98 results on '"Satoru Unzai"'
Search Results
2. A systematic mutational analysis identifies a 5‐residue proline tag that enhances the in vivo immunogenicity of a non‐immunogenic model protein
- Author
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Nafsoon Rahman, Mohammad Monirul Islam, Md. Golam Kibria, Satoru Unzai, and Yutaka Kuroda
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immunogenicity ,monomer ,peptide tag ,protein solubility ,SCP ,Biology (General) ,QH301-705.5 - Abstract
Poor immunogenicity of small proteins is a major hurdle in developing vaccines or producing antibodies for biopharmaceutical usage. Here, we systematically analyzed the effects of 10 solubility controlling peptide tags (SCP‐tags) on the immunogenicity of a non‐immunogenic model protein, bovine pancreatic trypsin inhibitor (BPTI‐19A; 6 kDa). CD, fluorescence, DLS, SLS, and AUC measurements indicated that the SCP‐tags did not change the secondary structure content nor the tertiary structures of the protein nor its monomeric state. ELISA results indicated that the 5‐proline (C5P) and 5‐arginine (C5R) tags unexpectedly increased the IgG level of BPTI‐19A by 240‐ and 73‐fold, respectively, suggesting that non‐oligomerizing SCP‐tags may provide a novel method for increasing the immunogenicity of a protein in a highly specific manner.
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- 2020
- Full Text
- View/download PDF
3. Structural insights into a 20.8-kDa tegumental-allergen-like (TAL) protein from Clonorchis sinensis
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Chang Hwa Jo, Jonghyeon Son, Sulhee Kim, Takashi Oda, Jaehoon Kim, Myoung-Ro Lee, Mamoru Sato, Hyun Tae Kim, Satoru Unzai, Sam-Yong Park, and Kwang Yeon Hwang
- Subjects
Medicine ,Science - Abstract
Abstract 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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- 2017
- Full Text
- View/download PDF
4. Molecular Landscape of the Ribosome Pre-initiation Complex during mRNA Scanning: Structural Role for eIF3c and Its Control by eIF5
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Eiji Obayashi, Rafael E. Luna, Takashi Nagata, Pilar Martin-Marcos, Hiroyuki Hiraishi, Chingakham Ranjit Singh, Jan Peter Erzberger, Fan Zhang, Haribabu Arthanari, Jacob Morris, Riccardo Pellarin, Chelsea Moore, Ian Harmon, Evangelos Papadopoulos, Hisashi Yoshida, Mahmoud L. Nasr, Satoru Unzai, Brytteny Thompson, Eric Aube, Samantha Hustak, Florian Stengel, Eddie Dagraca, Asokan Ananbandam, Philip Gao, Takeshi Urano, Alan G. Hinnebusch, Gerhard Wagner, and Katsura Asano
- Subjects
translation initiation ,ribosome ,eIF5 ,eIF3 ,eIF1 ,start codon selection ,ribosomal pre-initiation complex ,Biology (General) ,QH301-705.5 - Abstract
During eukaryotic translation initiation, eIF3 binds the solvent-accessible side of the 40S ribosome and recruits the gate-keeper protein eIF1 and eIF5 to the decoding center. This is largely mediated by the N-terminal domain (NTD) of eIF3c, which can be divided into three parts: 3c0, 3c1, and 3c2. The N-terminal part, 3c0, binds eIF5 strongly but only weakly to the ribosome-binding surface of eIF1, whereas 3c1 and 3c2 form a stoichiometric complex with eIF1. 3c1 contacts eIF1 through Arg-53 and Leu-96, while 3c2 faces 40S protein uS15/S13, to anchor eIF1 to the scanning pre-initiation complex (PIC). We propose that the 3c0:eIF1 interaction diminishes eIF1 binding to the 40S, whereas 3c0:eIF5 interaction stabilizes the scanning PIC by precluding this inhibitory interaction. Upon start codon recognition, interactions involving eIF5, and ultimately 3c0:eIF1 association, facilitate eIF1 release. Our results reveal intricate molecular interactions within the PIC, programmed for rapid scanning-arrest at the start codon.
- Published
- 2017
- Full Text
- View/download PDF
5. A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation.
- Author
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Huaying Zhao, Rodolfo Ghirlando, Carlos Alfonso, Fumio Arisaka, Ilan Attali, David L Bain, Marina M Bakhtina, Donald F Becker, Gregory J Bedwell, Ahmet Bekdemir, Tabot M D Besong, Catherine Birck, Chad A Brautigam, William Brennerman, Olwyn Byron, Agnieszka Bzowska, Jonathan B Chaires, Catherine T Chaton, Helmut Cölfen, Keith D Connaghan, Kimberly A Crowley, Ute Curth, Tina Daviter, William L Dean, Ana I Díez, Christine Ebel, Debra M Eckert, Leslie E Eisele, Edward Eisenstein, Patrick England, Carlos Escalante, Jeffrey A Fagan, Robert Fairman, Ron M Finn, Wolfgang Fischle, José García de la Torre, Jayesh Gor, Henning Gustafsson, Damien Hall, Stephen E Harding, José G Hernández Cifre, Andrew B Herr, Elizabeth E Howell, Richard S Isaac, Shu-Chuan Jao, Davis Jose, Soon-Jong Kim, Bashkim Kokona, Jack A Kornblatt, Dalibor Kosek, Elena Krayukhina, Daniel Krzizike, Eric A Kusznir, Hyewon Kwon, Adam Larson, Thomas M Laue, Aline Le Roy, Andrew P Leech, Hauke Lilie, Karolin Luger, Juan R Luque-Ortega, Jia Ma, Carrie A May, Ernest L Maynard, Anna Modrak-Wojcik, Yee-Foong Mok, Norbert Mücke, Luitgard Nagel-Steger, Geeta J Narlikar, Masanori Noda, Amanda Nourse, Tomas Obsil, Chad K Park, Jin-Ku Park, Peter D Pawelek, Erby E Perdue, Stephen J Perkins, Matthew A Perugini, Craig L Peterson, Martin G Peverelli, Grzegorz Piszczek, Gali Prag, Peter E Prevelige, Bertrand D E Raynal, Lenka Rezabkova, Klaus Richter, Alison E Ringel, Rose Rosenberg, Arthur J Rowe, Arne C Rufer, David J Scott, Javier G Seravalli, Alexandra S Solovyova, Renjie Song, David Staunton, Caitlin Stoddard, Katherine Stott, Holger M Strauss, Werner W Streicher, John P Sumida, Sarah G Swygert, Roman H Szczepanowski, Ingrid Tessmer, Ronald T Toth, Ashutosh Tripathy, Susumu Uchiyama, Stephan F W Uebel, Satoru Unzai, Anna Vitlin Gruber, Peter H von Hippel, Christine Wandrey, Szu-Huan Wang, Steven E Weitzel, Beata Wielgus-Kutrowska, Cynthia Wolberger, Martin Wolff, Edward Wright, Yu-Sung Wu, Jacinta M Wubben, and Peter Schuck
- Subjects
Medicine ,Science - Abstract
Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.
