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Now showing items 1 - 16 of 32

  • Analytical ultracentrifugation in structural biology

    Unzai, Satoru  

    Researchers in the field of structural biology, especially X-ray crystallography and protein nuclear magnetic resonance, are interested in knowing as much as possible about the state of their target protein in solution. Not only is this knowledge relevant to studies of biological function, it also facilitates determination of a protein structure using homogeneous monodisperse protein samples. A researcher faced with a new protein to study will have many questions even after that protein has been purified. Analytical ultracentrifugation (AUC) can provide all of this information readily from a small sample in a non-destructive way, without the need for labeling, enabling structure determination experiments without any wasting time and material on uncharacterized samples. In this article, I use examples to illustrate how AUC can contribute to protein structural analysis. Integrating information from a variety of biophysical experimental methods, such as X-ray crystallography, small angle X-ray scattering, electrospray ionization-mass spectrometry, AUC allows a more complete understanding of the structure and function of biomacromolecules.=20
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  • Computational design of a self-assembling symmetrical beta-propeller protein.

    Voet, Arnout R D   Noguchi, Hiroki   Addy, Christine   Simoncini, David   Terada, Daiki   Unzai, Satoru   Park, Sam-Yong   Zhang, Kam Y J   Tame, Jeremy R H  

    The modular structure of many protein families, such as beta-propeller proteins, strongly implies that duplication played an important role in their evolution, leading to highly symmetrical intermediate forms. Previous attempts to create perfectly symmetrical propeller proteins have failed, however. We have therefore developed a new and rapid computational approach to design such proteins. As a test case, we have created a sixfold symmetrical beta-propeller protein and experimentally validated the structure using X-ray crystallography. Each blade consists of 42 residues. Proteins carrying 2-10 identical blades were also expressed and purified. Two or three tandem blades assemble to recreate the highly stable sixfold symmetrical architecture, consistent with the duplication and fusion theory. The other proteins produce different monodisperse complexes, up to 42 blades (180 kDa) in size, which self-assemble according to simple symmetry rules. Our procedure is suitable for creating nano-building blocks from different protein templates of desired symmetry. =20
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  • Insight into structural diversity of influenza virus haemagglutinin

    Cho, Ki Joon   Lee, Ji-Hye   Hong, Kwang W.   Kim, Se-Ho   Park, Yiho   Lee, Jun Young   Kang, Seokha   Kim, Sella   Yang, Ji Hoon   Kim, Eui-Ki   Seok, Jong Hyeon   Unzai, Satoru   Park, Sam Yong   Saelens, Xavier   Kim, Chul-Joong   Lee, Joo-Yeon   Kang, Chun   Oh, Hee-Bok   Chung, Mi Sook   Kim, Kyung Hyun  

    Influenza virus infects host cells through membrane fusion, a process mediated by the low pH-induced conformational change of the viral surface glycoprotein haemagglutinin (HA). We determined the structures and biochemical properties of the HA proteins from A/Korea/01/2009 (KR01), a 2009 pandemic strain, and A/Thailand/CU44/2006 (CU44), a seasonal strain. The crystal structure of KR01 HA revealed a V-shaped head-to-head arrangement, which is not seen in other HA proteins including CU44 HA. We isolated a broadly neutralizing H1-specific monoclonal antibody GC0757. The KR01 HA-Fab0757 complex structure also exhibited a head-to-head arrangement of HA. Both native and Fab complex structures reveal a different spatial orientation of HA1 relative to HA2, indicating that HA is flexible and dynamic at neutral pH. Further, the KR01 HA exhibited significantly lower protein stability and increased susceptibility to proteolytic cleavage compared with other HAs. Our structures provide important insights into the conformational flexibility of HA.
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  • Role of domains within the autotransporter Hbp/Tsh

    Nishimura, Kaoru   Yoon, Young-Ho   Kurihara, Atsushi   Unzai, Satoru   Luirink, Joen   Park, Sam-Yong   Tame, Jeremy R. H.  

