rohan J. Williams, Salila Pengthaisong, Supaporn Baiya, Zalihe Hakki, Robert C. Robinson, Maria Hrmova, Carme Rovira, Spencer J. Williams, James R. Ketudat Cairns

    ACS CATALYSIS   5 ( 10 )   6041 - 6051   2015年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER CHEMICAL SOC  

    Hydrolysis of beta-D-mannosides by beta-mannosidases typically proceeds via a B-2,B-5 transition state conformation for the pyranoside ring, while that of beta-D-glucosides by beta-glucosidases proceeds through a distinct H-4(3) transition state conformation. However, rice Os7BGIu26 beta-glycosidase hydrolyzes 4-nitrophenyl beta-D-glucoside and beta-D-mannoside with similar efficiencies. The origin of this dual substrate specificity was investigated by kinetic, structural, and computational approaches. The glycosidase inhibitors glucoimidazole and mannoimidazole inhibited Os7BGIu26 with K-i values of 2.7 nM and 10.4 mu M, respectively. In X-ray crystal structures of complexes with Os7BGlu26, glucoimidazole bound to the active site in a E-4 conformation, while mannoimidazole bound in a B-2,B-5 conformation, suggesting different transition state conformations. Moreover, calculation of quantum mechanics/molecular mechanics (QM/MM) free energy landscapes showed that 4-nitrophenyl beta-D-glucoside adopts a S-1(3)/E-4 conformation in the Michaelis complex, while 4-nitrophenyl beta-D-mannoside adopts a S-1(5)/B-2,(5) conformation. The QM/MM simulations of Os7BGIu26 catalysis of hydrolysis also supported the itineraries of S-1(3) -> E-4/H-4(3)double dagger -> C-4(1) for beta-D-glucosides and S-1(5) -> B2,5 double dagger -> S-0(2) for beta-D-mannosides, thereby revealing that a single glycoside hydrolase can hydrolyze glycosides of different configurations via distinct transition state pyranoside conformations.

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  • Calcium-controlled conformational choreography in the N-terminal half of adseverin 査読

    Sakesit Chumnarnsilpa, Robert C. Robinson, Jonathan M. Grimes, Cedric Leyrat

    NATURE COMMUNICATIONS   6   8254   2015年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATURE PUBLISHING GROUP  

    Adseverin is a member of the calcium-regulated gelsolin superfamily of actin-binding proteins. Here we report the crystal structure of the calcium-free N-terminal half of adseverin (iA1-A3) and the Ca2+-bound structure of A3, which reveal structural similarities and differences with gelsolin. Solution small-angle X-ray scattering combined with ensemble optimization revealed a dynamic Ca2+-dependent equilibrium between inactive, intermediate and active conformations. Increasing calcium concentrations progressively shift this equilibrium from a main population of inactive conformation to the active form. Molecular dynamics simulations of iA1-A3 provided insights into Ca2+-induced destabilization, implicating a critical role for the A2 type II calcium-binding site and the A2A3 linker in the activation process. Finally, mutations that disrupt the A1/A3 interface increase Ca2+-independent F-actin severing by A1-A3, albeit at a lower efficiency than observed for gelsolin domains G1-G3. Together, these data address the calcium dependency of A1-A3 activity in relation to the calcium-independent activity of G1-G3.

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  • In search of the primordial actin filament 査読

    Umesh Ghoshdastider, Shimin Jiang, David Popp, Robert C. Robinson

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   112 ( 30 )   9150 - 9151   2015年7月

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    記述言語:英語   出版者・発行元:NATL ACAD SCIENCES  

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  • Synthetic Polyketide Enzymology: Platform for Biosynthesis of Antimicrobial Polyketides 査読

    Maybelle Kho Go, Jantana Wongsantichon, Vivian Wing Ngar Cheung, Jeng Yeong Chow, Robert C. Robinson, Wen Shan Yew

    ACS CATALYSIS   5 ( 7 )   4033 - 4042   2015年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER CHEMICAL SOC  

    Synthetic biology often employs enzymes in the biosynthesis of compounds for purposeful function. Here, we define synthetic enzymology as the application of enzymological principles in synthetic biology and describe its use as an enabling platform in synthetic biology' for the purposeful production of compounds of biomedical and commercial importance. In particular, we demonstrated the use of synthetic polyketide enzymology as a means to develop lead polyketide based compounds for antimicrobial therapeutics, as exemplified by the modular coupling of acid:CoA ligases to type III polyketide synthases in the biosynthesis and development of polyketide-based biochemicals. Using wild-type and rationally designed mutants of a type III polyketide synthase isolated from Oryza sativa (OsPKS), we produced a chemically diverse library of novel polyketides and identified two bioactive antimicrobials, 4-hydroxy-6-[(1E)-2-(4-hydroxyphenyl)ethenyl]-2H-pyran-2-one (bisnoryangonin) and 3,6,7-trihydroxy-2-(4-methoxybenzyl)-4H-1-benzopyran-4,5,8-trione (26OH), respectively, from a screen against a collection of Gram-positive and Gram-negative bacteria. The purification, crystallization, and structural resolution of recombinant OsPKS at 1.93 angstrom resolution are also reported. Using the described route of synthetic polyketide enzymology, a library of OsPKS mutants was generated as an additional means to increase the diversity of the polyketide product library. We expect the utility of synthetic enzymology to be extended to other classes of biomolecules and translated to various purposeful functions as the field of synthetic biology progresses.

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  • In search of the primordial actin filament 査読

    Umesh Ghoshdastider, Shimin Jiang, David Popp, Robert C. Robinson

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   112 ( 30 )   9150 - 9151   2015年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATL ACAD SCIENCES  

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    その他リンク: http://orcid.org/0000-0001-6367-6903

  • The evolution of compositionally and functionally distinct actin filaments 査読

    Peter W. Gunning, Umesh Ghoshdastider, Shane Whitaker, David Popp, Robert C. Robinson

    JOURNAL OF CELL SCIENCE   128 ( 11 )   2009 - 2019   2015年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:COMPANY OF BIOLOGISTS LTD  

    The actin filament is astonishingly well conserved across a diverse set of eukaryotic species. It has essentially remained unchanged in the billion years that separate yeast, Arabidopsis and man. In contrast, bacterial actin-like proteins have diverged to the extreme, and many of them are not readily identified from sequence-based homology searches. Here, we present phylogenetic analyses that point to an evolutionary drive to diversify actin filament composition across kingdoms. Bacteria use a one-filament-one-function system to create distinct filament systems within a single cell. In contrast, eukaryotic actin is a universal force provider in a wide range of processes. In plants, there has been an expansion of the number of closely related actin genes, whereas in fungi and metazoa diversification in tropomyosins has increased the compositional variety in actin filament systems. Both mechanisms dictate the subset of actin-binding proteins that interact with each filament type, leading to specialization in function. In this Hypothesis, we thus propose that different mechanisms were selected in bacteria, plants and metazoa, which achieved actin filament compositional variation leading to the expansion of their functional diversity.

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  • An ER-directed gelsolin nanobody targets the first step in amyloid formation in a gelsolin amyloidosis mouse model 査読

    Wouter Van Overbeke, Jantana Wongsantichon, Inge Everaert, Adriaan Verhelle, Olivier Zwaenepoel, Anantasak Loonchanta, Leslie D. Burtnick, Ariane De Ganck, Tino Hochepied, Jody Haigh, Claude Cuvelier, Wim Derave, Robert C. Robinson, Jan Gettemans

    HUMAN MOLECULAR GENETICS   24 ( 9 )   2492 - 2507   2015年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

    Hereditary gelsolin amyloidosis is an autosomal dominantly inherited amyloid disorder. A point mutation in the GSN gene (G654A being the most common one) results in disturbed calcium binding by the second gelsolin domain (G2). As a result, the folding of G2 is hampered, rendering the mutant plasma gelsolin susceptible to a proteolytic cascade. Consecutive cleavage by furin and MT1-MMP-like proteases generates 8 and 5 kDa amyloidogenic peptides that cause neurological, ophthalmological and dermatological findings. To this day, no specific treatment is available to counter the pathogenesis. Using GSN nanobody 11 as a molecular chaperone, we aimed to protect mutant plasma gelsolin from furin proteolysis in the trans-Golgi network. We report a transgenic, GSN nanobody 11 secreting mouse that was used for crossbreeding with gelsolin amyloidosis mice. Insertion of the therapeutic nanobody gene into the gelsolin amyloidosis mouse genome resulted in improved muscle contractility. X-ray crystal structure determination of the gelsolin G2:Nb11 complex revealed that Nb11 does not directly block the furin cleavage site. We conclude that nanobodies can be used to shield substrates from aberrant proteolysis and this approach might establish a novel therapeutic strategy in amyloid diseases.

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  • Yersinia effector YopO uses actin as bait to phosphorylate proteins that regulate actin polymerization 査読

    Wei Lin Lee, Jonathan M. Grimes, Robert C. Robinson

    NATURE STRUCTURAL & MOLECULAR BIOLOGY   22 ( 3 )   248 - 255   2015年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATURE PUBLISHING GROUP  

    Pathogenic Yersinia species evade host immune systems through the injection of Yersinia outer proteins (Yops) into phagocytic cells. One Yop, YopO, also known as YpkA, induces actin-filament disruption, impairing phagocytosis. Here we describe the X-ray structure of Yersinia enterocolitica YopO in complex with actin, which reveals that YopO binds to an actin monomer in a manner that blocks polymerization yet allows the bound actin to interact with host actin-regulating proteins. SILAC-MS and biochemical analyses confirm that actin-polymerization regulators such as VASP, EVL, WASP, gelsolin and the formin diaphanous 1 are directly sequestered and phosphorylated by YopO through formation of ternary complexes with actin. This leads to a model in which YopO at the membrane sequesters actin from polymerization while using the bound actin as bait to recruit, phosphorylate and misregulate host actin-regulating proteins to disrupt phagocytosis.

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  • Soluble overexpression and purification of bioactive human CCL2 in E-coli by maltose-binding protein 査読

    Thu Trang Thi Vu, Bon-Kyung Koo, Jung-A Song, Seon-Ha Chong, Cho Rong Park, Minh Tan Nguyen, Boram Jeong, Han-Bong Ryu, Jae Young Seong, Yeon Jin Jang, Robert Charles Robinson, Han Choe

    MOLECULAR BIOLOGY REPORTS   42 ( 3 )   651 - 663   2015年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPRINGER  

    Human chemokine (C-C motif) ligand 2 (hCCL2) is a small cytokine in the CC chemokine family that attracts monocytes, memory T lymphocytes, and natural killer cells to the site of tissue injury- or infection-induced inflammation. hCCL2 has been implicated in the pathogeneses of diseases characterized by monocytic infiltrates, including psoriasis, rheumatoid arthritis, atherosclerosis, multiple sclerosis, and insulin-resistant diabetes. The prokaryotic overexpression of hCCL2 has been investigated previously in an attempt to develop biomedical applications for this factor, but this has been hampered by protein misfolding and aggregation into inclusion bodies. In our present study, we screened 7 protein tags-Trx, GST, MBP, NusA, His8, PDI, and PDIb'a'aEuro"for their ability to allow the soluble overexpression of hCCL2. Three tags-MBP, His8, and PDI-solubilized more than half of the expressed hCCL2 fusion proteins. Lowering the expression temperature to 18 A degrees C significantly further improved the solubility of all fusion proteins. MBP was chosen for further study based on its solubility, expression level, ease of purification, and tag size. MBP-CCL2 was purified using conventional chromatography and cleaved using TEV or Factor Xa proteases. Biological activity was assessed using luciferase and cell migration assays. Factor Xa-cleaved hCCL2 was found to be active and TEV-cleaved hCCL2 showed relatively less activity. This is probably because the additional glycine residues present at the N-terminus of hCCL2 following TEV digestion interfere with the binding of hCCL2 to its receptor.

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  • Structural and Thermodynamic Insights into Chitooligosaccharide Binding to Human Cartilage Chitinase 3-like Protein 2 (CHI3L2 or YKL-39) 査読

    Araya Ranok, Jantana Wongsantichon, Robert C. Robinson, Wipa Suginta

    JOURNAL OF BIOLOGICAL CHEMISTRY   290 ( 5 )   2617 - 2629   2015年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Background: Human YKL-39 is currently recognized as a biomarker for osteoarthritis. Results: Crystal structures of YKL-39 reveal that chitooligosaccharide induces local conformational changes to stabilize sugarprotein complexes and that the protein contains five binding subsites for sugars. Conclusion: YKL-39 binds to chitooligosaccharide through enthalpic reactions. Significance: Our findings suggest how YKL-39 interacts with GlcNAc moieties of the natural ligands, which may possibly activate local tissue inflammation.
    Four crystal structures of human YKL-39 were solved in the absence and presence of chitooligosaccharides. The structure of YKL-39 comprises a major (/)(8) triose-phosphate isomerase barrel domain and a small + insertion domain. Structural analysis demonstrates that YKL-39 interacts with chitooligosaccharides through hydrogen bonds and hydrophobic interactions. The binding of chitin fragments induces local conformational changes that facilitate tight binding. Compared with other GH-18 members, YKL-39 has the least extended chitin-binding cleft, containing five subsites for sugars, namely (-3)(-2)(-1)(+1)(+2), with Trp-360 playing a prominent role in the sugar-protein interactions at the center of the chitin-binding cleft. Evaluation of binding affinities obtained from isothermal titration calorimetry and intrinsic fluorescence spectroscopy suggests that YKL-39 binds to chitooligosaccharides with K-d values in the micromolar concentration range and that the binding energies increase with the chain length. There were no significant differences between the K-d values of chitopentaose and chitohexaose, supporting the structural evidence for the five binding subsite topology. Thermodynamic analysis indicates that binding of chitooligosaccharide to YKL-39 is mainly driven by enthalpy.

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  • Arp2/3 complex regulates adipogenesis by controlling cortical actin remodelling 査読

    Wulin Yang, Shermaine Thein, Chun-Yan Lim, Russell E. Ericksen, Shigeki Sugii, Feng Xu, Robert C. Robinson, Jae Bum Kim, Weiping Han

    BIOCHEMICAL JOURNAL   464 ( 2 )   179 - 192   2014年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PORTLAND PRESS LTD  

    Extensive actin cytoskeleton remodelling occurs during adipocyte development. We have previously shown that disruption of stress fibres by the actin-severing protein cofilin is a requisite step in adipogenesis. However, it remains unclear whether actin nucleation and assembly into the cortical structure are essential for adipocyte development. In the present study we investigated the role of cortical actin assembly and of actin nucleation by the actin-related protein 2/3 (Arp2/3) complex in adipogenesis. Cortical actin structure formation started with accumulation of filamentous actin (F-actin) patches near the plasma membrane during adipogenesis. Depletion of Arp2/3 by knockdown of its subunits Arp3 or ARPC3 strongly impaired adipocyte differentiation, although adipogenesis-initiating factors were unaffected. Moreover, the assembly of F-actin-rich structures at the plasma membrane was suppressed and the cortical actin structure poorly developed after adipogenic induction in Arp2/3-deficient cells. Finally, we provide evidence that the cortical actin cytoskeleton is essential for efficient glucose transporter 4 (GLUT4) vesicle exocytosis and insulin signal transduction. These results show that the Arp2/3 complex is an essential regulator of adipocyte development through control of the formation of cortical actin structures, which may facilitate nutrient uptake and signalling events.

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  • Soluble prokaryotic expression and purification of crotamine using an N-terminal maltose-binding protein tag 査読

    Thu Trang Thi Vu, Boram Jeong, Jing Yu, Bon-Kyung Koo, Su-Hyun Jo, Robert Charles Robinson, Han Choe

    TOXICON   92   157 - 165   2014年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PERGAMON-ELSEVIER SCIENCE LTD  

    Crotamine is a peptide toxin found in the venom of the rattlesnake Crotalus durissus terrificus. Interestingly, crotamine demonstrates promising anticancer, antimicrobial, and antifungal activities. The crotamine peptide can also deliver plasmids into rapidly dividing cells, such as cancer and stem cells, and demonstrates potent analgesic effects. Efficiently producing crotamine in mammalian cells is difficult because it is both cell-permeable and cytotoxic. Prokaryotic expression of this peptide is also difficult to maintain because it does not fold properly in the cytoplasm, resulting in aggregation and in the formation of inclusion bodies.
    In our current study, we show for the first time that N-terminal fusion with three protein tags-N-utilization substance protein A (NusA), protein disulfide isomerase b'a' domain (PDIb'a'), and maltose-binding protein (MBP) enables the soluble overexpression of crotamine in the cytoplasm of Escherichia coli. MBP-tagged crotamine was purified using Ni affinity, anion exchange, and MBP chromatography. The tag was cleaved using TEV protease, and the final product was pure on a silver-stained gels. In total, 0.9 mg pure crotamine was obtained from each liter of bacterial culture with endotoxin level approximately 0.15 EU/mu g, which is low enough to use in biomedical applications. The identity and intramolecular disulfide bonds were confirmed using MALDI-TOF MS analysis. Purified crotamine inhibited the hKv1.3 channel (but not hKv1.5) in a dose-dependent manner with IC50 value of 67.2 +/- 44.7 nM (n = 10), indicating the correct protein folding. The crotamine product fused with MBP at its N-terminus also inhibited the hKv1.3 channel, suggesting that the N-terminus is not involved in the channel binding of the toxin. (C) 2014 Elsevier Ltd. All rights reserved.