- Published
- 2015
- Full Text
- View/download PDF
6. Crystal structure of human importin-α1 (Rch1), revealing a potential autoinhibition mode involving homodimerization.
- Author
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Hideyuki Miyatake, Akira Sanjoh, Satoru Unzai, Go Matsuda, Yuko Tatsumi, Yoichi Miyamoto, Naoshi Dohmae, and Yoko Aida
- Subjects
Medicine ,Science - Abstract
In this study, we determined the crystal structure of N-terminal importin-β-binding domain (IBB)-truncated human importin-α1 (ΔIBB-h-importin-α1) at 2.63 Å resolution. The crystal structure of ΔIBB-h-importin-α1 reveals a novel closed homodimer. The homodimer exists in an autoinhibited state in which both the major and minor nuclear localization signal (NLS) binding sites are completely buried in the homodimerization interface, an arrangement that restricts NLS binding. Analytical ultracentrifugation studies revealed that ΔIBB-h-importin-α1 is in equilibrium between monomers and dimers and that NLS peptides shifted the equilibrium toward the monomer side. This finding suggests that the NLS binding sites are also involved in the dimer interface in solution. These results show that when the IBB domain dissociates from the internal NLS binding sites, e.g., by binding to importin-β, homodimerization possibly occurs as an autoinhibition state.
- Published
- 2015
- Full Text
- View/download PDF
7. Blocking PSD95‐PDZ3's amyloidogenesis through point mutations that inhibit high‐temperature reversible oligomerization (RO)
- Author
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Tomonori Saotome, Sawaros Onchaiya, Subbaian Brindha, Taichi Mezaki, Satoru Unzai, Keiichi Noguchi, Jose C. Martinez, Shun‐ichi Kidokoro, and Yutaka Kuroda
- Subjects
Amyloid ,Protein Folding ,Alanine ,Calorimetry, Differential Scanning ,Circular Dichroism ,Temperature ,Point Mutation ,Thermodynamics ,Cell Biology ,Disks Large Homolog 4 Protein ,Molecular Biology ,Biochemistry - Abstract
The third PDZ domain of the postsynaptic density protein 95 (PSD95-PDZ3; 11 kDa, 103 residues) has a propensity to form amyloid fibrils at high temperatures. At neutral pH, PDZ3 is natively folded, but it exhibits a peculiar three-state thermal unfolding with a reversible oligomerization (RO) equilibrium at high temperatures, which is uncharacteristic in the unfolding of a small globular protein as PDZ3 is. Here, we examined the RO's role in PDZ3's amyloidogenesis at high-temperature using two variants (F340A and L342A) that suppress the high-temperature RO and five single-alanine-mutated variants, where we mutated surface-exposed hydrophobic residues to alanine. Circular Dichroism (CD), Analytical Ultracentrifuge (AUC), and other spectroscopic measurements confirmed the retention of the native structure at ambient temperature. Differential Scanning Calorimetry (DSC) was used to assess the presence or absence of the high-temperature RO, and the amyloidogenicity of the variants was measured by Thioflavin T (ThT) fluorescence and Transmission Electron Microscopy (TEM). By comparing the fraction of RO and the ThT signal, we found that mutations that suppressed the high-temperature RO strongly inhibited amyloidogenesis. On the other hand, all variants forming RO also formed amyloids under the same conditions as the wild-type PDZ3.
- Published
- 2022
8. Solution structure of Gaussia Luciferase with five disulfide bonds and identification of a putative coelenterazine binding cavity by heteronuclear NMR
- Author
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Toshio Yamazaki, Nan Wu, Naohiro Kobayashi, Yutaka Kuroda, Kengo Tsuda, Satoru Unzai, and Tomonori Saotome
- Subjects
0301 basic medicine ,Protein Folding ,Protein Conformation ,Stereochemistry ,lcsh:Medicine ,010402 general chemistry ,01 natural sciences ,Article ,Copepoda ,03 medical and health sciences ,Gaussia ,chemistry.chemical_compound ,Protein structure ,Protein Domains ,Coelenterazine ,Animals ,Luciferase ,Amino Acid Sequence ,Disulfides ,Luciferases ,lcsh:Science ,Nuclear Magnetic Resonance, Biomolecular ,Peptide sequence ,Multidisciplinary ,biology ,Chemistry ,lcsh:R ,Imidazoles ,biology.organism_classification ,0104 chemical sciences ,030104 developmental biology ,Heteronuclear molecule ,Pyrazines ,Helix ,Protein folding ,lcsh:Q ,Oxidoreductases ,Solution-state NMR - Abstract
Gaussia luciferase (GLuc) is a small luciferase (18.2 kDa; 168 residues) and is thus attracting much attention as a reporter protein, but the lack of structural information is hampering further application. Here, we report the first solution structure of a fully active, recombinant GLuc determined by heteronuclear multidimensional NMR. We obtained a natively folded GLuc by bacterial expression and efficient refolding using a Solubility Enhancement Petide (SEP) tag. Almost perfect assignments of GLuc’s 1H, 13C and 15N backbone signals were obtained. GLuc structure was determined using CYANA, which automatically identified over 2500 NOEs of which > 570 were long-range. GLuc is an all-alpha-helix protein made of nine helices. The region spanning residues 10–18, 36–81, 96–145 and containing eight out of the nine helices was determined with a Cα-atom RMSD of 1.39 Å ± 0.39 Å. The structure of GLuc is novel and unique. Two homologous sequential repeats form two anti-parallel bundles made by 4 helices and tied together by three disulfide bonds. The N-terminal helix 1 is grabbed by these 4 helices. Further, we found a hydrophobic cavity where several residues responsible for bioluminescence were identified in previous mutational studies, and we thus hypothesize that this is a catalytic cavity, where the hydrophobic coelenterazine binds and the bioluminescence reaction takes place.