    The autotransporter Tsh (temperature-sensitive haemagglutinin) secreted by avian pathogenic Escherichia coli was reported in 1994 and the almost identical Hbp (haemoglobin protease) was discovered some years later in isolates from patients suffering from peritoneal abscesses. However, the function of the protein remains uncertain. The crystal structure of Hbp shows that the protein carries a serine protease domain (domain 1) and a small domain of 75 residues called domain 2 which is inserted into the long beta-helix characteristic of autotransporter passenger proteins. In this paper, domain 1 is shown to bind calcium, although metal ions binding to this site do not seem to regulate protease activity. Tsh has been reported to bind red cells and components of the extracellular matrix, but it is demonstrated that these properties are not a consequence of the presence of domain 2.
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  • Structural basis for the dual RNA-recognition modes of human Tra2-beta RRM

    Tsuda, Kengo   Someya, Tatsuhiko   Kuwasako, Kanako   Takahashi, Mari   He, Fahu   Unzai, Satoru   Inoue, Makoto   Harada, Takushi   Watanabe, Satoru   Terada, Takaho   Kobayashi, Naohiro   Shirouzu, Mikako   Kigawa, Takanori   Tanaka, Akiko   Sugano, Sumio   Guentert, Peter   Yokoyama, Shigeyuki   Muto, Yutaka  

    Human Transformer2-beta (hTra2-beta) is an important member of the serine/arginine-rich protein family, and contains one RNA recognition motif (RRM). It controls the alternative splicing of several pre-mRNAs, including those of the calcitonin/calcitonin gene-related peptide (CGRP), the survival motor neuron 1 (SMN1) protein and the tau protein. Accordingly, the RRM of hTra2-beta specifically binds to two types of RNA sequences [the CAA and (GAA)(2) sequences]. We determined the solution structure of the hTra2-beta RRM (spanning residues Asn110-Thr201), which not only has a canonical RRM fold, but also an unusual alignment of the aromatic amino acids on the beta-sheet surface. We then solved the complex structure of the hTra2-beta RRM with the (GAA)(2) sequence, and found that the AGAA tetra-nucleotide was specifically recognized through hydrogen-bond formation with several amino acids on the N- and C-terminal extensions, as well as stacking interactions mediated by the unusually aligned aromatic rings on the beta-sheet surface. Further NMR experiments revealed that the hTra2-beta RRM recognizes the CAA sequence when it is integrated in the stem-loop structure. This study indicates that the hTra2-beta RRM recognizes two types of RNA sequences in different RNA binding modes.
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  • RBFOX and SUP-12 sandwich a G base to cooperatively regulate tissue-specific splicing.

    Kuwasako, Kanako   Takahashi, Mari   Unzai, Satoru   Tsuda, Kengo   Yoshikawa, Seiko   He, Fahu   Kobayashi, Naohiro   Guntert, Peter   Shirouzu, Mikako   Ito, Takuhiro   Tanaka, Akiko   Yokoyama, Shigeyuki   Hagiwara, Masatoshi   Kuroyanagi, Hidehito   Muto, Yutaka  

    Tissue-specific alternative pre-mRNA splicing is often cooperatively regulated by multiple splicing factors, but the structural basis of cooperative RNA recognition is poorly understood. In Caenorhabditis elegans, ligand binding specificity of fibroblast growth factor receptors (FGFRs) is determined by mutually exclusive alternative splicing of the sole FGFR gene, egl-15. Here we determined the solution structure of a ternary complex of the RNA-recognition motif (RRM) domains from the RBFOX protein ASD-1, SUP-12 and their target RNA from egl-15. The two RRM domains cooperatively interact with the RNA by sandwiching a G base to form the stable complex. Multichromatic fluorescence splicing reporters confirmed the requirement of the G and the juxtaposition of the respective cis elements for effective splicing regulation in vivo. Moreover, we identified a new target for the heterologous complex through an element search, confirming the functional significance of the intermolecular coordination. =20
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  • The nature of the TRAP-Anti-TRAP complex

    Watanabe, Masahiro   Heddle, Jonathan G.   Kikuchi, Kenichi   Unzai, Satoru   Akashi, Satoko   Park, Sam-Yong  