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  • Arp2/3 complex regulates adipogenesis by controlling cortical actin remodelling 査読

    Wulin Yang, Shermaine Thein, Chun-Yan Lim, Russell E. Ericksen, Shigeki Sugii, Feng Xu, Robert C. Robinson, Jae Bum Kim, Weiping Han

    BIOCHEMICAL JOURNAL   464   179 - 192   2014年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PORTLAND PRESS LTD  

    Extensive actin cytoskeleton remodelling occurs during adipocyte development. We have previously shown that disruption of stress fibres by the actin-severing protein cofilin is a requisite step in adipogenesis. However, it remains unclear whether actin nucleation and assembly into the cortical structure are essential for adipocyte development. In the present study we investigated the role of cortical actin assembly and of actin nucleation by the actin-related protein 2/3 (Arp2/3) complex in adipogenesis. Cortical actin structure formation started with accumulation of filamentous actin (F-actin) patches near the plasma membrane during adipogenesis. Depletion of Arp2/3 by knockdown of its subunits Arp3 or ARPC3 strongly impaired adipocyte differentiation, although adipogenesis-initiating factors were unaffected. Moreover, the assembly of F-actin-rich structures at the plasma membrane was suppressed and the cortical actin structure poorly developed after adipogenic induction in Arp2/3-deficient cells. Finally, we provide evidence that the cortical actin cytoskeleton is essential for efficient glucose transporter 4 (GLUT4) vesicle exocytosis and insulin signal transduction. These results show that the Arp2/3 complex is an essential regulator of adipocyte development through control of the formation of cortical actin structures, which may facilitate nutrient uptake and signalling events.

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  • Structural basis of thymosin-β4/profilin exchange leading to actin filament polymerization. 査読

    Xue B, Leyrat C, Grimes JM, Robinson RC

    Proceedings of the National Academy of Sciences of the United States of America   111 ( 43 )   E4596 - E4605   2014年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Structure of a Stapled Peptide Antagonist Bound to Nutlin-Resistant Mdm2 査読

    Sharon Min Qi Chee, Jantana Wongsantichon, Quah Soo Tng, Robert Robinson, Thomas L. Joseph, Chandra Verma, David P. Lane, Christopher J. Brown, Farid J. Ghadessy

    PLOS ONE   9 ( 8 )   e104914   2014年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PUBLIC LIBRARY SCIENCE  

    As key negative regulator of the p53 tumour suppressor, Mdm2 is an attractive therapeutic target. Small molecules such as Nutlin have been developed to antagonise Mdm2, resulting in p53-dependent death of tumour cells. We have recently described a mutation in Mdm2 (M62A), which precludes binding of Nutlin, but not p53. This Nutlin-resistant variant is not, however, refractory to binding and inhibition by stapled peptide antagonists targeting the same region of Mdm2. A detailed understanding of how stapled peptides are recalcitrant to Mdm2 mutations conferring Nutlin-resistance will aid in the further development of potent Mdm2 antagonists. Here, we report the 2.00 A crystal structure of a stapled peptide antagonist bound to Nutlin resistant Mdm2. The stapled peptide relies on an extended network of interactions along the hydrophobic binding cleft of Mdm2 for high affinity binding. Additionally, as seen in other stapled peptide structures, the hydrocarbon staple itself contributes to binding through favourable interactions with Mdm2. The structure highlights the intrinsic plasticity present in both Mdm2 and the hydrocarbon staple moiety, and can be used to guide future iterations of both small molecules and stapled peptides for improved antagonists of Mdm2.

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  • Single-molecule force spectroscopy reveals force-enhanced binding of calcium ions by gelsolin 査読

    Chunmei Lv, Xiang Gao, Wenfei Li, Bo Xue, Meng Qin, Leslie D. Burtnick, Hao Zhou, Yi Cao, Robert C. Robinson, Wei Wang

    NATURE COMMUNICATIONS   5   4623   2014年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATURE PUBLISHING GROUP  

    Force is increasingly recognized as an important element in controlling biological processes. Forces can deform native protein conformations leading to protein-specific effects. Protein-protein binding affinities may be decreased, or novel protein-protein interaction sites may be revealed, on mechanically stressing one or more components. Here we demonstrate that the calcium-binding affinity of the sixth domain of the actin-binding protein gelsolin (G6) can be enhanced by mechanical force. Our kinetic model suggests that the calcium-binding affinity of G6 increases exponentially with force, up to the point of G6 unfolding. This implies that gelsolin may be activated at lower calcium ion levels when subjected to tensile forces. The demonstration that cation-protein binding affinities can be force-dependent provides a new understanding of the complex behaviour of cation-regulated proteins in stressful cellular environments, such as those found in the cytoskeleton-rich leading edge and at cell adhesions.

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  • Identification of Polyketide Inhibitors Targeting 3-Dehydroquinate Dehydratase in the Shikimate Pathway of Enterococcus faecalis 査読

    Vivian Wing Ngar Cheung, Bo Xue, Maria Hernandez-Valladares, Maybelle Kho Go, Alvin Tung, Adeleke H. Aguda, Robert C. Robinson, Wen Shan Yew

    PLOS ONE   9 ( 7 )   e103598   2014年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PUBLIC LIBRARY SCIENCE  

    Due to the emergence of resistance toward current antibiotics, there is a pressing need to develop the next generation of antibiotics as therapeutics against infectious and opportunistic diseases of microbial origins. The shikimate pathway is exclusive to microbes, plants and fungi, and hence is an attractive and logical target for development of antimicrobial therapeutics. The Gram-positive commensal microbe, Enterococcus faecalis, is a major human pathogen associated with nosocomial infections and resistance to vancomycin, the "drug of last resort''. Here, we report the identification of several polyketide-based inhibitors against the E. faecalis shikimate pathway enzyme, 3-dehydroquinate dehydratase (DHQase). In particular, marein, a flavonoid polyketide, both inhibited DHQase and retarded the growth of Enterococcus faecalis. The purification, crystallization and structural resolution of recombinant DHQase from E. faecalis (at 2.2 angstrom resolution) are also reported. This study provides a route in the development of polyketide-based antimicrobial inhibitors targeting the shikimate pathway of the human pathogen E. faecalis.

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  • De Novo Design and Experimental Characterization of Ultrashort Self-Associating Peptides 査読

    James Smadbeck, Kiat Hwa Chan, George A. Khoury, Bo Xue, Robert C. Robinson, Charlotte A. E. Hauser, Christodoulos A. Floudas

    PLOS COMPUTATIONAL BIOLOGY   10 ( 7 )   e1003718   2014年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PUBLIC LIBRARY SCIENCE  

    Self-association is a common phenomenon in biology and one that can have positive and negative impacts, from the construction of the architectural cytoskeleton of cells to the formation of fibrils in amyloid diseases. Understanding the nature and mechanisms of self-association is important for modulating these systems and in creating biologically-inspired materials. Here, we present a two-stage de novo peptide design framework that can generate novel self-associating peptide systems. The first stage uses a simulated multimeric template structure as input into the optimization-based Sequence Selection to generate low potential energy sequences. The second stage is a computational validation procedure that calculates Fold Specificity and/or Approximate Association Affinity (K*(association)) based on metrics that we have devised for multimeric systems. This framework was applied to the design of self-associating tripeptides using the known self-associating tripeptide, Ac-IVD, as a structural template. Six computationally predicted tripeptides (Ac-LVE, Ac-YYD, Ac-LLE, Ac-YLD, Ac-MYD, Ac-VIE) were chosen for experimental validation in order to illustrate the self-association outcomes predicted by the three metrics. Self-association and electron microscopy studies revealed that Ac-LLE formed bead-like microstructures, Ac-LVE and Ac-YYD formed fibrillar aggregates, Ac-VIE and Ac-MYD formed hydrogels, and Ac-YLD crystallized under ambient conditions. An X-ray crystallographic study was carried out on a single crystal of Ac-YLD, which revealed that each molecule adopts a beta-strand conformation that stack together to form parallel beta-sheets. As an additional validation of the approach, the hydrogel-forming sequences of Ac-MYD and Ac-VIE were shuffled. The shuffled sequences were computationally predicted to have lower K*(association) values and were experimentally verified to not form hydrogels. This illustrates the robustness of the framework in predicting self-associating tripeptides. We expect that this enhanced multimeric de novo peptide design framework will find future application in creating novel self-associating peptides based on unnatural amino acids, and inhibitor peptides of detrimental self-aggregating biological proteins.

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  • Soluble expression and partial purification of recombinant human erythropoietin from E-coli 査読

    Taeck-Hyun Jeong, Young-Jin Son, Han-Bong Ryu, Bon-Kyung Koo, Seung-Mi Jeong, Phuong Hoang, Bich Hang Do, Jung-A Song, Seon-Ha Chong, Robert Charles Robinson, Han Choe

    PROTEIN EXPRESSION AND PURIFICATION   95   211 - 218   2014年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    Human erythropoietin (hEpo) is an essential regulator of erythrocyte production that induces the division and differentiation of erythroid progenitor cells in the bone marrow into mature erythrocytes. It is widely used for the treatment of anemia resulting from chronic kidney disease, chemotherapy, and cancer-related therapies. Active hEpo, and hEpo analogs, have been purified primarily from mammalian cells, which has several disadvantages, including low yields and high production costs. Although an Escherichia coli (E. coli) expression system may provide economic production of therapeutic proteins, it has not been used for the production of recombinant hEpo (rhEpo) because it aggregates in inclusion bodies in the E. coli cytoplasm and is not modified post-translationally. We investigated the soluble overexpression of active rhEpo with various protein tags in E. coli, and found out that several tags increased the solubility of rhEpo. Among them maltose binding protein (MBP)-tagged rhEpo was purified using affinity and gel filtration columns. Non-denaturing electrophoresis and MALDI-TOF MS analysis demonstrated that the purified rhEpo had two intra-disulfide bonds identical to those of the native hEpo. An in vitro proliferation assay showed that rhEpo purified from E. coli had similar biological activity as rhEpo derived from CHO cells. Therefore, we report for the first time that active rhEpo was overexpressed as a soluble form in the cytoplasm of E. coli and purified it in simple purification steps. We hope that our results offer opportunities for progress in rhEpo therapeutics. (C) 2014 Elsevier Inc. All rights reserved.

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  • Soluble Expression of Human Leukemia Inhibitory Factor with Protein Disulfide Isomerase in Escherichia coli and Its Simple Purification 査読

    A. Song Jung, Bon-Kyung Koo, Seon-Ha Chong, Kyunhoo Kim, Dong Kyu Choi, Thu Trang Thi Vu, Minh Tan Nguyen, Boram Jeong, Han-Bong Ryu, Injune Kim, Yeon Jin Jang, Robert Charles Robinson, Han Choe

    PLOS ONE   8 ( 12 )   e83781   2013年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PUBLIC LIBRARY SCIENCE  

    Human leukemia inhibitory factor (hLIF) is a multifunctional cytokine that is essential for maintaining the pluripotency of embryonic stem cells. hLIF may be also be useful in aiding fertility through its effects on increasing the implantation rate of fertilized eggs. Thus these applications in biomedical research and clinical medicine create a high demand for bioactive hLIF. However, production of active hLIF is problematic since eukaryotic cells demonstrate limited expression and prokaryotic cells produce insoluble protein. Here, we have adopted a hybrid protein disulfide isomerase design to increase the solubility of hLIF in Escherichia coli. Low temperature expression of hLIF fused to the b'a' domain of protein disulfide isomerase (PDIb'a') increased the soluble expression in comparison to controls. A simple purification protocol for bioactive hLIF was established that includes removal of the PDIb'a' domain by cleavage by TEV protease. The resulting hLIF, which contains one extra glycine residue at the N-terminus, was highly pure and demonstrated endotoxin levels below 0.05 EU/mu g. The presence of an intramolecular disulfide bond was identified using mass spectroscopy. This purified hLIF effectively maintained the pluripotency of a murine embryonic stem cell line. Thus we have developed an effective method to produce a pure bioactive version of hLIF in E. coli for use in biomedical research.

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  • The expanding superfamily of gelsolin homology domain proteins. 査読

    Ghoshdastider U, Popp D, Burtnick LD, Robinson RC

    Cytoskeleton (Hoboken, N.J.)   70 ( 11 )   775 - 795   2013年11月

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    掲載種別:研究論文(学術雑誌)  

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  • Structural analysis and insights into the glycon specificity of the rice GH1 Os7BGlu26 β-D-mannosidase. 査読

    Tankrathok A, Iglesias-Fernández J, Luang S, Robinson RC, Kimura A, Rovira C, Hrmova M, Ketudat Cairns JR

    Acta crystallographica. Section D, Biological crystallography   69 ( 10 )   2124 - 2135   2013年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Guardians of the actin monomer 査読

    Bo Xue, Robert C. Robinson

    EUROPEAN JOURNAL OF CELL BIOLOGY   92 ( 10-11 )   316 - 332   2013年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER GMBH, URBAN & FISCHER VERLAG  

    Actin is a universal force provider in eukaryotic cells. Biological processes harness the pressure generated from actin polymerization through dictating the time, place and direction of filament growth. As such, polymerization is initiated and maintained via tightly controlled filament nucleation and elongation machineries. Biological systems integrate force into their activities through recruiting and activating these machineries. In order that actin function as a common force generating polymerization motor, cells must maintain a pool of active, polymerization-ready monomeric actin, and minimize extemporaneous polymerization. Maintenance of the active monomeric actin pool requires the recycling of actin filaments, through depolymerization, nucleotide exchange and reloading of the polymerization machineries, while the levels of monomers are constantly monitored and supplemented, when needed, via the access of a reserve pool of monomers and through gene expression. Throughout its monomeric life, actin needs to be protected against gratuitous nucleation events. Here, we review the proteins that act as custodians of monomeric actin. We estimate their levels on a tissue scale, and calculate the implied concentrations of each actin complex based on reported binding affinities. These estimations predict that monomeric actin is rarely, if ever, alone. Thus, the guardians keep the volatility of actin in check, so that its explosive power is only released in the controlled environments of the nucleation and polymerization machineries. (C) 2013 Elsevier GmbH. All rights reserved.

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  • Structural analysis and insights into the glycon specificity of the rice GH1 Os7BGlu26 β-d-mannosidase 査読

    Anupong Tankrathok, Javier Iglesias-Fernández, Sukanya Luang, Robert C. Robinson, Atsuo Kimura, Carme Rovira, Maria Hrmova, James R. Ketudat Cairns

    Acta Crystallographica Section D: Biological Crystallography   69 ( 10 )   2124 - 2135   2013年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Rice Os7BGlu26 is a GH1 family glycoside hydrolase with a threefold higher k cat/K m value for 4-nitrophenyl β-d-mannoside (4NPMan) compared with 4-nitrophenyl β-d-glucoside (4NPGlc). To investigate its selectivity for β-d-mannoside and β-d-glucoside substrates, the structures of apo Os7BGlu26 at a resolution of 2.2014
    Å and of Os7BGlu26 with mannose at a resolution of 2.4514
    Å were elucidated from isomorphous crystals in space group P212121. The (β/)8-barrel structure is similar to other GH1 family structures, but with a narrower active-site cleft. The Os7BGlu26 structure with d-mannose corresponds to a product complex, with β-d-mannose in the 1 S 5 skew-boat conformation. Docking of the 1 S 3, 1 S 5, 2 S O and 3 S 1 pyranose-ring conformations of 4NPMan and 4NPGlc substrates into the active site of Os7BGlu26 indicated that the lowest energies were in the 1 S 5 and 1 S 3 skew-boat conformations. Comparison of these docked conformers with other rice GH1 structures revealed differences in the residues interacting with the catalytic acid/base between enzymes with and without β-d-mannosidase activity. The mutation of Tyr134 to Trp in Os7BGlu26 resulted in similar k cat/K m values for 4NPMan and 4NPGlc, while mutation of Tyr134 to Phe resulted in a 37-fold higher k cat/K m for 4NPMan than 4NPGlc. Mutation of Cys182 to Thr decreased both the activity and the selectivity for β-d-mannoside. It was concluded that interactions with the catalytic acid/base play a significant role in glycon selection. © 2013 International Union of Crystallography Printed in Singapore - all rights reserved. © 2013.