- Published
- 2020
9. Thermodynamic Analysis of Point Mutations Inhibiting High-Temperature Reversible Oligomerization of PDZ3
- Author
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Jose C. Martinez, Tomonori Saotome, Yutaka Kuroda, Shun-ichi Kidokoro, Satoru Unzai, Subbaian Brindha, and Taichi Mezaki
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chemistry.chemical_classification ,Protein Denaturation ,0303 health sciences ,Hot Temperature ,Future studies ,Calorimetry, Differential Scanning ,Globular protein ,Point mutation ,Temperature ,Biophysics ,Articles ,Crystal structure ,Alanine scanning ,Endothermic process ,03 medical and health sciences ,Crystallography ,0302 clinical medicine ,Differential scanning calorimetry ,chemistry ,Point Mutation ,Thermodynamics ,Denaturation (biochemistry) ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
Differential scanning calorimetry (DSC) indicated that PDZ3 undergoes a peculiar thermal denaturation, exhibiting two endothermic peaks because of the formation of reversible oligomers at high temperature (N↔I(6)↔D). This contrasts sharply with the standard two-state denaturation model observed for small, globular proteins. We performed an alanine scanning analysis by individually mutating three hydrophobic residues at the crystallographic oligomeric interface (Phe340, Leu342, and Ile389) and one away from the interface (Leu349, as a control). DSC analysis indicated that PDZ3-F340A and PDZ3-L342A exhibited a single endothermic peak. Furthermore, PDZ3-L342A underwent a perfect two-state denaturation, as evidenced by the single endothermic peak and confirmed by detailed DSC analysis, including global fitting of data measured at different protein concentrations. Reversible oligomerization (RO) at high temperatures by small globular proteins is a rare event. Furthermore, our present study showing that a point mutation, L342A, designed based on the crystal structure inhibited RO is surprising because RO occurs at a high-temperature. Future studies will determine how and why mutations designed using crystal structures determined at ambient temperatures influence the formation of RO at high temperatures, and whether high-temperature ROs are related to the propensity of proteins to aggregate or precipitate at lower temperatures, which would provide a novel and unique way of controlling protein solubility and aggregation.
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- 2020
10. Nanometer-Sized Aggregates Generated Using Short Solubility Controlling Peptide Tags Do Increase the In Vivo Immunogenicity of a Nonimmunogenic Protein
- Author
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Nafsoon Rahman, Satoru Unzai, Yutaka Kuroda, Shiho Miura, and Mohammad Monirul Islam
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chemistry.chemical_classification ,Immunogenicity ,Antibody titer ,Pharmaceutical Science ,Model protein ,Peptide ,02 engineering and technology ,021001 nanoscience & nanotechnology ,030226 pharmacology & pharmacy ,Bovine Pancreatic Trypsin Inhibitor ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,chemistry ,In vivo ,Drug Discovery ,Biophysics ,Molecular Medicine ,Solubility ,0210 nano-technology - Abstract
Subvisible aggregates of proteins are suspected to cause adverse immune response, and a recent FDA guideline has recommended the monitoring of micrometer-sized aggregates (2-10 μm) though recognizing that the underlying mechanism behind aggregation and immunogenicity remains unclear. Here, we report a correlation between the immunogenicity and the size of nanometer-scaled aggregates of a small 6.5 kDa model protein, bovine pancreatic trypsin inhibitor (BPTI) variant. BPTI-19A, a monomeric and nonimmunogenic protein, was oligomerized into subvisible aggregates with hydrodynamic radii (Rh) of 3-4 nm by attaching hydrophobic solubility controlling peptide (SCP) tags to its C-terminus. The results showed that the association of nonimmunogenic BPTI into nanometer-sized subvisible aggregates made it highly immunogenic, as assessed by the IgG antibody titers of the mice's sera. Overall, the study emphasizes that subvisible aggregates, as small as a few nanometers, which are presently ignored, are worth monitoring for deciphering the origin of undesired immunogenicity of therapeutic proteins.