    Tryptophan biosynthesis is subject to exquisite control in species of Bacillus and has become one of the best-studied model systems in gene regulation. The protein TRAP (trp RNA-binding attenuation protein) predominantly forms a ring-shaped 11-mer, which binds cognate RNA in the presence of tryptophan to suppress expression of the trp operon. TRAP is itself regulated by the protein Anti-TRAP, which binds to TRAP and prevents RNA binding. To date, the nature of this interaction has proved elusive. Here, we describe mass spectrometry and analytical centrifugation studies of the complex, and 2 crystal structures of the TRAP-Anti-TRAP complex. These crystal structures, both refined to 3.2-angstrom resolution, show that Anti-TRAP binds to TRAP as a trimer, sterically blocking RNA binding. Mass spectrometry shows that 11-mer TRAP may bind up to 5 AT trimers, and an artificial 12-mer TRAP may bind 6. Both forms of TRAP make the same interactions with Anti-TRAP. Crystallization of wild-type TRAP with Anti-TRAP selectively pulls the 12-mer TRAP form out of solution, so the crystal structure of wild-type TRAP-Anti-TRAP complex reflects a minor species from a mixed population.
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  • AU-rich RNA-binding induces changes in the quaternary structure of AUH

    Kurimoto, Kazuki   Kuwasako, Kanako   Sandercock, Alan M.   Unzai, Satoru   Robinson, Carol V.   Muto, Yutaka   Yokoyama, Shigeyuki  

    The human AU RNA binding protein/enoyl-Coenzyme A hydratase (AUH) is a 3-hydroxy-3-methylglutaconyl-CoA dehydratase in the leucine degradation pathway. It also possesses an RNA-binding activity to AUUU repeats, which involves no known conserved RNA-binding domains and is seemingly unrelated to the enzymatic activity. In this study, we performed mass spectrometric analyses to elucidate the oligomeric states of AUH in the presence and absence of RNA. With a short RNA (AUUU) or without RNA, AUH mainly exists as a trimer in solution. On the other hand, the AUH trimer dimerizes upon binding to one molecule of a long RNA containing 24 repeats of the AUUU motif, (AUUU)(24)A. AUH was crystallized with the long RNA. Although the RNA was disordered in the crystalline lattice, the AUH structure was determined as an asymmetric dimer of trimers with a kink in the alignment of the trimer axes, resulting in the formation of two clefts with significantly different sizes.
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  • Crystal structure of zinc-finger domain of Nanos and its functional implications

    Hara, Kodai   Hishiki, Asami   Kawaguchi, Shigeta   Shichijo, Naoki   Nakamura, Keishi   Unzai, Satoru   Tamaru, Yutaka   Shimizu, Toshiyuki   Sato, Mamoru  

    Nanos is an RNA-binding protein that is involved in the development and maintenance of germ cells. In combination with Pumilio, Nanos binds to the 3' untranslated region of a messenger RNA and represses its translation. Nanos has two conserved Cys-Cys-His-Cys zinc-finger motifs that are indispensable for its function. In this study, we have determined the crystal structure of the zinc-finger domain of zebrafish Nanos, for the first time revealing that Nanos adopts a novel zinc-finger structure. In addition, Nanos has a conserved basic surface that is directly involved in RNA binding. Our results provide the structural basis for further studies to clarify Nanos function.
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  • Crystal structures of penicillin-binding proteins 4 and 5 from Haemophilus influenzae.

    Kawai, Fumihiro   Clarke, Thomas B   Roper, David I   Han, Gab-Jo   Hwang, Kwang Yeon   Unzai, Satoru   Obayashi, Eiji   Park, Sam-Yong   Tame, Jeremy R H  

    We have determined high-resolution apo crystal structures of two low molecular weight penicillin-binding proteins (PBPs), PBP4 and PBP5, from Haemophilus influenzae, one of the most frequently found pathogens in the upper respiratory tract of children. Novel beta-lactams with notable antimicrobial activity have been designed, and crystal structures of PBP4 complexed with ampicillin and two of the novel molecules have also been determined. Comparing the apo form with those of the complexes, we find that the drugs disturb the PBP4 structure and weaken X-ray diffraction, to very different extents. PBP4 has recently been shown to act as a sensor of the presence of penicillins in Pseudomonas aeruginosa, and our models offer a clue to the structural basis for this effect. Covalently attached penicillins press against a phenylalanine residue near the active site and disturb the deacylation step. The ready inhibition of PBP4 by beta-lactams compared to PBP5 also appears to be related to the weaker interactions holding key residues in a catalytically competent position. Copyright (c) 2009. Elsevier Ltd. All rights reserved.
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  • A novel 3' splice site recognition by the two zinc fingers in the U2AF small subunit.