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  • Genetic variants and mutations of PPM1D control the response to DNA damage 査読

    Crissy Dudgeon, Sathyavageeswaran Shreeram, Kan Tanoue, Sharlyn J. Mazur, Ahmed Sayadi, Robert C. Robinson, Ettore Appella, Dmitry V. Bulavin

    CELL CYCLE   12 ( 16 )   2656 - 2664   2013年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:LANDES BIOSCIENCE  

    The Wip1 phosphatase is an oncogene that is overexpressed in a variety of primary human cancers. We were interested in identifying genetic variants that could change Wip1 activity. We identified 3 missense SNPs of the human Wip1 phosphatase, L120F, P322Q, and I496V confer a dominant-negative phenotype. On the other hand, in primary human cancers, PPM1D mutations commonly result in a gain-of-function phenotype, leading us to identify a hot-spot truncating mutation at position 525. Surprisingly, we also found a significant number of loss-of-function mutations of PPM1D in primary human cancers, both in the phosphatase domain and in the C terminus. Thus, PPM1D has evolved to generate genetic variants with lower activity, potentially providing a better fitness for the organism through suppression of multiple diseases. In cancer, however, the situation is more complex, and the presence of both activating and inhibiting mutations requires further investigation to understand their contribution to tumorigenesis.

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  • Gelsolin: The Tail of a Molecular Gymnast 査読

    Shalini Nag, Marten Larsson, Robert C. Robinson, Leslie D. Burtnick

    CYTOSKELETON   70 ( 7 )   360 - 384   2013年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

    Gelsolin superfamily members are Ca2+-dependent, multidomain regulators of the actin cytoskeleton. Calcium binding activates gelsolin by inducing molecular gymnastics (large-scale conformational changes) that expose actin interaction surfaces by releasing a series of latches. A specialized tail latch has distinguished gelsolin within the superfamily. Active gelsolin exhibits actin filament severing and capping, and actin monomer sequestering activities. Here, we analyze a combination of sequence, structural, biophysical and biochemical data to assess whether the molecular plasticity, regulation and actin-related properties of gelsolin are also present in other superfamily members. We conclude that all members of the superfamily will be able to transition between a compact conformation and a more open form, and that most of these open forms will interact with actin. Supervillin, which lacks the severing domain 1 and the F-actin binding-site on domain 2, is the clear exception. Eight calcium-binding sites are absolutely conserved in gelsolin, adseverin, advillin and villin, and compromised to increasing degrees in CapG, villin-like protein, supervillin and flightless I. Advillin, villin and supervillin each contain a potential tail latch, which is absent from CapG, adseverin and flightless I, and ambiguous in villin-like protein. Thus, calcium regulation will vary across the superfamily. Potential novel isoforms of the superfamily suggest complex regulation at the gene, transcript and protein levels. We review animal, clinical and cellular data that illuminate how the regulation of molecular flexibility in gelsolin-like proteins permits cells to exploit the force generated from actin polymerization to drive processes such as cell movement in health and disease. (c) 2013 Wiley Periodicals, Inc.

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  • Insight into Actin Organization and Function in Cytokinesis from Analysis of Fission Yeast Mutants 査読

    Dhivya Subramanian, Junqi Huang, Mayalagu Sevugan, Robert C. Robinson, Mohan K. Balasubramanian, Xie Tang

    GENETICS   194 ( 2 )   435 - +   2013年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:GENETICS SOCIETY AMERICA  

    Actin is a key cytoskeletal protein with multiple roles in cellular processes such as polarized growth, cytokinesis, endocytosis, and cell migration. Actin is present in all eukaryotes as highly dynamic filamentous structures, such as linear cables and branched filaments. Detailed investigation of the molecular role of actin in various processes has been hampered due to the multifunctionality of the protein and the lack of alleles defective in specific processes. The actin cytoskeleton of the fission yeast, Schizosaccharomyces pombe, has been extensively characterized and contains structures analogous to those in other cell types. In this study, primarily with the view to uncover actin function in cytokinesis, we generated a large bank of fission yeast actin mutants that affect the organization of distinct actin structures and/or discrete physiological functions of actin. Our screen identified 17 mutants with specific defects in cytokinesis. Some of these cytokinesis mutants helped in dissecting the function of specific actin structures during ring assembly. Further genetic analysis of some of these actin mutants revealed multiple genetic interactions with mutants previously known to affect the actomyosin ring assembly. We also characterize a mutant allele of actin that is suppressed upon overexpression of Cdc8p-tropomyosin, underscoring the utility of this mutant bank. Another 22 mutant alleles, defective in polarized growth and/or other functions of actin obtained from this screen, are also described in this article. This mutant bank should be a valuable resource to study the physiological and biochemical functions of actin.

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  • Structural Evidence of a Productive Active Site Architecture for an Evolved Quorum-Quenching GKL Lactonase 査読

    Bo Xue, Jeng Yeong Chow, Amgalanbaatar Baldansuren, Lai Lai Yap, Yunn Hwen Gan, Sergei A. Dikanov, Robert C. Robinson, Wen Shan Yew

    BIOCHEMISTRY   52 ( 13 )   2359 - 2370   2013年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER CHEMICAL SOC  

    The in vitro evolution and engineering of quorum-quenching lactonases with enhanced reactivities was achieved using a thermostable GKL enzyme as a template, yielding the E101G/R230C GKL mutant with increased catalytic activity and a broadened substrate range [Chow, J. Y., Xue, B., Lee, K. H., Tung, A., Wu, L., Robinson, R. C., and Yew, W. S. (2010) J. Biol. Chem. 285, 40911-40920]. This enzyme possesses the (beta/alpha)(8)-barrel fold and is a member of the PLL (phosphotriesterase-like lactonase) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acyl-homoserine lactones, which mediate the quorum-sensing pathways of bacteria. The structure of the evolved N-butyryl-L-homoserine lactone (substrate)-bound EIOIG/R230C GKL enzyme was determined, in the presence of the inactivating D266N mutation, to a resolution of 2.2 angstrom to provide an explanation for the observed rate enhancements. In addition, the substrate-bound structure of the catalytically inactive E101N/D266N mutant of the manganese-reconstituted enzyme was determinied to a resolution of 2.1 angstrom and the structure of the ligand-free, manganese-reconstituted E101N mutant to a resolution of 2.6 angstrom, and the structures of ligand-free zinc-reconstituted wild-type, EIOIN, R230D, and EIOIG/R230C mutants of GKL were determinied to resolutions of 2.1, 2.1, 1.9, and 2.0 angstrom, respectively. In particular, the structure of the evolved EIOIG/R230C mutant of GKL provides evidence of a catalytically productive active site architecture that contributes to the observed enhancement of catalysis. At high concentrations, wild-type and mutant GKL enzymes are differentially colored, with absorbance maxima in the range of 512-553 nm. The structures of the wild-type and mutant GKL provide a tractable link between the origins of the coloration and the charge-transfer complex between the alpha-cation and Tyr99 within the enzyme active site. Taken together, this study provides evidence of the modulability of enzymatic catalysis through subtle changes in enzyme active site architecture.

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  • Microtubule-like Properties of the Bacterial Actin Homolog ParM-R1 査読

    David Popp, Akihiro Narita, Lin Jie Lee, Marten Larsson, Robert C. Robinson

    JOURNAL OF BIOLOGICAL CHEMISTRY   287 ( 44 )   37078 - 37088   2012年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    In preparation for mammalian cell division, microtubules repeatedly probe the cytoplasm to capture chromosomes and assemble the mitotic spindle. Critical features of this microtubule system are the formation of radial arrays centered at the centrosomes and dynamic instability, leading to persistent cycles of polymerization and depolymerization. Here, we show that actin homolog, ParM-R1 that drives segregation of the R1 multidrug resistance plasmid from Escherichia coli, can also self-organize in vitro into asters, which resemble astral microtubules. ParM-R1 asters grow from centrosome-like structures consisting of interconnected nodes related by a pseudo 8-fold symmetry. In addition, we show that ParM-R1 is able to perform persistent microtubule-like oscillations of assembly and disassembly. In vitro, a whole population of ParM-R1 filaments is synchronized between phases of growth and shrinkage, leading to prolonged synchronous oscillations even at physiological ParM-R1 concentrations. These results imply that the selection pressure to reliably segregate DNA during cell division has led to common mechanisms within diverse segregation machineries.

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  • Novel Actin-like Filament Structure from Clostridium tetani 査読

    David Popp, Akihiro Narita, Lin Jie Lee, Umesh Ghoshdastider, Bo Xue, Ramanujam Srinivasan, Mohan K. Balasubramanian, Toshitsugu Tanaka, Robert C. Robinson

    JOURNAL OF BIOLOGICAL CHEMISTRY   287 ( 25 )   21121 - 21129   2012年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Eukaryotic F-actin is constructed from two protofilaments that gently wind around each other to form a helical polymer. Several bacterial actin-like proteins (Alps) are also known to form F-actin-like helical arrangements from two protofilaments, yet with varied helical geometries. Here, we report a unique filament architecture of Alp12 from Clostridium tetani that is constructed from four protofilaments. Through fitting of an Alp12 monomer homology model into the electron microscopy data, the filament was determined to be constructed from two antiparallel strands, each composed of two parallel protofilaments. These four protofilaments form an open helical cylinder separated by a wide cleft. The molecular interactions within single protofilaments are similar to F-actin, yet interactions between protofilaments differ from those in F-actin. The filament structure and assembly and disassembly kinetics suggest Alp12 to be a dynamically unstable force-generating motor involved in segregating the pE88 plasmid, which encodes the lethal tetanus toxin, and thus a potential target for drug design. Alp12 can be repeatedly cycled between states of polymerization and dissociation, making it a novel candidate for incorporation into fuel-propelled nanobiopolymer machines.

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  • Structural evidence for conformational changes of Delta class glutathione transferases after ligand binding 査読

    Jantana Wongsantichon, Robert C. Robinson, Albert J. Ketterman

    ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS   521 ( 1-2 )   77 - 83   2012年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE INC  

    We report four new crystal structures for Delta class glutathione transferases from insects. We compare these new structures as well as several previously reported structures to determine that structural transitions can be observed with ligand binding. These transitions occurred in the regions around the active site entrance, including alpha helix 2, C-terminus of alpha helix 4 including the loop to helix 5 and the C-terminus of helix 8. These structural movements have been reported or postulated to occur for several other glutathione transferase classes; however, this is the first report showing structural evidence of all these movements occurring, in this case in Delta class glutathione transferases. These fluctuations also can be observed occurring within a single structure as there is ligand bound in only one subunit and each subunit is undergoing different conformational transitions. The structural comparisons show reorganizations occur both pre- and post-GSH ligand binding communicated through the subunit interface of the quaternary assembly. Movements of these positions would allow 'breathing' of the active site for substrate entrance, topological rearrangement for varying substrate specificity and final product release. (C) 2012 Elsevier Inc. All rights reserved.

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  • Conformational dynamics of capping protein and interaction partners: Simulation studies 査読

    Suryani Lukman, Robert C. Robinson, David Wales, Chandra S. Verma

    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS   80 ( 4 )   1066 - 1077   2012年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

    Capping protein (CP) is important for the regulation of actin polymerization. CP binds to the barbed end of the actin filament and prevents actin polymerization. This interaction is modulated through competitive binding by regulatory proteins such as myotrophin (V-1) and the capping protein interacting (CPI) motif from CARMIL. The binding site of myotrophin overlaps with the region of CP that binds to the barbed end of actin filament, whereas CPI binds at a distant site. The binding of CPI to the myotrophin-CP complex dissociates myotrophin from CP. Detailed multicopy molecular dynamics simulations suggest that the binding of CPI shifts the conformational equilibria of CP away from states that favor myotrophin binding. This shift is underpinned by allosteric effects where CPI inhibits CP through suppression of flexibility and disruption of concerted motions that appear to mediate myotrophin binding. Accompanying these effects are changes in electrostatic interactions, notably those involving residue K142 beta, which appears to play a critical role in regulating flexibility. In addition, accessibility of the site on CP for binding the key hydrophobic residue W8 of myotrophin is modulated by CPI. These results provide insights into the modulation of CP by CPI and myotrophin and indicate the mechanism by which CPI drives the dissociation of the myotrophin-CP complex. Proteins 2012;. (c) 2011 Wiley Periodicals, Inc.

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  • Supramolecular cellular filament systems: How and why do they form? 査読

    David Popp, Robert C. Robinson

    CYTOSKELETON   69 ( 2 )   71 - 87   2012年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY  

    All cells, from simple bacteria to complex human tissues, rely on extensive networks of protein fibers to help maintain their proper form and function. These filament systems usually do not operate as single filaments, but form complex suprastructures, which are essential for specific cellular functions. Here, we describe the progress in determining the architectures of molecular filamentous suprastructures, the principles leading to their formation, and the mechanisms by which they may facilitate function. The complex eukaryotic cytoskeleton is tightly regulated by a large number of actin- or microtubule-associated proteins. In contrast, recently discovered bacterial actins and tubulins have few associated regulatory proteins. Hence, the quest to find basic principles that govern the formation of filamentous suprastructures is simplified in bacteria. Three common principles, which have been probed extensively during evolution, can be identified that lead to suprastructures formation: cationic counterion fluctuations; self-association into liquid crystals; and molecular crowding. The underlying physics of these processes will be discussed with respect to physiological circumstance. (c) 2012 Wiley Periodicals, Inc

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  • Structural Basis of Cooperativity in Human UDP-Glucose Dehydrogenase 査読

    Venkatachalam Rajakannan, Hui-Sun Lee, Seon-Ha Chong, Han-Bong Ryu, Ji-Young Bae, Eun-Young Whang, Jae-Wan Huh, Sung-Woo Cho, Lin-Woo Kang, Han Choe, Robert C. Robinson

    PLOS ONE   6 ( 10 )   2011年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PUBLIC LIBRARY SCIENCE  

    Background: UDP-glucose dehydrogenase (UGDH) is the sole enzyme that catalyzes the conversion of UDP-glucose to UDP-glucuronic acid. The product is used in xenobiotic glucuronidation in hepatocytes and in the production of proteoglycans that are involved in promoting normal cellular growth and migration. Overproduction of proteoglycans has been implicated in the progression of certain epithelial cancers, while inhibition of UGDH diminished tumor angiogenesis in vivo. A better understanding of the conformational changes occurring during the UGDH reaction cycle will pave the way for inhibitor design and potential cancer therapeutics.
    Methodology: Previously, the substrate-bound of UGDH was determined to be a symmetrical hexamer and this regular symmetry is disrupted on binding the inhibitor, UDP-alpha-D-xylose. Here, we have solved an alternate crystal structure of human UGDH (hUGDH) in complex with UDP-glucose at 2.8 angstrom resolution. Surprisingly, the quaternary structure of this substrate-bound protein complex consists of the open homohexamer that was previously observed for inhibitor-bound hUGDH, indicating that this conformation is relevant for deciphering elements of the normal reaction cycle.
    Conclusion: In all subunits of the present open structure, Thr131 has translocated into the active site occupying the volume vacated by the absent active water and partially disordered NAD(+) molecule. This conformation suggests a mechanism by which the enzyme may exchange NADH for NAD(+) and repolarize the catalytic water bound to Asp280 while protecting the reaction intermediates. The structure also indicates how the subunits may communicate with each other through two reaction state sensors in this highly cooperative enzyme.

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  • Structural Basis of Cooperativity in Human UDP-Glucose Dehydrogenase 査読

    Venkatachalam Rajakannan, Hui-Sun Lee, Seon-Ha Chong, Han-Bong Ryu, Ji-Young Bae, Eun-Young Whang, Jae-Wan Huh, Sung-Woo Cho, Lin-Woo Kang, Han Choe, Robert C. Robinson

    PLOS ONE   6 ( 10 )   e25226   2011年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PUBLIC LIBRARY SCIENCE  

    Background: UDP-glucose dehydrogenase (UGDH) is the sole enzyme that catalyzes the conversion of UDP-glucose to UDP-glucuronic acid. The product is used in xenobiotic glucuronidation in hepatocytes and in the production of proteoglycans that are involved in promoting normal cellular growth and migration. Overproduction of proteoglycans has been implicated in the progression of certain epithelial cancers, while inhibition of UGDH diminished tumor angiogenesis in vivo. A better understanding of the conformational changes occurring during the UGDH reaction cycle will pave the way for inhibitor design and potential cancer therapeutics.
    Methodology: Previously, the substrate-bound of UGDH was determined to be a symmetrical hexamer and this regular symmetry is disrupted on binding the inhibitor, UDP-alpha-D-xylose. Here, we have solved an alternate crystal structure of human UGDH (hUGDH) in complex with UDP-glucose at 2.8 angstrom resolution. Surprisingly, the quaternary structure of this substrate-bound protein complex consists of the open homohexamer that was previously observed for inhibitor-bound hUGDH, indicating that this conformation is relevant for deciphering elements of the normal reaction cycle.
    Conclusion: In all subunits of the present open structure, Thr131 has translocated into the active site occupying the volume vacated by the absent active water and partially disordered NAD(+) molecule. This conformation suggests a mechanism by which the enzyme may exchange NADH for NAD(+) and repolarize the catalytic water bound to Asp280 while protecting the reaction intermediates. The structure also indicates how the subunits may communicate with each other through two reaction state sensors in this highly cooperative enzyme.