- Published
- 2020
11. Author response for 'Blocking PSD95‐PDZ3's amyloidogenesis through point mutations that inhibit high‐temperature reversible oligomerization (RO)'
- Author
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null Tomonori Saotome, null Sawaros Onchaiya, null Brindha Subbaian, null Taichi Mezaki, null Satoru Unzai, null Keiichi Noguchi, null Jose C. Martinez, null Shun‐ichi Kidokoro, and null Yutaka Kuroda
- Published
- 2021
12. EGFR extracellular domain III expressed in Escherichia coli with SEP tag shows improved biophysical and functional properties and generate anti-sera inhibiting cancer cell growth
- Author
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Tomonori Saotome, Md. Golam Kibria, Satoru Unzai, Subbaian Brindha, and Yutaka Kuroda
- Subjects
0301 basic medicine ,Circular dichroism ,Protein Conformation ,Biophysics ,Peptide ,Enzyme-Linked Immunosorbent Assay ,medicine.disease_cause ,Biochemistry ,03 medical and health sciences ,0302 clinical medicine ,Antineoplastic Agents, Immunological ,Protein Domains ,Cell Line, Tumor ,Extracellular ,medicine ,Escherichia coli ,Animals ,Humans ,Epidermal growth factor receptor ,Particle Size ,Neutralizing antibody ,Molecular Biology ,chemistry.chemical_classification ,Chromatography, Reverse-Phase ,Mice, Inbred ICR ,biology ,Circular Dichroism ,Immune Sera ,Cell Biology ,Recombinant Proteins ,ErbB Receptors ,030104 developmental biology ,chemistry ,Solubility ,030220 oncology & carcinogenesis ,biology.protein ,Protein folding ,Female ,Antibody - Abstract
The epidermal growth factor receptor extracellular domain III (EGFR-ECDIII) protein is a promising target of anti-cancer research, and its production in Escherichia coli would thus represent significant benefits. However, despite its moderate size (19 kDa), the expression of EGFR-ECDIII in E.coli is hampered by the presence of multiple cysteines producing misfolded proteins with incorrect S-S bonds. In our study, we show that a short 12-residue solubility enhancing peptide (SEP) tag containing nine arginines (C9R) attached at the C-terminus of EGFR-ECDIII reduces the inclusion body formation and increases the final yield by six times (20 mg/L). EGFR-ECDIII-C9R purified from the soluble fraction eluted as a sharp single RP-HPLC peak, suggesting a single S-S bond pairing. Biophysical characterization using circular dichroism, fluorescence, and light scattering confirmed its native-like properties together with reversible thermal denaturation. The binding activity of EGFR-ECDIII-C9R to anti-EGFR-VHH7D12, a single-domain antibody with specific binding to the ECDIII, was assessed by sandwich ELISA. Further, we produced anti-EGFR-ECDIII-C9R antisera in mouse models and anti-sera inhibited A431 cancer cells’ growth. These results demonstrate that the SEP tag enables the rapid production of the multiple disulfide-bonded EGFR-ECDIII in E. coli having native-like biophysical properties and producing neutralizing antibodies.
- Published
- 2021
13. Reversible Oligomerization and Reverse Hydrophobic Effect Induced by Isoleucine Tags Attached at the C-Terminus of a Simplified BPTI Variant
- Author
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Golam Kibria, Shun-ichi Kidokoro, Satoru Unzai, Yutaka Kuroda, and Shigeyoshi Nakamura
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chemistry.chemical_classification ,Models, Molecular ,0303 health sciences ,Circular dichroism ,Chemistry ,Protein Stability ,030302 biochemistry & molecular biology ,Peptide ,Protein aggregation ,Biochemistry ,Hydrophobic effect ,03 medical and health sciences ,Protein Aggregates ,Differential scanning calorimetry ,Aprotinin ,Dynamic light scattering ,Biophysics ,Animals ,Denaturation (biochemistry) ,Chemical stability ,Cattle ,Isoleucine ,Protein Multimerization ,Peptides ,Hydrophobic and Hydrophilic Interactions - Abstract
Protein amorphous aggregation has become the focus of great attention, as it can impair the ability of cells to function properly. Here, we evaluated the effects of three peptide tags, consisting of one, three, and five consecutive isoleucines attached at the C-terminus end of a simplified bovine pancreatic trypsin inhibitor (BPTI) variant, BPTI-19A, on the thermal stability and oligomerization by circular dichroism spectrometry and differential scanning calorimetry in detail. All of the BPTI-19A variants exhibited a reversible and apparently two-state thermal transition like BPTI-19A at pH 4.7. The thermal transition of the five-isoleucine-tagged variant showed clear protein-concentration dependence, where the apparent denaturation temperature decreased as the protein concentration increased. Quantitative analysis indicated that this phenomenon originated from the presence of reversibly oligomerized (RO) states at high temperatures. The results also illustrated that the thermodynamic stability difference between the native and the monomeric denatured state in all the proteins was destabilized by the hydrophobic tags and was well explained by the reverse hydrophobic effect due to the tags. The existence of the RO states was confirmed by both analytical ultracentrifugation and dynamic light scattering. This indicated that the five-isoleucine hydrophobic tag is strong enough to induce intermolecular hydrophobic contact among the denatured molecules leading to oligomerization, and even one- or three-isoleucine tags are effective enough to generate intramolecular hydrophobic contact, thus provoking denaturation through the reverse hydrophobic effect.
- Published
- 2020
14. Solution structure of Gaussia Luciferase with five disulfide bonds and identification of a putative coelenterazine binding cavity by heteronuclear NMR
- Author
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Tomonori Saotome, Yutaka Kuroda, Kengo Tsuda, Satoru Unzai, Toshio Yamazaki, Naohiro Kobayashi, and Nan Wu
- Subjects
biology ,Chemistry ,Stereochemistry ,Cyana ,biology.organism_classification ,law.invention ,Gaussia ,chemistry.chemical_compound ,Heteronuclear molecule ,law ,Coelenterazine ,Helix ,Recombinant DNA ,Bioluminescence ,Luciferase - Abstract
Gaussia luciferase (GLuc) is the smallest luciferase (18.2kDa; 168 residues) reported so far and is thus attracting much attention as a reporter protein, but the lack of structural information is hampering further application. Here, we report the first solution structure of a fully active, recombinant GLuc determined by heteronuclear multidimensional NMR. We obtained a natively folded GLuc by bacterial expression and efficient refolding using a solubility tag. Almost perfect assignments of GLuc’s 1H, 13C and 15N backbone signals were obtained. GLuc structure was determined using CYANA, which automatically identified over 2500 NOEs of which > 570 were long-range. GLuc is an all-alpha-helix protein made of nine helices. The region spanning residues 10–18, 36-81, 96-145 and containing eight out of the nine helices was determined with a Cα-atom RMSD of 1.39 ű 0.39 Å. The structure of GLuc is novel and unique. Two homologous sequential repeats form two anti-parallel bundles made by 4 helices and tied together by three disulfide bonds. The N-terminal helix 1 is grabbed by these 4 helices. Further, we found a hydrophobic cavity where several residues responsible for bioluminescence were identified in previous mutational studies, and we thus hypothesize that this is a catalytic cavity, where the hydrophobic coelenterazine binds and the bioluminescence reaction takes place.