    Yoshida, Hisashi   Park, Sam-Yong   Oda, Takashi   Akiyoshi, Taeko   Sato, Mamoru   Shirouzu, Mikako   Tsuda, Kengo   Kuwasako, Kanako   Unzai, Satoru   Muto, Yutaka   Urano, Takeshi   Obayashi, Eiji  

    The pre-mRNA splicing reaction of eukaryotic cells has to be carried out extremely accurately, as failure to recognize the splice sites correctly causes serious disease. The small subunit of the U2AF heterodimer is essential for the determination of 3' splice sites in pre-mRNA splicing, and several single-residue mutations of the U2AF small subunit cause severe disorders such as myelodysplastic syndromes. However, the mechanism of RNA recognition is poorly understood. Here we solved the crystal structure of the U2AF small subunit (U2AF23) from fission yeast, consisting of an RNA recognition motif (RRM) domain flanked by two conserved CCCH-type zinc fingers (ZFs). The two ZFs are positioned side by side on the beta sheet of the RRM domain. Further mutational analysis revealed that the ZFs bind cooperatively to the target RNA sequence, but the RRM domain acts simply as a scaffold to organize the ZFs and does not itself contact the RNA directly. This completely novel and unexpected mode of RNA-binding mechanism by the U2AF small subunit sheds light on splicing errors caused by mutations of this highly conserved protein. =C2=A9 2015 Yoshida et al.; Published by Cold Spring Harbor Laboratory Press.
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  • Structural insights into a 20.8-kDa tegumental-allergen-like (TAL) protein from Clonorchis sinensis

    Jo, Chang Hwa   Son, Jonghyeon   Kim, Sulhee   Oda, Takashi   Kim, Jaehoon   Lee, Myoung-Ro   Sato, Mamoru   Kim, Hyun Tae   Unzai, Satoru   Park, Sam-Yong   Hwang, Kwang Yeon  

    Survival of Clonorchis sinensis, a cause of human clonorchiasis, requires tegument proteins, which are localized to the tegumental outer surface membrane. These proteins play an important role in a host response and parasite survival. Thus, these proteins are interesting molecular targets for vaccine and drug development. Here, we have determined two crystal structures of the calmodulin like domain (amino acid [aa] positions 1-81) and dynein light chain (DLC)-like domain (aa 83-177) of a 20.8-kDa tegumental-allergen-like protein from Clonorchis sinensis (CsTAL3). The calmodulin like domain has two Ca2+-binding sites (named CB1 and CB2), but Ca2+ binds to only one site, CB1. The DLC-like domain has a dimeric conformation; the interface is formed mainly by hydrogen bonds between the main chain atoms. In addition, we have determined full-length structure of CsTAL3 in solution and showed the conformational change of CsTAL3 induced by Ca2+ ion binding using small-angle X-ray scattering analysis and molecular dynamics simulations. The Ca2+-bound form has a more extended conformation than the Ca2+-free from does. These structural and biochemical analyses will advance the understanding of the biology of this liver fluke and may contribute to our understanding of the molecular mechanism of calcium-responsive and tegumental-allergen-like proteins.
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  • Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium

    Ohki, Mio   Sugiyama, Kanako   Kawai, Fumihiro   Tanaka, Hitomi   Nihei, Yuuki   Unzai, Satoru   Takebe, Masumi   Matsunaga, Shigeru   Adachi, Shin-ichi   Shibayama, Naoya   Zhou, Zhiwen   Koyama, Ryuta   Ikegaya, Yuji   Takahashi, Tetsuo   Tame, Jeremy R. H.  

    Cyclic-AMP is one of the most important second messengers, regulating many crucial cellular events in both prokaryotes and eukaryotes, and precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a small homodimer eminently suitable for this task, requiring only a simple flavin chromophore within a blue light using flavin (BLUF) domain. These domains, one of the most studied types of biological photoreceptor, respond to blue light and either regulate the activity of an attached enzyme domain or change its affinity for a repressor protein. BLUF domains were discovered through studies of photo-induced movements of Euglena gracilis, a unicellular flagellate, and gene expression in the purple bacterium Rhodobacter sphaeroides, but the precise details of light activation remain unknown. Here, we describe crystal structures and the light regulation mechanism of the previously undescribed OaPAC, showing a central coiled coil transmits changes from the light-sensing domains to the active sites with minimal structural rearrangement. Site-directed mutants show residues essential for signal transduction over 45 angstrom across the protein. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase, showing a rapid and stable response to light over many hours and activation cycles. The structures determined in this study will assist future efforts to create artificial light-regulated control modules as part of a general optogenetic toolkit.
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  • Expression, purification and crystallization of Swi5 and the Swi5-Sfr1 complex from fission yeast