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  • Many ways to build an actin filament 査読

    David Popp, Robert C. Robinson

    MOLECULAR MICROBIOLOGY   80 ( 2 )   300 - 308   2011年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

    P>Cells rely on extensive networks of protein fibres to help maintain their proper form and function. For species ranging from bacteria to humans, this 'cytoskeleton' is integrally involved in diverse processes including movement, DNA segregation, cell division and transport of molecular cargoes. The most abundant cytoskeletal filament-forming protein, F-actin, is remarkably well conserved across eukaryotic species. From yeast to human - an evolutionary distance of over one billion years - only about 10% of residues in actin have changed and the filament structure has been highly conserved. Surprisingly, recent structural data show this to be not the case for filamentous bacterial actins, which exhibit highly divergent helical symmetries in conjunction with structural plasticity or polymorphism, and dynamic properties that may make them uniquely suited for the specific cellular processes in which they participate. Bacterial actin filaments often organize themselves into complex structures within the prokaryotic cell, driven by molecular crowding and cation association, to form bundles (ParM) or interwoven sheets (MreB). The formation of supramolecular structures is essential for bacterial cytoskeleton function. We discuss the underlying physical principles that lead to complex structure formation and the implications these have on the physiological functions of cytoskeletal proteins.

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  • The structural basis of oligosaccharide binding by rice BGlu1 beta-glucosidase 査読

    Watchalee Chuenchor, Salila Pengthaisong, Robert C. Robinson, Jirundon Yuvaniyama, Jisnuson Svasti, James R. Ketudat Cairns

    JOURNAL OF STRUCTURAL BIOLOGY   173 ( 1 )   169 - 179   2011年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    Rice BGlu1 beta-glucosidase is an oligosaccharide exoglucosidase that binds to six beta-(1 -> 4)-linked glucosyl residues in its active site cleft. Here, we demonstrate that a BGlu1 E176Q active site mutant can be effectively rescued by small nucleophiles, such as acetate, azide and ascorbate, for hydrolysis of aryl glycosides in a pH-independent manner above pH 5, consistent with the role of El 76 as the catalytic acid-base. Cellotriose, cellotetraose, cellopentaose, cellohexaose and laminaribiose are not hydrolyzed by the mutant and instead exhibit competitive inhibition. The structures of the BGlu1 E176Q its complexes with cellotetraose, cellopentaose and laminaribiose, and its covalent intermediate with 2-deoxy-2-fluoroglucoside were determined at 1.65, 1.95, 1.80, 2.80, and 1.90 angstrom resolution, respectively. The Q176 N epsilon was found to hydrogen bond to the glycosidic oxygen of the scissile bond, thereby explaining its high activity. The enzyme interacts with cellooligosaccharides through direct hydrogen bonds to the nonreducing terminal glucosyl residue. However, interaction with the other glucosyl residues is predominantly mediated through water molecules, with the exception of a direct hydrogen bond from N245 to glucosyl residue 3, consistent with the apparent high binding energy at this residue. Hydrophobic interactions with the aromatic sidechain of W358 appear to orient glucosyl residues 2 and 3, while Y341 orients glucosyl residues 4 and 5. In contrast, laminaribiose has its second glucosyl residue positioned to allow direct hydrogen bonding between its 02 and Q176 O epsilon and 01 and N245. These are the first GH1 glycoside hydrolase family structures to show oligosaccharide binding in the hydrolytic configuration. (C) 2010 Elsevier Inc. All rights reserved.

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  • Directed Evolution of a Thermostable Quorum-quenching Lactonase from the Amidohydrolase Superfamily 査読

    Jeng Yeong Chow, Bo Xue, Kang Hao Lee, Alvin Tung, Long Wu, Robert C. Robinson, Wen Shan Yew

    JOURNAL OF BIOLOGICAL CHEMISTRY   285 ( 52 )   40911 - 40920   2010年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    A thermostable quorum-quenching lactonase from Geobacillus kaustophilus HTA426 (GI: 56420041) was used as an initial template for in vitro directed evolution experiments. This enzyme belongs to the phosphotriesterase-like lactonase (PLL) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acylhomoserine lactones (AHLs) that are involved in virulence pathways of quorum-sensing pathogenic bacteria. Here we have determined the N-butyryl-L-homoserine lactone-liganded structure of the catalytically inactive D266N mutant of this enzyme to a resolution of 1.6 angstrom. Using a tunable, bioluminescence-based quorum-quenching molecular circuit, the catalytic efficiency was enhanced, and the AHL substrate range increased through two point mutations on the loops at the C-terminal ends of the third and seventh beta-strands. This E101N/R230I mutant had an increased value of k(cat)/K-m of 72-fold toward 3-oxo-N-dodecanoyl-L-homoserine lactone. The evolved mutant also exhibited lactonase activity toward N-butyryl-L-homoserine lactone, an AHL that was previously not hydrolyzed by the wild-type enzyme. Both the purified wild-type and mutant enzymes contain a mixture of zinc and iron and are colored purple and brown, respectively, at high concentrations. The origin of this coloration is suggested to be because of a charge transfer complex involving the beta-cation and Tyr-99 within the enzyme active site. Modulation of the charge transfer complex alters the lactonase activity of the mutant enzymes and is reflected in enzyme coloration changes. We attribute the observed enhancement in catalytic reactivity of the evolved enzyme to favorable modulations of the active site architecture toward productive geometries required for chemical catalysis.

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  • Bacterial cytoskeleton suprastructures and their physical origin. 査読

    Popp D, Robinson RC

    Communicative & integrative biology   3 ( 5 )   451 - 453   2010年9月

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    掲載種別:研究論文(学術雑誌)  

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  • The Structure of Native G-Actin 査読

    Hui Wang, Robert C. Robinson, Leslie D. Burtnick

    CYTOSKELETON   67 ( 7 )   456 - 465   2010年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

    Heat shock proteins act as cytoplasmic chaperones to ensure correct protein folding and prevent protein aggregation. The presence of stoichiometric amounts of one such heat shock protein, Hsp27, in supersaturated solutions of unmodified G-actin leads to crystallization, in preference to polymerization, of the actin. Hsp27 is not evident in the resulting crystal structure. Thus, for the first time, we present the structure of G-actin in a form that is devoid of polymerization-deterring chemical modifications or binding partners, either of which may alter its conformation. The structure contains a calcium ion and ATP within a closed nucleotide-binding cleft, and the D-loop is disordered. This native G-actin structure invites comparison with the current F-actin model in order to understand the structural implications for actin polymerization. In particular, this analysis suggests a mechanism by which the bound cation coordinates conformational change and ATP-hydrolysis. (C) 2010 Wiley-Liss, Inc.

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  • Structural contributions of Delta class glutathione transferase active-site residues to catalysis 査読

    Jantana Wongsantichon, Robert C. Robinson, Albert J. Ketterman

    BIOCHEMICAL JOURNAL   428   25 - 32   2010年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PORTLAND PRESS LTD  

    GST (glutathione transferase) is a dimeric enzyme recognized for biotransformation of xenobiotics and endogenous toxic compounds. In the present study, residues forming the hydrophobic substrate-binding site (H-site) of a Delta class enzyme were investigated in detail for the first time by site-directed mutagenesis and crystallographic studies. Enzyme kinetics reveal that Tyr(111) indirectly stabilizes GSH binding, Tyr(119) modulates hydrophobic substrate binding and Phe(123) indirectly modulates catalysis. Mutations at Tyr(111) and Phe(123) also showed evidence for positive co-operativity for GSH and 1-chloro-2,4-dinitrobenzene respectively, strongly suggesting a role for these residues in manipulating subunit-subunit communication. In the present paper we report crystal structures of the wild-type enzyme, and two mutants, in complex with S-hexylglutathione. This study has identified an aromatic 'zipper' in the H-site contributing a network of aromatic pi-pi interactions. Several residues of the cluster directly interact with the hydrophobic substrate, whereas others indirectly maintain conformational stability of the dimeric structure through the C-terminal domain (domain II). The Y119E mutant structure shows major main-chain rearrangement of domain II. This reorganization is moderated through the 'zipper' that contributes to the H-site remodelling, thus illustrating a role in co-substrate binding modulation. The F123A structure shows molecular rearrangement of the H-site in one subunit, but not the other, explaining weakened hydrophobic substrate binding and kinetic co-operativity effects of Phe(123) mutations. The three crystal structures provide comprehensive evidence of the aromatic 'zipper' residues having an impact upon protein stability, catalysis and specificity. Consequently, 'zipper' residues appear to modulate and co-ordinate substrate processing through permissive flexing.

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  • Structural contributions of Delta class glutathione transferase active-site residues to catalysis 査読

    Jantana Wongsantichon, Robert C. Robinson, Albert J. Ketterman

    BIOCHEMICAL JOURNAL   428 ( 1 )   25 - 32   2010年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PORTLAND PRESS LTD  

    GST (glutathione transferase) is a dimeric enzyme recognized for biotransformation of xenobiotics and endogenous toxic compounds. In the present study, residues forming the hydrophobic substrate-binding site (H-site) of a Delta class enzyme were investigated in detail for the first time by site-directed mutagenesis and crystallographic studies. Enzyme kinetics reveal that Tyr(111) indirectly stabilizes GSH binding, Tyr(119) modulates hydrophobic substrate binding and Phe(123) indirectly modulates catalysis. Mutations at Tyr(111) and Phe(123) also showed evidence for positive co-operativity for GSH and 1-chloro-2,4-dinitrobenzene respectively, strongly suggesting a role for these residues in manipulating subunit-subunit communication. In the present paper we report crystal structures of the wild-type enzyme, and two mutants, in complex with S-hexylglutathione. This study has identified an aromatic 'zipper' in the H-site contributing a network of aromatic pi-pi interactions. Several residues of the cluster directly interact with the hydrophobic substrate, whereas others indirectly maintain conformational stability of the dimeric structure through the C-terminal domain (domain II). The Y119E mutant structure shows major main-chain rearrangement of domain II. This reorganization is moderated through the 'zipper' that contributes to the H-site remodelling, thus illustrating a role in co-substrate binding modulation. The F123A structure shows molecular rearrangement of the H-site in one subunit, but not the other, explaining weakened hydrophobic substrate binding and kinetic co-operativity effects of Phe(123) mutations. The three crystal structures provide comprehensive evidence of the aromatic 'zipper' residues having an impact upon protein stability, catalysis and specificity. Consequently, 'zipper' residues appear to modulate and co-ordinate substrate processing through permissive flexing.

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  • Filament Structure, Organization, and Dynamics in MreB Sheets 査読

    David Popp, Akihiro Narita, Kayo Maeda, Tetsuro Fujisawa, Umesh Ghoshdastider, Mitsusada Iwasa, Yuichiro Maeda, Robert C. Robinson

    JOURNAL OF BIOLOGICAL CHEMISTRY   285 ( 21 )   15858 - 15865   2010年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    In vivo fluorescence microscopy studies of bacterial cells have shown that the bacterial shape-determining protein and actin homolog, MreB, forms cable-like structures that spiral around the periphery of the cell. The molecular structure of these cables has yet to be established. Here we show by electron microscopy that Thermatoga maritime MreB forms complex, several mu m long multilayered sheets consisting of diagonally interwoven filaments in the presence of either ATP or GTP. This architecture, in agreement with recent rheological measurements on MreB cables, may have superior mechanical properties and could be an important feature for maintaining bacterial cell shape. MreB polymers within the sheets appear to be single-stranded helical filaments rather than the linear protofilaments found in the MreB crystal structure. Sheet assembly occurs over a wide range of pH, ionic strength, and temperature. Polymerization kinetics are consistent with a cooperative assembly mechanism requiring only two steps: monomer activation followed by elongation. Steady-state TIRF microscopy studies of MreB suggest filament treadmilling while high pressure small angle x-ray scattering measurements indicate that the stability of MreB polymers is similar to that of F-actin filaments. In the presence of ADP or GDP, long, thin cables formed in which MreB was arranged in parallel as linear protofilaments. This suggests that the bacterial cell may exploit various nucleotides to generate different filament structures within cables for specific MreB-based functions.

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  • Polymeric Structures and Dynamic Properties of the Bacterial Actin AlfA 査読

    David Popp, Akihiro Narita, Umesh Ghoshdastider, Kayo Maeda, Yuichiro Maeda, Toshiro Oda, Tetsuro Fujisawa, Hirufumi Onishi, Kazuki Ito, Robert C. Robinson

    JOURNAL OF MOLECULAR BIOLOGY   397 ( 4 )   1031 - 1041   2010年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

    AlfA is a recently discovered DNA segregation protein from Bacillus subtilis that is distantly related to actin and the bacterial actin homologues ParM and MreB. Here we show that AlfA mostly forms helical 7/3 filaments, with a repeat of about 180 angstrom, that are arranged in three-dimensional bundles. Other polymorphic structures in the form of two-dimensional rafts or paracrystalline nets were also observed. Here AlfA adopted a 16/7 helical symmetry, with a repeat of about 387 A. Thin polymers consisting of several intertwining filaments also formed. Observed helical symmetries of AlfA filaments differed from those of other members of the actin family: F-actin, ParM, or MreB. Both ATP and guanosine 5'-triphosphate are able to promote rapid AlfA filament formation with almost equal efficiencies. The helical structure is only preserved under physiological salt concentrations and at a pH between 6.4 and 7.4, the physiological range of the cytoplasm of B. subtilis. Polymerization kinetics are extremely rapid and compatible with a cooperative assembly mechanism requiring only two steps: monomer activation followed by elongation, making AlfA one of the most efficient polymerizing motors within the actin family. Phosphate release lags behind polymerization, and time-lapse total internal reflection fluorescence images of AlfA bundles are consistent with treadmilling rather than dynamic microtubule-like instability. High-pressure small angle X-ray scattering experiments reveal that the stability of AlfA filaments is intermediate between the stability of ParM and the stability of F-actin. These results emphasize that actin-like polymerizing machineries have diverged to produce a variety of filament geometries with diverse properties that are tailored for specific biological processes. (C) 2010 Elsevier Ltd. All rights reserved.

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  • Structural characterization of a capping protein interaction motif defines a family of actin filament regulators. 査読

    Hernandez-Valladares M, Kim T, Kannan B, Tung A, Aguda AH, Larsson M, Cooper JA, Robinson RC

    Nature structural & molecular biology   17 ( 4 )   497 - 503   2010年4月

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    掲載種別:研究論文(学術雑誌)  

    DOI: 10.1038/nsmb.1792

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  • Suprastructures and Dynamic Properties of Mycobacterium tuberculosis FtsZ 査読

    David Popp, Mitsusada Iwasa, Harold P. Erickson, Akihiro Narita, Yuichiro Maeda, Robert C. Robinson

    JOURNAL OF BIOLOGICAL CHEMISTRY   285 ( 15 )   11281 - 11289   2010年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Tuberculosis causes the most death in humans by any bacterium. Drug targeting of bacterial cytoskeletal proteins requires detailed knowledge of the various filamentous suprastructures and dynamic properties. Here, we have investigated by high resolution electron microscopy the assembly of cell division protein and microtubule homolog FtsZ from Mycobacterium tuberculosis (MtbFtsZ) in vitro in the presence of various monovalent salts, crowding agents and polycations. Supramolecular structures, including two-dimensional rings, three-dimensional toroids, and multistranded helices formed in the presence of molecular crowding, were similar to those observed by fluorescence microscopy in bacteria in vivo. Dynamic properties of MtbFtsZ filaments were visualized by light scattering and real time total internal reflection fluorescence microscopy. Interestingly, MtbFtsZ revealed a form of dynamic instability at steady state. Cation-induced condensation phenomena of bacterial cytomotive polymers have not been investigated in any detail, although it is known that many bacteria can contain high amounts of polycations, which may modulate the prokaryotic cytoskeleton. We find that above a threshold concentration of polycations which varied with the valence of the cation, ionic strength, and pH, MtbFtsZ mainly formed sheets. The general features of these cation-induced condensation phenomena could be explained in the framework of the Manning condensation theory. Chirality and packing defects limited the dimensions of sheets and toroids at steady state as predicted by theoretical models. In first approximation simple physical principles seem to govern the formation of MtbFtsZ suprastructures.

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  • Structural characterization of a capping protein interaction motif defines a family of actin filament regulators 査読

    Maria Hernandez-Valladares, Taekyung Kim, Balakrishnan Kannan, Alvin Tung, Adeleke H. Aguda, Marten Larsson, John A. Cooper, Robert C. Robinson

    NATURE STRUCTURAL & MOLECULAR BIOLOGY   17 ( 4 )   497 - U145   2010年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATURE PUBLISHING GROUP  

    Capping protein (CP) regulates actin dynamics by binding the barbed ends of actin filaments. Removal of CP may be one means to harness actin polymerization for processes such as cell movement and endocytosis. Here we structurally and biochemically investigated a CP interaction (CPI) motif present in the otherwise unrelated proteins CARMIL and CD2AP. The CPI motif wraps around the stalk of the mushroom-shaped CP at a site distant from the actin-binding interface, which lies on the top of the mushroom cap. We propose that the CPI motif may act as an allosteric modulator, restricting CP to a low-affinity, filament-binding conformation. Structure-based sequence alignments extend the CPI motif-containing family to include CIN85, CKIP-1, CapZIP and a relatively uncharacterized protein, WASHCAP (FAM21). Peptides comprising these CPI motifs are able to inhibit CP and to uncap CP-bound actin filaments.