- Published
- 2020
15. Thermodynamic analysis of point mutations inhibiting high-temperature reversible oligomer of PDZ3
- Author
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Satoru Unzai, T. Mezaki, Tomonori Saotome, B. Subbaian, Yutaka Kuroda, Shun-ichi Kidokoro, and Jose C. Martinez
- Subjects
Alanine ,chemistry.chemical_classification ,chemistry.chemical_compound ,Crystallography ,Circular dichroism ,Differential scanning calorimetry ,chemistry ,Globular protein ,Denaturation (biochemistry) ,Alanine scanning ,Endothermic process ,Oligomer - Abstract
Differential scanning calorimetry (DSC) indicated that PDZ3 undergoes a peculiar thermal denaturation exhibiting two endothermic peaks due to the formation of reversible oligomers at high temperature (N↔I6↔D). This contrasts sharply with the standard 2-state denaturation model observed for small, globular proteins. We performed an alanine scanning analysis by individually mutating three hydrophobic residues at the crystallographic oligomeric interface (Phe340, Leu342, Ile389) and one away from the interface (Leu349, as a control). DSC analysis indicated that PDZ3-F340A and PDZ3-L342A exhibited a single endothermic peak. Furthermore, PDZ3-L342A underwent a perfect 2-state denaturation, as evidenced by the single endothermic peak, and confirmed by detailed DSC analysis, including global fitting of data measured at different protein concentrations. Reversible oligomerization (RO) at high temperatures by small globular proteins is a rare event. While we designed the mutations based on our previous study showing that a point mutation Val380 to a nonhydrophobic amino acid inhibited RO in DEN4 ED3, the results are nevertheless surprising since high-temperature RO involves proteins in a denatured state, as assessed by circular dichroism. Future studies will determine how and why mutations designed using crystal structures determined at ambient temperatures influence the formation of RO at high temperatures, and whether high-temperature ROs are related to the propensity of proteins to aggregate or precipitate at lower temperatures, which would provide a novel and unique way of controlling protein solubility and aggregation.SignificanceDespite being a small globular protein, which normaly undergo a two-state unfolding, the thermal denaturation of PSD95-PDZ3, monitored by DSC, exhibited two endothermic peaks. The second peak resulted from a reversible oligomerization (RO) at high temperatures, which is, on its own, a rare phenomenon. In this study, we show that the substitution of a single hydrophobic residue to an alanine at the interface of the crystallographic tetramers inhibited high-temperature RO, resulting in a single endothermic peak. Future studies are required to determine why mutations designed using crystal structures determined at ambient temperatures can inhibit high-temperature RO, and whether the ROs are precursor of irreversible aggregation, If so, the present observations will provide an entirely new basis for creating aggregation-resistant proteins.
- Published
- 2020
16. A systematic mutational analysis identifies a 5-residue proline tag that enhances the in vivo immunogenicity of a non-immunogenic model protein 240 folds
- Author
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Satoru Unzai, Golam Kibria, Mohammad Monirul Islam, Yutaka Kuroda, and Nafsoon Rahman
- Subjects
chemistry.chemical_classification ,Titer ,Circular dichroism ,chemistry ,Biochemistry ,biology ,In vivo ,Immunogenicity ,biology.protein ,Peptide ,Proline ,Antibody ,Protein secondary structure - Abstract
Small proteins are generally non-immunogenic, which can be a major hurdle in developing protein and peptide vaccines or producing antibodies for biopharmaceutical usage. For improving a protein’s immunogenicity, we previously proposed to use short Solubility Controlling Peptide (SCP) tags that oligomerize proteins into soluble aggregates. Here, we systematically analyzed the effect of SCP-tags that do not induce oligomerization on the immunogenicity of a small, non-immunogenic, model protein, Bovine Pancreatic Trypsin Inhibitor (BPTI-19A; 6 kDa). We assessed the effect of the following ten SCP-tags: Six tags made of five consecutive Arg, Lys, His, Asp, Asn, Pro; one made of seven Pro; two tags made of consecutive Arg-lle and Asn-Ile, all attached at the C-terminus of BPTI-19A; and a 5-proline tag attached at the N-terminus. Circular dichroism, fluorescence, dynamic light scattering measurements, and analytical ultra-centrifugation indicated that the addition of the SCP-tags did not change the secondary structure content nor the tertiary structures of the protein nor its monomeric state. On the other hand, the C-terminus 5-proline (C5P) tag unexpectedly increased the immunogenicity (IgG level) of BPTI-19A by up to 240 fold as assessed by ELISA. Additionally, the 5-arginine tag (C5R) increased the titer by up to 73 fold. The titer increase lasted for several weeks, and the effect was cumulative to that of the Freund’s adjuvant, which is commonly used to boost a protein’s immunogenicity. Altogether, SCP-tags that do not oligomerize proteins substantially increased the immunogenicity of a non-immunogenic protein, suggesting that the 5-proline and the 5-arginine SCP-tags may provide a novel tool for facilitating the production of antibodies or improving the effectiveness of protein-based vaccines.
- Published
- 2020
- Full Text
- View/download PDF
17. Generation of 'nanometer-size aggregates' using Solubility Controlling Peptide tags and their ability to increase a protein’s immunogenicity in vivo
- Author
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Mohammad Monirul Islam, Nafsoon Rahman, Yutaka Kuroda, Satoru Unzai, and Shiho Miura
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chemistry.chemical_classification ,chemistry.chemical_compound ,Immune system ,Monomer ,Chemistry ,In vivo ,Immunogenicity ,Antibody titer ,Biophysics ,Nanometer size ,Peptide ,Solubility - Abstract
Sub-visible aggregates of proteins are suspected to cause adverse immune response, and a recent FDA guideline has recommended the monitoring of micrometer-size aggregates (2-10 μm) though recognizing that the underlying mechanism behind aggregation and immunogenicity remains unclear. Here, we report a correlation between the immunogenicity and the size of nanometer-scale aggregates of a small 6.5 kDa model protein, Bovine Pancreatic Trypsin Inhibitor (BPTI) variant. BPTI-19A, a monomeric and non-immunogenic protein, was oligomerized into sub-visible aggregates with hydrodynamic radii (Rh) of 3~4 nm by attaching hydrophobic solubility controlling peptide (SCP) tags to its C-terminus. The results showed that the association of non-immunogenic BPTI into nanometer-size aggregates made it highly immunogenic, as assessed by the IgG antibody titers of the mice’s sera. Overall, the study emphasizes that sub-visible aggregates, as small as a few nanometers, which are presently ignored, are worth monitoring for deciphering the origin of undesired immunogenicity of therapeutic proteins.