    Kuwabara, Naoyuki   Hashimoto, Hiroshi   Yamada, Noriyo   Unzai, Satoru   Ikeguchi, Mitsunori   Sato, Mamoru   Murayama, Yasuto   Iwasaki, Hiroshi  

    The assembly of the presynaptic filament of recombinases represents the most important step in homologous recombination. The formation of the filament requires assistance from mediator proteins. Swi5 and Sfr1 have been identified as mediators in fission yeast and these proteins form a complex that stimulates strand exchange. Here, the expression, purification and crystallization of Swi5 and its complex with an N-terminally truncated form of Sfr1 (delta N180Sfr1) are presented. Analytical ultracentrifugation of the purified samples showed that Swi5 and the protein complex exist as tetramers and heterodimers in solution, respectively. Swi5 was crystallized in two forms belonging to space groups C2 and R3 and the crystals diffracted to 2.7 A resolution. Swi5-delta N180Sfr1 was crystallized in space group P2(1)2(1)2 and the crystals diffracted to 2.3 A resolution. The crystals of Swi5 and Swi5-delta N180Sfr1 are likely to contain one tetramer and two heterodimers in the asymmetric unit, respectively.
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  • Oligomerization of Hmo1 mediated by box A is essential for DNA binding in vitro and in vivo

    Kasahara, Koji   Higashino, Ayako   Unzai, Satoru   Yoshikawa, Hirofumi   Kokubo, Tetsuro  

    Hmo1, a member of HMGB family proteins in Saccharomyces cerevisiae, binds to and regulates the transcription of genes encoding ribosomal RNA and ribosomal proteins. The functional motifs of Hmo1 include two HMG-like motifs, box A and box B, and a C-terminal tail. To elucidate the molecular roles of the HMG-like boxes in DNA binding in vivo, we analyzed the DNA-binding activity of various Hmo1 mutants using ChIP or reporter assays that enabled us to conveniently detect Hmo1 binding to the promoter of RPS5, a major target gene of Hmo1. Our mutational analyses showed that box B is a bona fide DNA-binding motif and that it also plays other important roles in cell growth. However, box A, especially its first alpha-helix, contributes to DNA binding of Hmo1 by inducing self-assembly of Hmo1. Intriguingly, box A mediated formation of oligomers of more than two proteins on DNA in vivo. Furthermore, duplication of the box B partially alleviates the requirement for box A. These findings suggest that the principal role of box A is to assemble multiple box B in the appropriate orientation, thereby stabilizing the binding of Hmo1 to DNA and nucleating specific chromosomal architecture on its target genes.
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  • RNA and protein complexes of trp RNA-binding attenuation protein characterized by mass spectrometry.

    Akashi, Satoko   Watanabe, Masahiro   Heddle, Jonathan G   Unzai, Satoru   Park, Sam-Yong   Tame, Jeremy R H  

    We have characterized both wild-type and mutant TRAP (trp RNA-binding attenuation protein) from Bacillus stearothermophilus , and their complexes with RNA or its regulator anti-TRAP protein (AT), by electrospray ionization mass spectrometry (ESI-MS). Wild-type TRAP mainly forms homo-11mer rings. The mutant used carries three copies of the TRAP monomer on a single polypeptide chain so that it associates to form a 12mer ring with four polypeptide molecules. Mass spectra showed that both the wild-type TRAP 11mer and the mutant TRAP 12mer can bind a cognate single-stranded RNA molecule with a molar ratio of 1:1. The crystal structure of wild-type TRAP complexed with AT shows a TRAP 12mer ring surrounded by six AT trimers. However, nanoESI-MS of wild-type TRAP mixed with AT shows four species with different binding stoichiometries, and the complex observed by crystallography represents only a minor species in solution; most of the TRAP remains in an 11mer ring form. Mass spectra of mutant TRAP showed only a single species, TRAP 12mer + six copies of AT trimer, which is observed by crystallography. These results suggest that crystallization selects only the most symmetrical TRAP-AT complex from the solution, whereas ESI-MS can take a "snapshot" of all the species in solution.
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