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  • Structure and Filament Dynamics of the pSK41 Actin-like ParM Protein IMPLICATIONS FOR PLASMID DNA SEGREGATION 査読

    David Popp, Weijun Xu, Akihiro Narita, Anthony J. Brzoska, Ronald A. Skurray, Neville Firth, Umesh Goshdastider, Yuichiro Maeda, Robert C. Robinson, Maria A. Schumacher

    JOURNAL OF BIOLOGICAL CHEMISTRY   285 ( 13 )   10130 - 10140   2010年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Type II plasmid partition systems utilize ParM NTPases in coordination with a centromere-binding protein called ParR to mediate accurate DNA segregation, a process critical for plasmid retention. The Staphylococcus aureus pSK41 plasmid is a medically important plasmid that confers resistance to multiple antibiotics, disinfectants, and antiseptics. In the first step of partition, the pSK41 ParR binds its DNA centromere to form a superhelical partition complex that recruits ParM, which then mediates plasmid separation. pSK41 ParM is homologous to R1 ParM, a known actin homologue, suggesting that it may also form filaments to drive partition. To gain insight into the partition function of ParM, we examined its ability to form filaments and determined the crystal structure of apoParM to 1.95 angstrom. The structure shows that pSK41 ParM belongs to the actin/Hsp70 superfamily. Unexpectedly, however, pSK41 ParM shows the strongest structural homology to the archaeal actin-like protein Thermoplasma acidophilum Ta0583, rather than its functional homologue, R1 ParM. Consistent with this divergence, we find that regions shown to be involved in R1 ParM filament formation are not important in formation of pSK41 ParM polymers. These data are also consonant with our finding that pSK41 ParM forms 1-start 10/4 helices very different from the 37/17 symmetry of R1 ParM. The polymerization kinetics of pSK41 ParM also differed from that of R1 ParM. These results indicate that type II NTPases utilize different polymeric structures to drive plasmid segregation.

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  • Molecular mechanism of bundle formation by the bacterial actin ParM 査読

    David Popp, Akihiro Narita, Mitsusada Iwasa, Yuichiro Maeda, Robert C. Robinson

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   391 ( 4 )   1598 - 1603   2010年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    The actin homolog ParM plays a microtubule-like role in segregating DNA prior to bacterial cell division. Fluorescence and cryo-electron microscopy have shown that ParM forms filament bundles between separating DNA plasmids in vivo. Given the lack of ParM bundling proteins it remains unknown how ParM bundles form at the molecular level. Here we show using time-lapse TIRF microscopy, under in vitro molecular crowding conditions, that ParM-bundle formation consists of two distinct phases. At the onset of polymerization bundle thickness and shape are determined in the form of nuclei of short helically disordered filaments arranged in a liquid-like lattice. These nuclei then undergo an elongation phase whereby they rapidly increase in length. At steady state, ParM bundles fuse into one single large aggregate. This behavior had been predicted by theory but has not been observed for any other cytomotive biopolymer, including F-actin. We employed electron micrographs of ParM rafts, which are 2-D analogs of 3-D bundles, to identify the main molecular interfilament contacts within these suprastructures. The interface between filaments is similar for both parallel and anti-parallel orientations and the distribution of filament polarity is random within a bundle. We Suggest that the interfilament interactions are not due to the interactions of specific residues but rather to long-range, counter ion mediated, electrostatic attractive forces. A randomly oriented bundle ensures that the assembly is rigid and that DNA may be captured with equal efficiency at both ends of the bundle via the ParR binding protein. (C) 2009 Elsevier Inc. All rights reserved.

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  • Helix Straightening as an Activation Mechanism in the Gelsolin Superfamily of Actin Regulatory Proteins 査読

    Hui Wang, Sakesit Chumnarnsilpa, Anantasak Loonchanta, Qiang Li, Yang-Mei Kuan, Sylvie Robine, Marten Larsson, Ivana Mihalek, Leslie D. Burtnick, Robert C. Robinson

    JOURNAL OF BIOLOGICAL CHEMISTRY   284 ( 32 )   21265 - 21269   2009年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Villin and gelsolin consist of six homologous domains of the gelsolin/cofilin fold (V1-V6 and G1-G6, respectively). Villin differs from gelsolin in possessing at its C terminus an unrelated seventh domain, the villin headpiece. Here, we present the crystal structure of villin domain V6 in an environment in which intact villin would be inactive, in the absence of bound Ca(2+) or phosphorylation. The structure of V6 more closely resembles that of the activated form of G6, which contains one bound Ca(2+), rather than that of the calcium ion-free form of G6 within intact inactive gelsolin. Strikingly apparent is that the long helix in V6 is straight, as found in the activated form of G6, as opposed to the kinked version in inactive gelsolin. Molecular dynamics calculations suggest that the preferable conformation for this helix in the isolated G6 domain is also straight in the absence of Ca(2+) and other gelsolin domains. However, the G6 helix bends in intact calcium ion-free gelsolin to allow interaction with G2 and G4. We suggest that a similar situation exists in villin. Within the intact protein, a bent V6 helix, when triggered by Ca(2+), straightens and helps push apart adjacent domains to expose actin-binding sites within the protein. The sixth domain in this superfamily of proteins serves as a keystone that locks together a compact ensemble of domains in an inactive state. Perturbing the keystone initiates reorganization of the structure to reveal previously buried actin-binding sites.

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  • Ca2+ binding by domain 2 plays a critical role in the activation and stabilization of gelsolin 査読

    Shalini Nag, Qing Ma, Hui Wang, Sakesit Chumnarnsilpa, Wei Lin Lee, Marten Larsson, Balakrishnan Kannan, Maria Hernandez-Valladarez, Leslie D. Burtnick, Robert C. Robinson

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   106 ( 33 )   13713 - 13718   2009年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATL ACAD SCIENCES  

    Gelsolin consists of six homologous domains (G1-G6), each containing a conserved Ca-binding site. Occupation of a subset of these sites enables gelsolin to sever and cap actin filaments in a Ca-dependent manner. Here, we present the structures of Ca-free human gelsolin and of Ca-bound human G1-G3 in a complex with actin. These structures closely resemble those determined previously for equine gelsolin. However, the G2 Ca-binding site is occupied in the human G1-G3/actin structure, whereas it is vacant in the equine version. In-depth comparison of the Ca-free and Ca-activated, actin-bound human gelsolin structures suggests G2 and G6 to be cooperative in binding Ca2+ and responsible for opening the G2-G6 latch to expose the F-actin-binding site on G2. Mutational analysis of the G2 and G6 Ca-binding sites demonstrates their interdependence in maintaining the compact structure in the absence of calcium. Examination of Ca binding by G2 in human G1-G3/actin reveals that the Ca2+ locks the G2-G3 interface. Thermal denaturation studies of G2-G3 indicate that Ca binding stabilizes this fragment, driving it into the active conformation. The G2 Ca-binding site is mutated in gelsolin from familial amyloidosis (Finnish-type) patients. This disease initially proceeds through protease cleavage of G2, ultimately to produce a fragment that forms amyloid fibrils. The data presented here support a mechanism whereby the loss of Ca binding by G2 prolongs the lifetime of partially activated, intermediate conformations in which the protease cleavage site is exposed.

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  • The crystal structure of the C-terminus of adseverin reveals the actin-binding interface 査読

    Sakesit Chumnarnsilpa, Wei Lin Lee, Shalini Nag, Balakrishnan Kannan, Marten Larsson, Leslie D. Burtnick, Robert C. Robinson

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   106 ( 33 )   13719 - 13724   2009年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATL ACAD SCIENCES  

    Adseverin is a member of the calcium-regulated gelsolin superfamily of actin severing and capping proteins. Adseverin comprises 6 homologous domains (A1-A6), which share 60% identity with the 6 domains from gelsolin (G1-G6). Adseverin is truncated in comparison to gelsolin, lacking the C-terminal extension that masks the F-actin binding site in calcium-free gelsolin. Biochemical assays have indicated differences in the interaction of the C-terminal halves of adseverin and gelsolin with actin. Gelsolin contacts actin through a major site on G4 and a minor site on G6, whereas adseverin uses a site on A5. Here, we present the X-ray structure of the activated C-terminal half of adseverin (A4-A6). This structure is highly similar to that of the activated form of the C-terminal half of gelsolin (G4-G6), both in arrangement of domains and in the 3 bound calcium ions. Comparative analysis of the actin-binding surfaces observed in the G4-G6/actin structure suggests that adseverin in this conformation will also be able to interact with actin through A4 and A6, whereas the A5 surface is obscured. A single residue mutation in A4-A6 located at the predicted A4/actin interface completely abrogates actin sequestration. A model of calcium-free adseverin, constructed from the structure of gelsolin, predicts that in the absence of a gelsolin-like C-terminal extension the interaction between A2 and A6 provides the steric inhibition to prevent interaction with F-actin. We propose that calcium binding to the N terminus of adseverin dominates the activation process to expose the F-actin binding site on A2.

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  • The 1.9 X-Ray Structure of Egg-white Lysozyme from Taiwanese Soft-Shelled Turtle (Trionyx Sinensis Wiegmann) Exhibits Structural Differences from the Standard Chicken-Type Lysozyme 査読

    Jaruwan Siritapetawee, Sompong Thammasirirak, Robert C. Robinson, Jirundon Yuvaniyama

    JOURNAL OF BIOCHEMISTRY   145 ( 2 )   193 - 198   2009年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

    Lysozyme from Taiwanese soft-shelled turtle (SSTLB) has been purified from turtle egg white and crystallized. The crystals diffract X-rays beyond 2 resolution and belong to the orthorhombic P2(1)2(1)2(1) space group containing one molecule per asymmetric unit. The structure was elucidated using pheasant egg-white lysozyme as the molecular replacement search template. The overall structure of SSTLB is very similar to that of hen egg-white lysozyme (HEWL). Nevertheless, Pro104 in the substrate subsite A and other amino acids in the substrate subsites E and F differ from those of HEWL. These substitutions result in local conformational differences in the substrate binding sites of the two enzymes, effecting substrate binding and transglycosylation efficiency, translating into differing ranges of products.

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  • Crystal structures of Vibrio harveyi chitinase A complexed with chitooligosaccharides: Implications for the catalytic mechanism 査読

    Chomphunuch Songsiriritthigul, Supansa Pantoom, Adeleke H. Aguda, Robert C. Robinson, Wipa Suginta

    JOURNAL OF STRUCTURAL BIOLOGY   162 ( 3 )   491 - 499   2008年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    This research describes four X-ray structures of Vibrio harveyi chitinase A and its catalytically inactive mutant (E315M) in the presence and absence of substrates. The overall structure of chitinase A is that of a typical family-18 glycosyl hydrolase comprising three distinct domains: (i) the amino-terminal chitin-binding domain; (ii) the main catalytic (alpha/beta)(8) TIM-barrel domain; and (iii) the small (alpha + beta) insertion domain. The catalytic cleft of chitinase A has a long, deep groove, which contains six chitooligosaccharide ring-binding subsites (-4)(-3)(-2)(-1)(+1)(+2). The binding cleft of the ligand-free E315M is partially blocked by the C-terminal (His)(6)-tag. Structures of E315M-chitooligosaccharide complexes display a linear conformation of pentaNAG, but a bent conformation of hexaNAG. Analysis of the final 2F(o) - F-c omit map of E315M-NAG6 reveals the existence of the linear conformation of the hexaNAG at a lower occupancy with respect to the bent conformation. These crystallographic data provide evidence that the interacting sugars undergo conformational changes prior to hydrolysis by the wild-type enzyme. (c) 2008 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.jsb.2008.03.008

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  • Structural insights into rice BGlu1 beta-glucosidase oligosaccharide hydrolysis and transglycosylation 査読

    Watchalee Chuenchor, Salila Pengthaisong, Robert C. Robinson, Jirundon Yuvaniyama, Worrapoj Oonanant, David R. Bevan, Asim Esen, Chun-Jung Chen, Rodjana Opassiri, Jisnuson Svasti, James R. Ketudat Cairns

    JOURNAL OF MOLECULAR BIOLOGY   377 ( 4 )   1200 - 1215   2008年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

    The structures of rice BGlu1 beta-glucosiclase, a plant beta-glucosidase active in hydrolyzing cell wall-derived oligosaccharides, and its covalent intermediate with 2-deoxy-2-fluoroglucoside have been solved at 2.2 angstrom and 1.55 angstrom resolution, respectively. The structures were similar to the known structures of other glycosyl hydrolase family 1 (GHI) beta-glucosidases, but showed several differences in the loops around the active site, which lead to an open active site with a narrow slot at the bottom, compatible with the hydrolysis of long beta-1,4-linked oligosaccharides. Though this active site structure is somewhat similar to that of the Paenibacillus polyinyxa beta-glucosidase B, which hydrolyzes similar ohgosaccharides, molecular docking studies indicate that the residues interacting with the substrate beyond the conserved -1 site are completely different, reflecting the independent evolution of plant and microbial GH1 exo-beta-glucanase/beta-glucosidases. The complex with the 2-fluoroglucoside included a glycerol molecule, which appears to be in a position to make a nucleophilic attack on the anomeric carbon in a transglycosylation reaction. The coordination of the hydroxyl groups suggests that sugars are positioned as acceptors for transglycosylation by their interactions with E176, the catalytic acid/base, and Y131, which is conserved in barley BGQ60/beta-II beta-glucosidase, that has oligosaccharide hydrolysis and transglycosylation activity similar to rice BGlu1. As the rice and barley enzymes have different preferences for cellobiose and cellotriose, residues that appeared to interact with docked oligosaccharides were mutated to those of the barley enzyme to see if the relative activities of rice BGlu1 toward these substrates could be changed to those of BGQ60. Although no single residue appeared to be responsible for these differences, 1179, N190 and N245 did appear to interact with the substrates. (C) 2008 Elsevier Ltd. All rights reserved.

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  • Expression, crystallization and preliminary crystallographic data analysis of filamin A repeats 14-16 査読

    Adeleke Halilu Aguda, Amos Malle Sakwe, Lars Rask, Robert Charles Robinson

    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS   63 ( Pt 4 )   291 - 293   2007年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:BLACKWELL PUBLISHING  

    Human filamin A is a 280 kDa protein involved in actin-filament cross-linking. It is structurally divided into an actin-binding headpiece (ABD) and a rod domain containing 24 immunoglobulin-like (Ig) repeats. A fragment of human filamin A (Ig repeats 14-16) was cloned and expressed in Escherichia coli and the purified protein was crystallized in 1.6 M ammonium sulfate, 2% PEG 1000 and 100 mM HEPES pH 7.5. The crystals diffracted to 1.95 angstrom and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.63, b = 52.10, c = 98.46 angstrom, alpha = beta = gamma = 90 degrees.

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  • Expression, crystallization and preliminary crystallographic data analysis of filamin A repeats 14-16 査読

    Adeleke Halilu Aguda, Amos Malle Sakwe, Lars Rask, Robert Charles Robinson

    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS   63   291 - 293   2007年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:BLACKWELL PUBLISHING  

    Human filamin A is a 280 kDa protein involved in actin-filament cross-linking. It is structurally divided into an actin-binding headpiece (ABD) and a rod domain containing 24 immunoglobulin-like (Ig) repeats. A fragment of human filamin A (Ig repeats 14-16) was cloned and expressed in Escherichia coli and the purified protein was crystallized in 1.6 M ammonium sulfate, 2% PEG 1000 and 100 mM HEPES pH 7.5. The crystals diffracted to 1.95 angstrom and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.63, b = 52.10, c = 98.46 angstrom, alpha = beta = gamma = 90 degrees.

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  • Crystallographic and in silico analysis of the sialoside-binding characteristics of the Siglec sialoadhesin 査読

    Nathan R. Zaccai, Andrew P. May, Robert C. Robinson, Leslie D. Burtnick, Paul R. Crocker, Reinhard Brossmer, Soerge Keim, E. Yvonne Jones

    JOURNAL OF MOLECULAR BIOLOGY   365 ( 5 )   1469 - 1479   2007年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD  

    The Siglec family of receptors mediates cell-surface interactions through recognition of sialylated glycoconjugates. Previously reported structures of the N-terminal domain of the Siglec sialoadhesin (SnD1) in complex with various sialic acid analogs revealed the structural template for sialic acid binding. To characterize further the carbohydrate-binding properties, we have determined the crystal structures of SnD1 in the absence of ligand, and in complex with 2-benzyl-Neu5NPro and 2-benzyl-Neu5NAc. These structures reveal that SnD1 undergoes very few structural changes on ligand binding and detail how two novel classes of sialic acid analogs bind, one of which unexpectedly can induce Siglec dimerization. In conjunction with in silico analysis, this set of structures informs us about the design of putative ligands with enhanced binding affinities and specificities to different Siglecs, and provides data with which to test the effectiveness of different computational drug design protocols. (c) 2006 Elsevier Ltd. All rights reserved.