- Published
- 2019
- Full Text
- View/download PDF
18. Molecular Landscape of the Ribosome Pre-initiation Complex during mRNA Scanning: Structural Role for eIF3c and Its Control by eIF5
- Author
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Asokan Ananbandam, Hisashi Yoshida, Florian Stengel, Mahmoud L. Nasr, Brytteny Thompson, Takeshi Urano, Fan Zhang, Philip Gao, Haribabu Arthanari, Takashi Nagata, Riccardo Pellarin, Gerhard Wagner, Alan G. Hinnebusch, Jacob Morris, Rafael E. Luna, Katsura Asano, Evangelos Papadopoulos, Chelsea Moore, Eric Aube, Pilar Martin-Marcos, Hiroyuki Hiraishi, Eiji Obayashi, Eddie Dagraca, Jan P. Erzberger, Chingakham Ranjit Singh, Satoru Unzai, Ian Harmon, Samantha Hustak, Berkeley California Institute for Quantitative Biosciences [Berkeley], University of California (UC), University of California [San Francisco] (UC San Francisco), Institute for Molecular Systems Biology [ETH Zurich] (IMSB), Department of Biology [ETH Zürich] (D-BIOL), Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich)- Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich), Division of Biology, Kansas State University, Kansas State University, This work was supported by a grant from the NIH (R01 GM64781), a pilot grant from University of Kansas COBRE-PSF (P30 GM 110761), NSF Research Grant (1412250), and an Innovative Award from KSU Terry Johnson Cancer Center to K.A., CA68262 and GM47467 to G.W., an intramural grant from NICHD, NIH, to A.G.H., and JSPS KAKENHI 15H01634 and 26440026 to T.N., We thank Hiroshi Matsuo and Erin Adamson for comments, Ivan Topisirovic for proofreading, and Michaela Flax and Speed Rogers for technical assistance., University of California, and University of California [San Francisco] (UCSF)
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Models, Molecular ,MESH: Eukaryotic Initiation Factor-5 ,0301 basic medicine ,Magnetic Resonance Spectroscopy ,MESH: Eukaryotic Initiation Factor-3 ,Eukaryotic Initiation Factor-3 ,ribosomal pre-initiation complex ,Eukaryotic Initiation Factor-1 ,MESH: Amino Acid Sequence ,MESH: Saccharomyces cerevisiae Proteins ,Start codon ,Eukaryotic initiation factor ,Eukaryotic Initiation Factor-5 ,Peptide Chain Initiation, Translational ,lcsh:QH301-705.5 ,Shine-Dalgarno sequence ,MESH: Protein Subunits ,MESH: Saccharomyces cerevisiae ,[SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics ,EIF1 ,ribosome ,eIF1 ,eIF3 ,MESH: Eukaryotic Initiation Factor-1 ,eIF5 ,MESH: Models, Molecular ,Protein Binding ,MESH: Peptide Chain Initiation, Translational ,Saccharomyces cerevisiae Proteins ,MESH: Mutation ,Saccharomyces cerevisiae ,Biology ,start codon selection ,translation initiation ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,ddc:570 ,MESH: Protein Binding ,Initiation factor ,Amino Acid Sequence ,RNA, Messenger ,MESH: RNA, Messenger ,Binding Sites ,MESH: Magnetic Resonance Spectroscopy ,[SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology ,Molecular biology ,Ribosomal binding site ,Protein Subunits ,A-site ,Internal ribosome entry site ,030104 developmental biology ,lcsh:Biology (General) ,MESH: Binding Sites ,Ribosomal pre-initiation complex ,Ribosome ,Start codon selection ,Translation initiation ,Mutation ,Biophysics ,Ribosomes ,MESH: Ribosomes - Abstract
During eukaryotic translation initiation, eIF3 binds the solvent-accessible side of the 40S ribosome and recruits the gate-keeper protein eIF1 and eIF5 to the decoding center. This is largely mediated by the N-terminal domain (NTD) of eIF3c, which can be divided into three parts: 3c0, 3c1, and 3c2. The N-terminal part, 3c0, binds eIF5 strongly but only weakly to the ribosome-binding surface of eIF1, whereas 3c1 and 3c2 form a stoichiometric complex with eIF1. 3c1 contacts eIF1 through Arg-53 and Leu-96, while 3c2 faces 40S protein uS15/S13, to anchor eIF1 to the scanning pre-initiation complex (PIC). We propose that the 3c0:eIF1 interaction diminishes eIF1 binding to the 40S, whereas 3c0:eIF5 interaction stabilizes the scanning PIC by precluding this inhibitory interaction. Upon start codon recognition, interactions involving eIF5, and ultimately 3c0:eIF1 association, facilitate eIF1 release. Our results reveal intricate molecular interactions within the PIC, programmed for rapid scanning-arrest at the start codon., Cell Reports, 18 (11), ISSN:2666-3864, ISSN:2211-1247
- Published
- 2017
19. Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium
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Sam-Yong Park, Kanako Sugiyama, Yuuki Nihei, Jeremy R. H. Tame, Tetsuo Takahashi, Zhiwen Zhou, Mineo Iseki, Shin-ichi Adachi, Satoru Unzai, Shigeru Matsunaga, Masumi Takebe, Naoya Shibayama, Fumihiro Kawai, Ryuta Koyama, Yuji Ikegaya, Hitomi Tanaka, and Mio Ohki
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0301 basic medicine ,Euglena gracilis ,Light ,ved/biology.organism_classification_rank.species ,Protein domain ,Flavin group ,Biology ,Second Messenger Systems ,Cyclase ,Structure-Activity Relationship ,03 medical and health sciences ,Rhodobacter sphaeroides ,Bacterial Proteins ,Protein Domains ,Cyclic AMP ,Humans ,Luciferase ,Coiled coil ,Multidisciplinary ,030102 biochemistry & molecular biology ,ved/biology ,Biological Sciences ,biology.organism_classification ,Enzyme Activation ,HEK293 Cells ,030104 developmental biology ,Biochemistry ,Oscillatoria ,Second messenger system ,Biophysics ,Adenylyl Cyclases - Abstract
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 Å 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.