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  • Models of the actin-bound forms of the beta-thymosins 査読

    Bo Xue, Adeleke Halilu Aguda, Robert Charles Robinson

    THYMOSINS IN HEALTH AND DISEASE: FIRST INTERNATIONAL SYMPOSIUM   1112   56 - 66   2007年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:BLACKWELL PUBLISHING  

    recent years two structures have been reported that demonstrate how the two halves of a beta-thymosin repeat bind to actin monomers. Here we assess the validity of these structures and construct minimally biased models of the P-thymosin:actin complexes. The models reveal that the beta-thymosins interact with actin throughout their length and that all the conserved residues are functional in this interface. These models are judged to be in excellent agreement with published biochemical and functional data. In particular, the models are consistent with the actin monomer sequestering and actin filament binding properties of beta-thymosins. The models also correctly predict competition between thymosin-beta 4 with DNase I or profilin in binding actin while allowing ternary complexes at higher concentrations.

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  • Targeted molecular dynamics simulation studies of calcium binding and conformational change in the C-terminal half of gelsolin 査読

    HS Lee, RC Robinson, CH Joo, H Lee, YK Kim, H Choe

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   342 ( 3 )   702 - 709   2006年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    Gelsolin consists of six related domains (G1-G6) and the C-terminal half (G4-G6) acts as a calcium sensor during the activation of the whole molecule, a process that involves large domain movements. In this study, we used targeted Molecular dynamics Simulations to elucidate the conformational transitions of G4-G6 at an atomic level. Domains G4 and G6 are initially ruptured, followed by a rotation of G6 by similar to 90 degrees, which is the dominant conformational change. During this period, local conformational changes occur at the G4 and G5 calcium-binding sites, facilitating large changes in interdomain distances. Alterations in the binding affinities of the calcium ions in these three domains appear to be related to local conformational changes at their binding sites. Analysis of the relative stabilities of the G4-G6-bound calcium ions suggests that they bind first to G6. then to G4, and finally to G5. (c) 2006 Elsevier Inc. All rights reserved.

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  • Calcium ion exchange in crystalline gelsolin 査読

    S Chumnarnsilpa, A Loonchanta, B Xue, H Choe, D Urosev, H Wang, U Lindberg, LD Burtnick, RC Robinson

    JOURNAL OF MOLECULAR BIOLOGY   357 ( 3 )   773 - 782   2006年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD  

    Gelsolin is a calcium and pH-sensitive modulator of actin filament length. Here, we use X-ray crystallography to examine the extraction and exchange of calcium ions from their binding sites in different crystalline forms of the activated N and C-terminal halves of gelsolin, G1-G3 and G4-G6, respectively. We demonstrate that the combination of calcium and low pH activating conditions do not induce conformational changes in G4-G6 beyond those elicited by calcium alone. EGTA is able to remove calcium ions bound to the type I and type II metal ion-binding sites in G4-G6. Constrained by crystal contacts and stabilized by interdomain interaction surfaces, the gross structure of calcium-depleted G4-G6 remains that of the activated form. However, high-resolution details of changes in the ion-binding sites may represent the initial steps toward restoration of the arrangement of domains found in the calcium-free inactive form of gelsolin in solution. Furthermore, bathing crystals with the trivalent calcium ion mimic, Tb3+, results in anomalous scattering data that permit unequivocal localization of terbium ions in each of the proposed type I and type II ion-binding sites of both halves of gelsolin. In contrast to predictions based on solution studies, we find that no calcium ion is immune to exchange. (c) 2006 Elsevier Ltd. All rights reserved.

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  • The structure of gelsolin bound to ATP 査読

    D Urosev, Q Ma, ALC Tan, RC Robinson, LD Burtnick

    JOURNAL OF MOLECULAR BIOLOGY   357 ( 3 )   765 - 772   2006年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD  

    Calcium activation of the actin-modifying properties of gelsolin is sensitive to ATP. Here, we show that soaking calcium-free gelsolin crystals in ATP-containing media results in ATP occupying a site that spans the two pseudosymmetrical halves of the protein. ATP binding involves numerous polar and hydrophobic contacts and is identical for the two copies of gelsolin related by non-crystallographic symmetry within the crystal. The gamma-phosphate of ATP participates in several charge-charge interactions consistent with the preference of gelsolin for ATP, as a binding partner, over ADP. In addition, disruption of the ATP-binding site through Ca2+ activation of gelsolin reveals why ATP binds more tightly to the inactive molecule, and suggests how the binding of ATP may modulate the sensitivity of gelsolin to calcium ions. Similarities between the ATP and PIP2 interactions with the C-terminal half of gelsolin are evident from their overlapping binding sites and in that both molecules bind more tightly in the absence of calcium ions. We propose a model for how PIP2 may bind to calcium-free gelsolin based on the ATP-binding site. (c) 2006 Elsevier Ltd. All rights reserved.

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  • The structural basis of actin interaction with multiple WH2/beta-thymosin motif-containing proteins 査読

    AH Aguda, B Xue, E Irobi, T Preat, RC Robinson

    STRUCTURE   14 ( 3 )   469 - 476   2006年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:CELL PRESS  

    Participation of actin in cellular processes relies on the dynamics of filament assembly. Filament elongation is fed by monomeric actin in complex with either profilin or a Wiscott-Aldrich syndrome protein (WASP) homology domain 2 (WH2)/beta-thymosin (beta T) domain. WH2/beta T motif repetition (typified by ciboulot) or combination with nonrelated domains (as found in N-WASP) results in proteins that yield their actin to filament elongation. Here, we report the crystal structures of actin bound hybrid proteins, constructed between gelsolin and WH2/beta T domains from ciboulot or N-WASP. We observe the C-terminal half of ciboulot domain 2 bound to actin. In solution, we show that cibolout domains 2 and 3 bind to both G- and F-actin, and that whole ciboulot forms a complex with two actin monomers. In contrast, the analogous portion of N-WASP WH2 domain 2 is detached from actin, indicating that the C-terminal halves of the PT and WH2 motifs are not functionally analogous.

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  • Expression, purification, crystallization, and preliminary X-ray analysis of the human UDP-glucose dehydrogenase 査読

    Jae Wan Huh, Robert Charles Robinson, Han Sam Lee, Jae Il Lee, Yong Seok Heo, Hyun Tae Kim, Hyun Ju Lee, Sung Woo Cho, Han Choe

    PROTEIN AND PEPTIDE LETTERS   13 ( 8 )   859 - 862   2006年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:BENTHAM SCIENCE PUBL LTD  

    UDP-glucose dehydrogenase (UGDH) catalyzes the synthesis of UDP-glucuronic acid from UDP-glucose resulting in the formation of proteoglycans that are involved in promoting normal cellular growth and migration. Overproduction of proteoglycans has been implicated in the progression of certain epithelial cancers. Here, human UGDH (hUGDH) was purified and crystallized from a solution of 0.2 M ammonium sulfate, 0.1 M Na cacodylate, pH 6.5, and 21% PEG 8000. Diffraction data were collected to a resolution of 2.8 angstrom. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1) with unit-cell parameters a = 173.25, b = 191.16, c = 225.94 angstrom, and alpha = beta = gamma = 90.0 degrees. Based on preliminary analysis of the diffraction data, we propose that the biological unit of hUGDH is a tetramer.

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  • Expression, purification, crystallization and preliminary crystallographic analysis of chitinase A from Vibrio carchariae 査読

    C Songsiriritthigul, J Yuvaniyama, RC Robinson, A Vongsuwan, H Prinz, W Suginta

    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS   61   895 - 898   2005年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:INT UNION CRYSTALLOGRAPHY  

    Chitinase A of Vibrio carchariae was expressed in Escherichia coli M15 host cells as a 575-amino-acid fragment with full enzymatic activity using the pQE60 expression vector. The yield of the highly purified recombinant protein was approximately 70 mg per litre of bacterial culture. The molecular mass of the expressed protein was determined by HPLC/ESI-MS to be 63 770, including the hexahistidine tag. Crystals of recombinant chitinase A were grown to a suitable size for X-ray structure analysis in a precipitant containing 10% (v/v) PEG 400, 0.1 M sodium acetate pH 4.6 and 0.125 M CaCl2. The crystals belonged to the tetragonal space group P422, with two molecules per asymmetric unit and unit-cell parameters a = b = 127.64, c = 171.42 angstrom. A complete diffraction data set was collected to 2.14 angstrom resolution using a Rigaku/MSC R-AXIS IV++ detector system mounted on an RU-H3R rotating-anode X-ray generator.

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  • Expression, purification, crystallization and preliminary crystallographic analysis of chitinase A from Vibrio carchariae 査読

    C Songsiriritthigul, J Yuvaniyama, RC Robinson, A Vongsuwan, H Prinz, W Suginta

    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS   61 ( Pt 10 )   895 - 898   2005年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:INT UNION CRYSTALLOGRAPHY  

    Chitinase A of Vibrio carchariae was expressed in Escherichia coli M15 host cells as a 575-amino-acid fragment with full enzymatic activity using the pQE60 expression vector. The yield of the highly purified recombinant protein was approximately 70 mg per litre of bacterial culture. The molecular mass of the expressed protein was determined by HPLC/ESI-MS to be 63 770, including the hexahistidine tag. Crystals of recombinant chitinase A were grown to a suitable size for X-ray structure analysis in a precipitant containing 10% (v/v) PEG 400, 0.1 M sodium acetate pH 4.6 and 0.125 M CaCl2. The crystals belonged to the tetragonal space group P422, with two molecules per asymmetric unit and unit-cell parameters a = b = 127.64, c = 171.42 angstrom. A complete diffraction data set was collected to 2.14 angstrom resolution using a Rigaku/MSC R-AXIS IV++ detector system mounted on an RU-H3R rotating-anode X-ray generator.

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  • The state of the filament 査読

    AH Aguda, LD Burtnick, RC Robinson

    EMBO REPORTS   6 ( 3 )   220 - 226   2005年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATURE PUBLISHING GROUP  

    Movement is a defining characteristic of life. Macroscopic motion is driven by the dynamic interactions of myosin with actin filaments in muscle. Directed polymerization of actin behind the advancing membrane of a eukaryotic cell generates microscopic movement. Despite the fundamental importance of actin in these processes, the structure of the actin filament remains unknown. The Holmes model of the actin filament was published 15 years ago, and although it has been widely accepted, no high-resolution structural data have yet confirmed its veracity. Here, we review the implications of recently determined structures of F-actin-binding proteins for the structure of the actin filament and suggest a series of in silico tests for actin-filament models. We also review the significance of these structures for the arp2/3-mediated branched filament.

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  • Arabidopsis VILLIN1 generates actin filament cables that are resistant to depolymerization 査読

    S Huang, RC Robinson, LY Gao, T Matsumoto, A Brunet, L Blanchoin, CJ Staiger

    PLANT CELL   17 ( 2 )   486 - 501   2005年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC PLANT BIOLOGISTS  

    Dynamic cytoplasmic streaming, organelle positioning, and nuclear migration use molecular tracks generated from actin filaments arrayed into higher-order structures like actin cables and bundles. How these arrays are formed and stabilized, against cellular depolymerizing forces remains an open question. Villin and fimbrin are the best characterized actin-filament bundling or cross-linking proteins in plants and each is encoded by a multigene family of five members in Arabidopsis thaliana. The related villins and gelsolins are conserved proteins that are constructed from a core of six homologous gelsolin domains. Gelsolin is a calcium-regulated actin filament severing, nucleating and barbed end capping factor. Villin has a seventh domain at its C terminus, the villin headpiece, which can bind to an actin filament, conferring the ability to crosslink or bundle actin filaments. Many, but not all, villins retain the ability to sever, nucleate, and cap filaments. Here we have identified a putative calcium-insensitive villin isoform through comparison of sequence alignments between human gelsolin and plant villins with x-ray crystallography data for vertebrate gelsolin. VILLIN1 (VLN1) has the least well-conserved type 1 and type 2 calcium binding sites among the Arabidopsis VILLIN isoforms. Recombinant VLN1 binds to actin filaments with high affinity (K-d similar to1 muM) and generates bundled filament networks; both properties are independent of the free Ca2+ concentration. Unlike human plasma gelsolin, VLN1 does not nucleate the assembly of filaments from monomer, does not block the polymerization of profilin-actin onto barbed ends, and does not stimulate depolymerization or sever preexisting filaments. In kinetic assays with ADF/cofilin, villin appears to bind first to growing filaments and protects filaments against ADF-mediated depolymerization. We propose that VLN1 is a major regulator of the formation and stability of actin filament bundles in plant cells and that it functions to maintain the cable network even in the presence of stimuli that result in depolymerization of other actin arrays.

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  • Structural basis of actin sequestration by thymosin-beta 4: implications for WH2 proteins 査読

    E Irobi, AH Aguda, M Larsson, C Guerin, HL Yin, LD Burtnick, L Blanchoin, RC Robinson

    EMBO JOURNAL   23 ( 18 )   3599 - 3608   2004年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATURE PUBLISHING GROUP  

    The WH2 (Wiscott - Aldridge syndrome protein homology domain 2) repeat is an actin interacting motif found in monomer sequestering and filament assembly proteins. We have stabilized the prototypical WH2 family member, thymosin-beta4 (Tbeta4), with respect to actin, by creating a hybrid between gelsolin domain 1 and the C-terminal half of Tbeta4 (G1-Tbeta4). This hybrid protein sequesters actin monomers, severs actin filaments and acts as a leaky barbed end cap. Here, we present the structure of the G1-Tbeta4: actin complex at 2Angstrom resolution. The structure reveals that Tbeta4 sequesters by capping both ends of the actin monomer, and that exchange of actin between Tb4 and profilin is mediated by a minor overlap in binding sites. The structure implies that multiple WH2 motif-containing proteins will associate longitudinally with actin filaments. Finally, we discuss the role of the WH2 motif in arp2/3 activation.

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  • Structure of the N-terminal half of gelsolin bound to actin: roles in severing, apoptosis and FAF 査読

    LD Burtnick, D Urosev, E Irobi, K Narayan, RC Robinson

    EMBO JOURNAL   23 ( 14 )   2713 - 2722   2004年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATURE PUBLISHING GROUP  

    The actin filament-severing functionality of gelsolin resides in its N-terminal three domains (G1 - G3). We have determined the structure of this fragment in complex with an actin monomer. The structure reveals the dramatic domain rearrangements that activate G1 - G3, which include the replacement of interdomain interactions observed in the inactive, calcium-free protein by new contacts to actin, and by a novel G2 - G3 interface. Together, these conformational changes are critical for actin filament severing, and we suggest that their absence leads to the disease Finnish-type familial amyloidosis. Furthermore, we propose that association with actin drives the calcium-independent activation of isolated G1 G3 during apoptosis, and that a similar mechanism operates to activate native gelsolin at micromolar levels of calcium. This is the first structure of a filament-binding protein bound to actin and it sets stringent, high-resolution limitations on the arrangement of actin protomers within the filament.