- Published
- 2016
20. Revisiting a Classical Allosteric Model - Examination of an Obliterated Interface
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Satoru Unzai and Antonio Tsuneshige
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Physics ,Classical mechanics ,Interface (Java) ,Allosteric model ,Biophysics - Published
- 2020
21. Analytical ultracentrifugation in structural biology
- Author
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Satoru Unzai
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0301 basic medicine ,030102 biochemistry & molecular biology ,Computer science ,Biophysics ,A protein ,Small sample ,Computational biology ,Review ,Structure and function ,Analytical Ultracentrifugation ,03 medical and health sciences ,030104 developmental biology ,Structural biology ,Structural Biology ,Homogeneous ,Target protein ,Experimental methods ,Molecular Biology - Abstract
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.
- Published
- 2017
22. A novel 3 ' splice site recognition by the two zinc fingers in the U2AF small subunit
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Takashi Oda, Sam-Yong Park, Taeko Akiyoshi, Eiji Obayashi, Kengo Tsuda, Satoru Unzai, Mikako Shirouzu, Takeshi Urano, Hisashi Yoshida, Yutaka Muto, Mamoru Sato, and Kanako Kuwasako
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Models, Molecular ,Amino Acid Motifs ,DNA Mutational Analysis ,Biology ,Splicing Factor U2AF ,Schizosaccharomyces ,Genetics ,splice ,Protein Structure, Quaternary ,Gene ,Ribonucleoprotein ,Zinc finger ,Binding Sites ,RNA recognition motif ,RNA ,Nuclear Proteins ,Zinc Fingers ,Cell biology ,Protein Structure, Tertiary ,RNA splicing ,3 ' splice site ,U2AF small subunit ,Ribonucleoproteins ,RNA Splice Sites ,Developmental Biology ,Research Paper ,Protein Binding - Abstract
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 β 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.
- Published
- 2015
23. Heat-induced native dimerization prevents amyloid formation by variable domain from immunoglobulin light-chain REI
- Author
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Mikio Kataoka, Satoru Unzai, Daizo Hamada, Marina Nawata, Shouhei Mine, Yuta Kobayashi, Hironari Kamikubo, Hirotaka Tsutsumi, Tsutomu Nakamura, and Koichi Uegaki
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0301 basic medicine ,Models, Molecular ,Circular dichroism ,Amyloid ,Protein Folding ,Hot Temperature ,Stereochemistry ,Dimer ,Mutant ,Immunoglobulin Variable Region ,Gene Expression ,Immunoglobulin light chain ,Crystallography, X-Ray ,Biochemistry ,Protein Structure, Secondary ,03 medical and health sciences ,chemistry.chemical_compound ,X-Ray Diffraction ,Scattering, Small Angle ,medicine ,Native state ,Escherichia coli ,Humans ,Protein Interaction Domains and Motifs ,Amino Acid Sequence ,Cloning, Molecular ,Molecular Biology ,Protein Unfolding ,Sequence Homology, Amino Acid ,Chemistry ,Protein Stability ,Amyloidosis ,Cell Biology ,computer.file_format ,medicine.disease ,Protein Data Bank ,Recombinant Proteins ,Kinetics ,030104 developmental biology ,Monomer ,Mutation ,Thermodynamics ,Immunoglobulin Light Chains ,Protein Multimerization ,computer ,Sequence Alignment - Abstract
Amyloid light-chain (AL) amyloidosis is a protein-misfolding disease characterized by accumulation of immunoglobulin light chains (LCs) into amyloid fibrils. Dimerization of a full length or variable domain (VL) of LC serves to stabilize the native state and prevent the formation of amyloid fibrils. We here analyzed the thermodynamic properties of dimerization and unfolding reactions by nonamyloidogenic VL from REI LC or its monomeric Y96K mutant using sedimentation velocity and circular dichroism. The data indicate that the equilibrium shifts to native dimerization for wild-type REI VL by elevating temperature due to the negative enthalpy change for dimer dissociation (−81.2 kJ·mol−1). The Y96K mutation did not affect the stability of the monomeric native state but increased amyloidogenicity. These results suggest that the heat-induced native homodimerization is the major factor preventing amyloid formation by wild-type REI VL. Heat-induced native oligomerization may be an efficient strategy to avoid the formation of misfolded aggregates particularly for thermostable proteins that are used at elevated temperatures under conditions where other proteins tend to misfold. Database Structural data are available in the Protein Data Bank under the accession numbers 5XP1 and 5XQY.
- Published
- 2017
24. Insight into structural diversity of influenza virus haemagglutinin
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Ki Joon Cho, Junyoung Lee, Jong Hyeon Seok, Sam-Yong Park, Mi Sook Chung, Hee-Bok Oh, Xavier Saelens, Kwang W. Hong, Yiho Park, Kyung Hyun Kim, Ji-Hye Lee, Joo-Yeon Lee, Chun Kang, Se-Ho Kim, Sella Kim, Satoru Unzai, Ji Hoon Yang, Eui-Ki Kim, Chul-Joong Kim, and Seokha Kang
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Models, Molecular ,Conformational change ,Protein Conformation ,Viral protein ,medicine.drug_class ,Hemagglutinin Glycoproteins, Influenza Virus ,Biology ,Antibodies, Viral ,Crystallography, X-Ray ,Cleavage (embryo) ,medicine.disease_cause ,Monoclonal antibody ,Virus ,Virology ,medicine ,Humans ,chemistry.chemical_classification ,Strain (chemistry) ,Protein Stability ,Antibodies, Monoclonal ,Lipid bilayer fusion ,Orthomyxoviridae ,Antibodies, Neutralizing ,chemistry ,Proteolysis ,Glycoprotein - Abstract
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.