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  • From the first to the second domain of gelsolin: a common path on the surface of actin? 査読

    E Irobi, LD Burtnick, D Urosev, K Narayan, RC Robinson

    FEBS LETTERS   552 ( 2-3 )   86 - 90   2003年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    We present the 2.6. resolution crystal structure of a complex formed between G-actin and gelsolin fragment Met25-GIn160 (G1+). The structure differs from those of other gelsolin domain 1 (G1) complexes in that an additional six amino acid residues from the crucial linker region into gelsolin domain 2 (G2) are visible and are attached securely to the surface of actin. The linker segment extends away from G1 up the face of actin in a direction that infers G2 will bind along the same long-pitch helical strand as the actin bound to G1. This is consistent with a mechanism whereby G2 attaches gelsolin to the side of a filament and then directs G1 toward a position where it would disrupt actin-actin contacts. Alignment of the sequence of the structurally important residues within the G1-G2 linker with those of WH2 (WASp homology domain 2) domain protein family members (e.g. WASp (Wiscott-Aldridge syndrome protein) and thymosin beta4) suggests that the opposing activities of filament assembly and disassembly may exploit a common patch on the surface of actin. (C) 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

    DOI: 10.1016/S0014-5793(03)00934-7

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  • Activation in isolation: exposure of the actin-binding site in the C-terminal half of gelsolin does not require actin 査読

    K Narayan, S Chumnarnsilpa, H Choe, E Irobi, D Urosev, U Lindberg, CE Schutt, LD Burtnick, RC Robinson

    FEBS LETTERS   552 ( 2-3 )   82 - 85   2003年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    Gelsolin requires activation to carry out its severing and capping activities on F-actin. Here, we present the structure of the isolated C-terminal half of gelsolin (G4-G6) at 2.0 resolution in the presence of Ca2+ ions. This structure completes a triptych of the states of activation of G4-G6 that illuminates its role in the function of gelsolin. Activated G4-G6 displays an open conformation, with the actin-binding site on G4 fully exposed and all three type-2 Ca2+ sites occupied. Neither actin nor the type-1 Ca2+, which normally is sandwiched between actin and G4, is required to achieve this conformation. (C) 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

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  • Selective interaction of megalin with postsynaptic density-95 (PSD-95)-like membrane-associated guanylate kinase (MAGUK) proteins 査読

    M Larsson, G Hjalm, AM Sakwe, A Engstrom, AS Hoglund, E Larsson, RC Robinson, C Sundberg, L Rash

    BIOCHEMICAL JOURNAL   373 ( Pt 2 )   381 - 391   2003年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PORTLAND PRESS  

    Megalin is an integral membrane receptor belonging to the low-density lipoprotein receptor family. In addition to its role as an endocytotic receptor, megalin has also been proposed to have signalling functions. Using interaction cloning in yeast, we identified the membrane-associated guanylate kinase family member postsynaptic density-95 (PSD-95) as an interaction partner for megalin. PSD-95 and a truncated version of megalin were coimmunoprecipitated from HEK-293 cell lysates overexpressing the two proteins, which confirmed the interaction. The two proteins were found to be co-localized in these cells by confocal microscopy. Immunocytochemical studies showed that cells in the parathyroid, proximal tubuli of the kidney and placenta express both megalin and PSD-95. We found that the interaction between the two proteins is mediated by the binding of the C-terminus of megalin, which has a type I PSD-95/Drosophila discs-large/zona occludens 1 (PDZ)-binding motif, to the PDZ2 domain of PSD-95. The PSD-95-like membrane-associated guanylate kinase ('MAGUK') family contains three additional members: PSD-93, synapse-associated protein 97 (SAP97) and SAP102. We detected these proteins, apart from SAP102, in parathyroid chief cells, a cell type having a marked expression of megalin. The PDZ2 domains of PSD-93 and SAP102 were also shown to interact with megalin, whereas no interaction was detected for SAP97. The SAP97 PDZ2 domain differed at four positions from the other members of the PSD-95 subfamily. One of these residues was Thr(389), located in the alphaB-helix and part of the hydrophobic pocket of the PDZ2 domain. Surface plasmon resonance experiments revealed that mutation of SAP97 Thr(389) to alanine, as with the other PSD-95-like membrane-associated guanylate kinases, induced binding to megalin.

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  • Selective interaction of megalin with postsynaptic density-95 (PSD-95)-like membrane-associated guanylate kinase (MAGUK) proteins 査読

    M Larsson, G Hjalm, AM Sakwe, A Engstrom, AS Hoglund, E Larsson, RC Robinson, C Sundberg, L Rash

    BIOCHEMICAL JOURNAL   373   381 - 391   2003年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PORTLAND PRESS  

    Megalin is an integral membrane receptor belonging to the low-density lipoprotein receptor family. In addition to its role as an endocytotic receptor, megalin has also been proposed to have signalling functions. Using interaction cloning in yeast, we identified the membrane-associated guanylate kinase family member postsynaptic density-95 (PSD-95) as an interaction partner for megalin. PSD-95 and a truncated version of megalin were coimmunoprecipitated from HEK-293 cell lysates overexpressing the two proteins, which confirmed the interaction. The two proteins were found to be co-localized in these cells by confocal microscopy. Immunocytochemical studies showed that cells in the parathyroid, proximal tubuli of the kidney and placenta express both megalin and PSD-95. We found that the interaction between the two proteins is mediated by the binding of the C-terminus of megalin, which has a type I PSD-95/Drosophila discs-large/zona occludens 1 (PDZ)-binding motif, to the PDZ2 domain of PSD-95. The PSD-95-like membrane-associated guanylate kinase ('MAGUK') family contains three additional members: PSD-93, synapse-associated protein 97 (SAP97) and SAP102. We detected these proteins, apart from SAP102, in parathyroid chief cells, a cell type having a marked expression of megalin. The PDZ2 domains of PSD-93 and SAP102 were also shown to interact with megalin, whereas no interaction was detected for SAP97. The SAP97 PDZ2 domain differed at four positions from the other members of the PSD-95 subfamily. One of these residues was Thr(389), located in the alphaB-helix and part of the hydrophobic pocket of the PDZ2 domain. Surface plasmon resonance experiments revealed that mutation of SAP97 Thr(389) to alanine, as with the other PSD-95-like membrane-associated guanylate kinases, induced binding to megalin.

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  • The calcium activation of gelsolin: Insights from the 3 angstrom structure of the G4-G6/actin complex 査読

    H Choe, LD Burtnick, M Mejillano, HL Yin, RC Robinson, S Choe

    JOURNAL OF MOLECULAR BIOLOGY   324 ( 4 )   691 - 702   2002年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD  

    Gelsolin participates in the reorganization of the actin cytoskeleton that is required during such phenomena as cell movement, cytokinesis, and apoptosis. It consists of six structurally similar domains, G1-G6, which are arranged at resting intracellular levels of calcium ion so as to obscure the three actin-binding surfaces. Elevation of Ca2+ concentrations releases latches within the constrained structure and produces large shifts in the relative positioning of the domains, permitting gelsolin to bind to and sever actin filaments. How Ca2+ is able to activate gelsolin has been a major question concerning the function of this protein. We present the improved structure of the C-terminal half of gelsolin bound to monomeric actin at 3.0 Angstrom resolution. Two classes of Ca2+-binding site are evident on gelsolin: type 1 sites share coordination of Ca2+ with actin, while type 2 sites are wholly contained within gelsolin. This structure of the complex reveals the locations of two novel metal ion-binding sites in domains G5 and G6, respectively. We identify both as type 2 sites. The absolute conservation of the type 2 calcium-ligating residues across the six,domains of gelsolin suggests that this site exists in each of the domains. In total, gelsolin has the potential to bind eight calcium ions, two type 1 and six type 2. The function of the type 2 sites is to facilitate structural rearrangements within gelsolin as part of the activation and actin-binding and severing processes. We propose the novel type 2 site in G6 to be the critical site that initiates overall activation of gelsolin by releasing the tail latch that locks calcium-free gelsolin in a conformation unable to bind actin. (C) 2002 Elsevier Science Ltd. All rights reserved.

    DOI: 10.1016/S0022-2836(02)01131-2

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  • Crystal structure of Arp2/3 complex 査読

    RC Robinson, K Turbedsky, DA Kaiser, JB Marchand, HN Higgs, S Choe, TD Pollard

    SCIENCE   294 ( 5547 )   1679 - 1684   2001年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER ASSOC ADVANCEMENT SCIENCE  

    We determined a crystal structure of bovine Arp2/3 complex, an assembly of seven proteins that initiates actin polymerization in eukaryotic cells, at 2.0 angstrom resolution. Actin-related protein 2 (Arp2) and Arp3 are folded like actin, with distinctive surface features. Subunits ARPC2 p34 and ARPC4 p20 in the core of the complex associate through long carboxyl-terminal a helices and have similarly folded amino-terminal alpha/beta domains. ARPC1 p40 is a seven-blade beta propeller with an insertion that may associate with the side of an actin filament. ARPC3 p21 and ARPC5 p16 are globular alpha -helical subunits. We predict that WASp/Scar proteins activate Arp2/3 complex by bringing Arp2 into proximity with Arp3 for nucleation of a branch on the side of a preexisting actin filament.

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  • Peptides from the amino terminal mdm-2-binding domain of p53, designed from conformational analysis, are selectively cytotoxic to transformed cells 査読

    M Kanovsky, A Raffo, L Drew, R Rosal, T Do, FK Friedman, P Rubinstein, J Visser, R Robinson, PW Brandt-Rauf, J Michl, RL Fine, MR Pincus

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   98 ( 22 )   12438 - 12443   2001年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATL ACAD SCIENCES  

    We have synthesized three peptides from the mdm-2 binding domain of human p53, residues 12-26 (PPLSQETFSDLWKLL), residues 12-20, and 17-26. To enable transport of the peptides across the cell membrane and at the same time to maximize the active mdm-2 binding a-helical conformation for these peptides, each was attached at its carboxyl terminus to the penetratin sequence, KKWKMRRNQFWVKVQRG, that contains many positively charged residues that stabilize an a-helix when present on its carboxyl terminal end. All three peptides were cytotoxic to human cancer cells in culture, whereas a control, unrelated peptide attached to the same penetratin sequence had no effect on these cell lines. The same three cytotoxic peptides had no effect on the growth of normal cells, including human cord blood-derived stem cells. These peptides were as effective in causing cell death in p53-null cancer cells as in those having mutant or normal p53. Peptide-induced cell death is not accompanied by expression of apoptosis-associated proteins such as Bax and waf(p21). Based on these findings, we conclude that the antiproliferative effects of these p53-derived peptides are not completely dependent on p53 activity and may prove useful as general anticancer agents.

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  • The disintegration of a molecule: The role of gelsolin in FAF, familial amyloidosis (Finnish type) 査読

    RC Robinson, SY Choe, LD Burtnick

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   98 ( 5 )   2117 - 2118   2001年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATL ACAD SCIENCES  

    DOI: 10.1073/pnas.051635098

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  • Structure and function of gelsolin 査読

    Burtnick L. D, Robinson R. C, Choe S

    Results Probl Cell Differ   32   201 - 11   2001年

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    掲載種別:研究論文(学術雑誌)  

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  • Phosphorylation of Acanthamoeba actophorin (ADF/cofilin) blocks interaction with actin without a change in atomic structure 査読

    L Blanchoin, RC Robinson, S Choe, TD Pollard

    JOURNAL OF MOLECULAR BIOLOGY   295 ( 2 )   203 - 211   2000年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS LTD  

    LIM-kinase activated by GST-Pak1 phosphorylates Acanthamoeba actophorin stoichiometrically and specifically on serine 1. The atomic structure of phosphorylated actophorin determined by X-ray crystallography is essentially identical with the structure of unphosphorylated actophorin. We compared biochemical properties of phosphorylated actophorin, unphosphorylated actophorin and mutants of actophorin with serine 1 replaced by aspartic acid or alanine. Phosphorylation strongly inhibits interaction of actophorin with Mg-ADP- or Mg-ATP-actin monomers and Mg-ADP-actin filaments, so Ser1 phosphorylation directly blocks interaction of actin-depolymerizing factor (ADF)/cofilin proteins with actin. About 30% of actophorin is phosphorylated in live amoebas grown in suspension culture. Phosphorylation of ADF/cofilin proteins by LIM-kinase or other enzymes will tend to stabilize actin filaments by inhibiting the ability of these proteins to sever and depolymerize older actin filaments that have hydrolyzed their bound ATP and dissociated the phosphate. (C) 2000 Academic Press.

    DOI: 10.1006/jmbi.1999.3336

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  • Domain movement in gelsolin: A calcium-activated switch 査読

    RC Robinson, M Mejillano, VP Le, LD Burtnick, HL Yin, S Choe

    SCIENCE   286 ( 5446 )   1939 - 1942   1999年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER ASSOC ADVANCEMENT SCIENCE  

    The actin-binding protein gelsolin is involved in remodeling the actin cytoskeleton during growth-factor signaling, apoptosis, cytokinesis, and cell movement. Calcium-activated gelsolin severs and caps actin filaments, The 3.4 angstrom x-ray structure of the carboxyl-terminal half of gelsolin (G4-G6) in complex with actin reveals the basis for gelsolin activation. Calcium binding induces a conformational rearrangement in which domain G6 is flipped over and translated by about 40 angstroms relative to G4 and G5. The structural reorganization tears apart the continuous beta sheet core of G4 and G6. This exposes the actin-binding site on G4, enabling severing and capping of actin filaments to proceed.

    DOI: 10.1126/science.286.5446.1939

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  • The structures of the neurotrophin 4 homodimer and the brain-derived neurotrophic factor/neurotrophin 4 heterodimer reveal a common Trk-binding site 査読

    RC Robinson, C Radziejewski, G Spraggon, J Greenwald, MR Kostura, LD Burtnick, DI Stuart, S Choe, EY Jones

    PROTEIN SCIENCE   8 ( 12 )   2589 - 2597   1999年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:CAMBRIDGE UNIV PRESS  

    The neurotrophins are growth factors that are involved in the development and survival of neurons. Neurotrophin release by a target tissue results in neuron growth along the neurotrophin concentration gradient, culminating in the eventual innervation of the target tissue. These activities are mediated through trk cell surface receptors. We have determined the structures of the heterodimer formed between brain-derived neurotrophic factor (BDNF) and neurotrophin 4 (NT4), as well as the structure of homodimer of NT4. We also present the structure of the Neurotrophin 3 homodimer, which is refined to higher resolution than previously published. These structures provide the first views of the architecture of the NT4 protomer. Comparison of the surface of a model of the BDNF homodimer with the structures of the neurotrophin homodimers reveals common features that may be important in the binding between the neurotrophins and their receptors. In particular, there exists an analogous region on the surface of each neurotrophin that is likely to be involved in trk receptor binding. Variations in sequence on the periphery of this common region serve to confer trk receptor specificity.

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  • An ingenious filter: The structural basis for ion channel selectivity 査読

    S Choe, R Robinson

    NEURON   20 ( 5 )   821 - 823   1998年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:CELL PRESS  

    DOI: 10.1016/s0896-6273(00)80462-6

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  • Crystal structure of the N-terminal domain of sialoadhesin in complex with 3 ' sialyllactose at 1.85 angstrom resolution 査読

    AP May, RC Robinson, M Vinson, PR Crocker, EY Jones

    MOLECULAR CELL   1 ( 5 )   719 - 728   1998年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:CELL PRESS  

    The structure of the functional N-terminal domain from the extracellular region of the cell surface receptor sialoadhesin has been determined in complex with the oligosaccharide 3' sialyllactose. This provides structural information for the siglec family of proteins. The structure conforms to the V-set immunoglobulin-like fold but contains several distinctive features, including an intra-beta sheet disulphide and a splitting of the standard beta strand G into two shorter strands. These novel features appear important in adapting the V-set fold for sialic acid-mediated recognition. Analysis of the complex with 3' sialyllactose highlights three residues, conserved throughout the siglec family, as key features of the sialic acid-binding template. The complex is representative of the functional recognition interaction with carbohydrate and as such provides detailed information for a heterotypic cell adhesion interaction.

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  • The crystal structure of plasma gelsolin: Implications for actin severing, capping, and nucleation 査読

    LD Burtnick, EK Koepf, J Grimes, EY Jones, DI Stuart, PJ McLaughlin, RC Robinson

    CELL   90 ( 4 )   661 - 670   1997年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:CELL PRESS  

    The structure of gelsolin has been determined by crystallography and comprises six structurally related domains that, in a Ca2+-free environment, pack together to form a compact globular structure in which the putative actin-binding sequences are not sufficiently exposed to enable binding to occur. We propose that binding Ca2+ can release the connections that join the N- and C-terminal halves of gelsolin, enabling each half to bind actin relatively independently. Domain shifts are proposed in response to Ca2+ as bases for models of how gelsolin acts to sever, cap, or nucleate F-actin filaments. The structure also invites discussion of polyphosphoinositide binding to segment 2 and suggests how mutation at Asp-187 could initiate a series of events that lead to deposition of amyloid plaques, as observed in victims of familial amyloidosis (Finnish type).

    DOI: 10.1016/s0092-8674(00)80527-9

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  • Expression, crystallization, and preliminary X-ray analysis of a sialic acid-binding fragment of sialoadhesin in the presence and absence of ligand 査読

    AP May, RC Robinson, RT Aplin, P Bradfield, PR Crocker, EY Jones

    PROTEIN SCIENCE   6 ( 3 )   717 - 721   1997年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:CAMBRIDGE UNIV PRESS  

    Sialoadhesin is a macrophage-restricted cell surface receptor, consisting of 17 immunoglobulin domains, which mediates cell adhesion via the recognition of specific sialylated glycoconjugates. A functional fragment of sialoadhesin, comprising the N-terminal immunoglobulin domain, has been expressed in Chinese hamster ovary cells as both native (SnD1) and selenomethionyl (Se-SnD1) stop protein. The successful production of 86% selenomethionine-incorporated protein represents a rare example of production of selenium-labeled protein in mammalian cells. SnD1 and Se-SnD1 have been crystallized in the absence of ligand, and SnD1 has also been crystallized in the presence of its ligand 2,3 sialyllactose. The ligand-free crystals of SnD1 and Se-SnD1 were isomorphous, of space group P3(1)21 or P3(2)21, with unit cell dimensions a = b = 38.9 Angstrom, c = 152.6 Angstrom, alpha = beta = 90 degrees, gamma = 120 degrees, and diffracted to a maximum resolution of 2.6 Angstrom. Cocrystals containing 2,3 sialyllactose diffracted to 1.85 Angstrom at a synchrotron source and belong to space group P2(1)2(1)2(1), with unit cell dimensions a = 40.9 Angstrom, b = 97.6 Angstrom, c = 101.6 Angstrom, alpha = beta = gamma = 90 degrees.

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  • Crystals of the neurotrophins 査読

    RC Robinson, C Radziejewski, DI Stuart, EY Jones

    PROTEIN SCIENCE   5 ( 5 )   973 - 977   1996年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:CAMBRIDGE UNIV PRESS  

    The neurotrophins show a high degree of amino acid sequence homology, share similar solution properties, and display distinct but parallel functionalities. Here we report the crystallization and preliminary X-ray characterization of three neurotrophins: brain-derived neurotrophin, neurotrophin 3, and the heterodimer between brain-derived neurotrophin and neurotrophin 4. These findings are related to other published crystal parameters for neurotrophins, leading to the observation that, although crystal packing is highly variant, neurotrophins share common solubilities with respect to crystal growth.

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  • The structure of horse plasma gelsolin to 2.5 angstrom. 査読

    LD Burtnick, RC Robinson, EK Koepf

    BIOPHYSICAL JOURNAL   70 ( 2 )   SUA12 - SUA12   1996年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:CELL PRESS  

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  • STRUCTURE OF THE BRAIN-DERIVED NEUROTROPHIC FACTOR NEUROTROPHIN 3 HETERODIMER 査読

    RC ROBINSON, C RADZIEJEWSKI, DI STUART, EY JONES

    BIOCHEMISTRY   34 ( 13 )   4139 - 4146   1995年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER CHEMICAL SOC  

    The development and sustenance of specific neuronal populations in the peripheral and central nervous systems are controlled through the binding of neurotrophic factors to high-affinity cell surface receptors. The neurotrophins (nerve growth factor, NGF; brain-derived neurotrophic factor, BDNF; neurotrophin 3, NT3; and neurotrophin 4, NT4) are dimeric molecules which share approximately 50% sequence identity. The crystal structure of the murine NGF homodimer [McDonald et al. (1991) Nature 354, 411-414] indicated that the dimer interface corresponds to regions of high sequence conservation throughout the neurotrophin family. This potential compatibility was duly exploited for the production in vitro of noncovalent heterodimers between the different neurotrophins [Radziejewski, C., and Robinson, R. C. (1993) Biochemistry 32, 13350-13356; Jungbluth et al. (1994) fur. J. Biochem. 221, 677-685]. Here, we report the X-ray structure at 2.3 Angstrom resolution of one such heterodimer, between human BDNF, and human NT3. The NGF, BDNF, and NT3 protomers share the same topology and are structurally equivalent in regions which contribute to the dimer interface in line with the propensity of the neurotrophins to form heterodimers. Analysis of the structure of regions of the BDNF/NT3 heterodimer involved in receptor specificity led us to conclude that heterodimer binding to p75 involves distant binding sites separately located on each protomer of the heterodimer. In contrast, heterodimer interactions with the trk receptors probably utilize hybrid binding sites comprised of residues contributed by both protomers in the heterodimer. The existence of such hybrid binding sites for the trk receptor provides an explanation for the lower activity of the BDNF/NT3 heterodimer in comparison to the homodimers. Finally, we discuss possible functional roles for the heterodimers in vivo.

    DOI: 10.1021/bi00013a001

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  • CRYSTAL-STRUCTURE OF AN INTEGRIN-BINDING FRAGMENT OF VASCULAR CELL-ADHESION MOLECULE-1 AT 1.8 ANGSTROM RESOLUTION 査読

    EY JONES, K HARLOS, MJ BOTTOMLEY, RC ROBINSON, PC DRISCOLL, RM EDWARDS, JM CLEMENTS, TJ DUDGEON, DI STUART

    NATURE   373 ( 6514 )   539 - 544   1995年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:MACMILLAN MAGAZINES LTD  

    THE cell-surface glycoprotein vascular cell adhesion molecule-1 (VCAM-1; ref. 1) mediates intercellular adhesion(2) by specific binding to the integrin very-late antigen-4 (VLA-4, alpha(4) beta(1); ref. 3). VCAM-1, with the intercellular adhesion molecules ICAM-1, ICAM-2, ICAM-3 and the mucosal vascular addressin MAd-CAM-1, forms an integrin-binding subgroup of the immunoglobulin superfamily. In addition to their clinical relevance in inflammation, these molecules act as cellular receptors for viral and parasitic agents(2). The predominant form of VCAM-1 in vivo has an amino-terminal extracellular region comprising seven immunoglobulin-like domains. Functional studies(4-7) have identified a conserved integrin-binding motif in domains 1 and 4, variants of which are present in the N-terminal domain of all members of the immunoglobulin superfamily subgroup. We report here the crystal structure of a VLA-4-binding fragment composed of the first two domains of VCAM-1. The integrin-binding motif (Q38IDSPL) is highly exposed and forms the N-terminal region of the loop between beta-strands C and D of domain 1. This motif exhibits a distinctive conformation which we predict will be common to all the integrin-binding IgSF molecules. These, and additional data, map VLA-4 binding to the face of the CFG beta-sheet, the surface previously identified(8) as the site for intercellular adhesive interactions between members of the immunoglobulin superfamily.

    DOI: 10.1038/373539a0

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  • The crystal structure of murine leukemia inhibitory factor 査読

    RC Robinson, LM Grey, D Staunton, DI Stuart, JK Heath, EY Jones

    INTERLEUKIN-6 TYPE CYTOKINES   762   179 - 188   1995年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NEW YORK ACAD SCIENCES  

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  • CRYSTALLIZATION AND PRELIMINARY-X-RAY DIFFRACTION CHARACTERIZATION OF BOTH A NATIVE AND SELENOMETHIONYL VLA-4 BINDING FRAGMENT OF VCAM-1 査読

    MJ BOTTOMLEY, RC ROBINSON, PC DRISCOLL, K HARLOS, DI STUART, RT APLIN, JM CLEMENTS, EY JONES, TJ DUDGEON

    JOURNAL OF MOLECULAR BIOLOGY   244 ( 4 )   464 - 468   1994年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS LTD  

    Soluble fragments of the extracellular region of vascular cell adhesion molecule 1 (VCAM-1) expressed in Escherichia coli retain functional adhesive activity An integrin (VLA-4) binding fragment consisting of the N-terminal two immunoglobulin-like domains (VCAM-d1,2) has been crystallized. The crystals belong to space group P2(1)2(1)2(1) with cell dimensions of 52.7 Angstrom b = 66.5 Angstrom, c = 113.2 Angstrom and contain two molecules in the crystallographic asymmetric unit. A batch of protein produced in the standard E. coli strain (HW1110), but grown in the presence of selenomethionine enriched media, showed 85% incorporation of selenium in place of sulphur at methionine residues. The selenomethionyl VCAM-d1,2 was crystallized by microseeding techniques initially using the native crystals for nucleation. Both native and selenomethionyl crystals diffract X-rays to a minimum Bragg spacing of 1.8 Angstrom.

    DOI: 10.1006/jmbi.1994.1743

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  • THE CRYSTAL-STRUCTURE AND BIOLOGICAL FUNCTION OF LEUKEMIA INHIBITORY FACTOR - IMPLICATIONS FOR RECEPTOR-BINDING 査読

    RC ROBINSON, LM GREY, D STAUNTON, H VANKELECOM, AB VERNALLIS, JF MOREAU, DI STUART, JK HEATH, EY JONES

    CELL   77 ( 7 )   1101 - 1116   1994年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:CELL PRESS  

    The structure of murine leukemia inhibitory factor (LIF) has been determined by X-ray crystallography at 2.0 Angstrom resolution. The main chain fold conforms to the four alpha-helix bundle topology previously observed for several members of the hematopoietic cytokine family. Of these, LIF shows closest structural homology to granulocyte colony-stimulating factor and growth hormone (GH). Sequence alignments for the functionally related molecules oncostatin M and ciliary neurotrophic factor, when mapped to the LIF structure, indicate regions of conserved surface character. Analysis of the biological function and receptor specificity of a series of human-mouse LIF chimeras implicate two regions of receptor interaction that are located in the fourth helix and the preceding loop. A model for receptor binding based on the structure of the GH ligand-receptor complex requires additional, novel features to account for these data.

    DOI: 10.1016/0092-8674(94)90449-9

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  • HETERODIMERS OF THE NEUROTROPHIC FACTORS - FORMATION, ISOLATION, AND DIFFERENTIAL STABILITY 査読

    C RADZIEWJEWSKI, RC ROBINSON

    BIOCHEMISTRY   32 ( 48 )   13350 - 13356   1993年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER CHEMICAL SOC  

    We have determined that all four known members of the neurotrophin family, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and neurotrophin 4 (NT-4), are capable of forming noncovalent heterodimers. The formation of these heterodimers was accomplished by homodimer subunit exchange promoted by treatment with guanidine hydrochloride, urea, low pH, or acetonitrile. In some cases (BDNF and mouse NGF; BDNF and NT-4), generation of the heterodimers was achieved by incubating homodimer mixtures in a neutral buffer at ambient temperature. The formation of heterodimers was in each case detected by nondenaturing gel electrophoresis at pH 7.4. High-performance cation-exchange chromatography was used to separate neurotrophin heterodimers from their parental homodimers. Heterodimers between BDNF and NT-3, BDNF and NT-4, and NT-3 and NT-4 are stable and show only a very small increase in homodimer content after 24 h of incubation at 37-degrees-C. In contrast, heterodimers containing NGF subunits undergo gradual rearrangement to the homodimers. Our studies indicate that low pH, acetonitrile, and urea merely increase the neurotrophin subunit exchange rate and decrease the time needed to reach an equilibrium between a heterodimer and its two parental homodimers.

    DOI: 10.1021/bi00211a049

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  • DIMERIC STRUCTURE AND CONFORMATIONAL STABILITY OF BRAIN-DERIVED NEUROTROPHIC FACTOR AND NEUROTROPHIN-3 査読

    C RADZIEJEWSKI, RC ROBINSON, PS DISTEFANO, JW TAYLOR

    BIOCHEMISTRY   31 ( 18 )   4431 - 4436   1992年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER CHEMICAL SOC  

    We have examined the molecular structure of the related neurotrophic factors brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) by physical methods, including gel filtration, velocity sedimentation, sedimentation equilibrium, urea gel electrophoresis, fluorescence spectroscopy, and far-ultraviolet circular dichroism. The results of these studies indicate that at physiologically relevant concentrations both recombinant proteins exist as tightly associated dimers. The dimers are stable even in 8 M solutions of urea. In solutions of guanidine hydrochloride, BDNF and NT-3 undergo slow unfolding between 3 and 5 M concentration of denaturant. Circular dichroism spectroscopy revealed approximately 70% beta-sheet and 20% beta-turn content in the native structure of both neurotrophic factors. In this respect, BDNF and NT-3 resemble other polypeptide growth factors whose receptors are also integral protein-tyrosine kinases.

    DOI: 10.1021/bi00133a007

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  • STABILIZATION OF THE STRUCTURE OF HORSE PLASMA VITAMIN-D BINDING-PROTEIN BY DISULFIDE BONDS 査読

    RC ROBINSON, LD BURTNICK

    BIOCHEMISTRY AND CELL BIOLOGY-BIOCHIMIE ET BIOLOGIE CELLULAIRE   70 ( 1 )   10 - 15   1992年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATL RESEARCH COUNCIL CANADA  

    Vitamin D binding protein (DBP) was isolated from horse plasma in a four-step procedure that involved Affi-Gel Blue affinity chromatography, gel filtration, hydroxylapatite chromatography, and anion exchange high-pressure liquid chromatography. The yield of DBP from 80 mL of plasma was 6-7 mg. Horse plasma DBP closely resembles other plasma DBPs, being a tryptophan-free protein of M(r) 53 000. It is able to bind to and block the polymerization of monomeric actin. The secondary structure of DBP was calculated from circular dichroism measurements to be 39% alpha-helix, 42% beta-sheet, and 19% random coil. Circular dichroism and fluorescence studies revealed that the disulfide bonds of DBP contribute substantial structural stabilization to the molecule with respect to thermal denaturation. The thermal stability of DBP can be used to advantage. Incorporation of a brief treatment at 70-degrees-C into the preparative scheme enables omission of one chromatographic step, without detectable alteration of the purified product.

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  • Actin filament severing activity exhibited by gelsolin-family proteins - a TIRF microscopy study

    B. Kannan, R. C. Robinson

    MOLECULAR BIOLOGY OF THE CELL   26   2015年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:AMER SOC CELL BIOLOGY  

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  • Single Molecule Force Spectroscopy Reveals Force-Enhanced Binding of Calcium Ions by Gelsolin

    Chunmei Lv, Xiang Gao, Wenfei Li, Robert Robinson, Meng Qin, Leslie Burtnick, Hao Zhou, Yi Cao, Wei Wang

    BIOPHYSICAL JOURNAL   106 ( 2 )   678A - 678A   2014年1月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:CELL PRESS  

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  • Developing polyketide-based anti-microbial therapeutics using synthetic enzymology

    Vivian Wing Ngar Cheung, Maybelle Darlene Kho Go, Robert Robinson, Jantana Wongsantichon, Wen Shan Yew

    FASEB JOURNAL   26   2012年4月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:FEDERATION AMER SOC EXP BIOL  

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  • Combinatorial biosynthesis of unnatural polyketides using a type III polyketide synthase from Oryza sativa

    Jeng Yeong Chow, Jantana Wongsantichon, Robert Robinson, Yunn Hwen Gan, Wen Shan Yew

    FASEB JOURNAL   26   2012年4月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:FEDERATION AMER SOC EXP BIOL  

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  • Conformational dynamics of capping protein and interaction partners: Simulation studies (vol 80, pg 1066, 2012)

    Suryani Lukman, Robert C. Robinson, David Wales, Chandra S. Verma

    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS   80 ( 5 )   1500 - 1500   2012年3月

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    記述言語:英語   出版者・発行元:WILEY-BLACKWELL  

    DOI: 10.1002/prot.24064

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  • Actin Polymerization Dynamics - Insights from In vitro TIRF Microscopy

    Balakrishnan Kannan, Marten Larsson, Wei Lin Lee, Maria Hernandez-Valladares, Robert C. Robinson

    BIOPHYSICAL JOURNAL   100 ( 3 )   300 - 301   2011年2月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:CELL PRESS  

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  • Structure and filament dynamics of the pSK41 actin-like ParM protein. IMPLICATIONS FOR PLASMID DNA SEGREGATION (vol 285, pg 10130, 2010)

    David Popp, Weijun Xu, Akihiro Narita, Anthony J. Brzoska, Ronald A. Skurray, Neville Firth, Umesh Ghoshdastider, Yuichiro Maeda, Robert C. Robinson, Maria A. Schumacher

    JOURNAL OF BIOLOGICAL CHEMISTRY   285 ( 23 )   18122 - 18122   2010年6月

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    記述言語:英語   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

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  • Single-Molecule Study of Actin Filament Severing by Gelsolin using Total Internal Reflection Fluorescence Microscopy

    Balakrishnan Kannan, Wei Lin Lee, Robert C. Robinson

    BIOPHYSICAL JOURNAL   98 ( 3 )   155A - 155A   2010年1月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:CELL PRESS  

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  • How the active apoptotic fragment of gelsolin binds to actin

    D Urosev, E Irobi, K Narayan, RC Robinson, LD Burtnick

    BIOPHYSICAL JOURNAL   86 ( 1 )   68A - 68A   2004年1月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:BIOPHYSICAL SOCIETY  

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  • Calcium-activation of gelsolin.

    D Urosev, R Robinson, H Choe, H Yin, S Choe, LD Burtnick

    BIOPHYSICAL JOURNAL   82 ( 1 )   382A - 382A   2002年1月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:BIOPHYSICAL SOCIETY  

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  • Atomic structure of bovine Arp2/3 complex

    K Turbedsky, R Robinson, DA Kaiser, HN Higgs, JB Marchand, S Choe, TD Pollard

    MOLECULAR BIOLOGY OF THE CELL   12   396A - 396A   2001年11月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:AMER SOC CELL BIOLOGY  

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  • Crystal structure of a G-actin/half-gelsolin complex.

    LD Burtnick, RC Robinson, M Mejillano, H Yin, S Choe

    BIOPHYSICAL JOURNAL   78 ( 1 )   18A - 18A   2000年1月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:BIOPHYSICAL SOCIETY  

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  • Acanthamoeba actophorin (ADF/cofilin) depolymerizes actin filaments capped with Arp2/3 and gelsolin as well as only barbed ends capped filaments. Effect of phosphorylation on actophorin interaction with actin.

    L Blanchoin, RD Mullins, RC Robinson, S Choe, TD Pollard

    MOLECULAR BIOLOGY OF THE CELL   10   24A - 24A   1999年11月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:AMER SOC CELL BIOLOGY  

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  • THE STRUCTURE OF THE AMINO-TERMINAL IG-LIKE SIALIC ACID BINDING DOMAIN OF SIALOADHESIN.

    A. May, R. C. Robinson, P. Bradfield, M. Vinson, P. R. Crocker, E. Y. Jones

    ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES   52   C193 - C193   1996年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:INT UNION CRYSTALLOGRAPHY  

    DOI: 10.1107/S0108767396091611

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