- Published
- 2013
25. Structures of haemoglobin from woolly mammoth in liganded and unliganded states
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Jeremy R. H. Tame, Sam-Yong Park, Satoru Unzai, Kevin L. Campbell, Chien Ho, and Hiroki Noguchi
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Models, Molecular ,Woolly mammoth ,Protein Conformation ,Elephants ,Molecular Sequence Data ,Crystallography, X-Ray ,medicine.disease_cause ,Oxygen affinity ,Hemoglobins ,Mammoths ,chemistry.chemical_compound ,Structural Biology ,medicine ,Animals ,Humans ,Amino Acid Sequence ,Globin gene ,Escherichia coli ,Mammoth ,biology ,Ecology ,Oxygen transport ,DNA ,General Medicine ,biology.organism_classification ,chemistry ,Biochemistry ,Organic phosphates ,Sequence Alignment - Abstract
The haemoglobin (Hb) of the extinct woolly mammoth has been recreated using recombinant genes expressed inEscherichia coli. The globin gene sequences were previously determined using DNA recovered from frozen cadavers. Although highly similar to the Hb of existing elephants, the woolly mammoth protein shows rather different responses to chloride ions and temperature. In particular, the heat of oxygenation is found to be much lower in mammoth Hb, which appears to be an adaptation to the harsh high-latitude climates of the Pleistocene Ice Ages and has been linked to heightened sensitivity of the mammoth protein to protons, chloride ions and organic phosphates relative to that of Asian elephants. To elucidate the structural basis for the altered homotropic and heterotropic effects, the crystal structures of mammoth Hb have been determined in the deoxy, carbonmonoxy and aquo-met forms. These models, which are the first structures of Hb from an extinct species, show many features reminiscent of human Hb, but underline how the delicate control of oxygen affinity relies on much more than simple overall quaternary-structure changes.
- Published
- 2012
26. AU-rich RNA-binding induces changes in the quaternary structure of AUH
- Author
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Alan M. Sandercock, Shigeyuki Yokoyama, Satoru Unzai, Kanako Kuwasako, Yutaka Muto, Kazuki Kurimoto, and Carol V. Robinson
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Protein Conformation ,Stereochemistry ,Dimer ,RNA-binding protein ,Trimer ,Biochemistry ,Mass Spectrometry ,chemistry.chemical_compound ,Structural Biology ,Humans ,Protein Structure, Quaternary ,Enoyl-CoA Hydratase ,Molecular Biology ,chemistry.chemical_classification ,Chemistry ,RNA-Binding Proteins ,RNA ,Lyase ,Enzyme ,Dehydratase ,Protein quaternary structure ,Protein Multimerization ,Crystallization ,Ultracentrifugation ,Protein Binding - Abstract
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)24A. 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. Proteins 2009. © 2008 Wiley-Liss, Inc.
- Published
- 2016
27. Oligomerization of Hmo1 mediated by box A is essential for DNA binding in vitro and in vivo
- Author
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Hirofumi Yoshikawa, Ayako Higashino, Satoru Unzai, Koji Kasahara, and Tetsuro Kokubo
- Subjects
0301 basic medicine ,Saccharomyces cerevisiae Proteins ,Saccharomyces cerevisiae ,Mutant ,Plasma protein binding ,Biology ,03 medical and health sciences ,chemistry.chemical_compound ,Transcription (biology) ,Ribosomal protein ,Gene duplication ,Genetics ,DNA, Fungal ,Gene ,030102 biochemistry & molecular biology ,High Mobility Group Proteins ,Cell Biology ,biology.organism_classification ,Cell biology ,DNA-Binding Proteins ,030104 developmental biology ,chemistry ,HMG-Box Domains ,Mutation ,DNA ,Protein Binding - Abstract
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 α-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.
- Published
- 2016
28. Crystal structure of MytiLec, a galactose-binding lectin from the mussel Mytilus galloprovincialis with cytotoxicity against certain cancer cell types
- Author
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Jeremy R. H. Tame, Hiroki Noguchi, Yuki Fujii, Satoru Unzai, Daiki Terada, Imtiaj Hasan, Yasuhiro Ozeki, Fumihiro Kawai, and Sam-Yong Park
- Subjects
Models, Molecular ,0301 basic medicine ,Cell Survival ,Calorimetry ,Biology ,Crystallography, X-Ray ,Ligands ,Protein Engineering ,Article ,Protein Structure, Secondary ,03 medical and health sciences ,chemistry.chemical_compound ,C-type lectin ,Cell Line, Tumor ,Lectins ,Animals ,Humans ,Cytotoxic T cell ,Cloning, Molecular ,Binding site ,Protein Structure, Quaternary ,Cytotoxicity ,Melibiose ,Mytilus ,Binding Sites ,Multidisciplinary ,030102 biochemistry & molecular biology ,Lectin ,Burkitt Lymphoma ,Recombinant Proteins ,Raji cell ,Hemagglutinins ,030104 developmental biology ,chemistry ,Biochemistry ,biology.protein ,Protein quaternary structure ,Trisaccharides ,Ultracentrifugation - Abstract
MytiLec is a lectin, isolated from bivalves, with cytotoxic activity against cancer cell lines that express globotriaosyl ceramide, Galα(1,4)Galβ(1,4)Glcα1-Cer, on the cell surface. Functional analysis shows that the protein binds to the disaccharide melibiose, Galα(1,6)Glc and the trisaccharide globotriose, Galα(1,4)Galβ(1,4)Glc. Recombinant MytiLec expressed in bacteria showed the same haemagglutinating and cytotoxic activity against Burkitt’s lymphoma (Raji) cells as the native form. The crystal structure has been determined to atomic resolution, in the presence and absence of ligands, showing the protein to be a member of the β-trefoil family, but with a mode of ligand binding unique to a small group of related trefoil lectins. Each of the three pseudo-equivalent binding sites within the monomer shows ligand binding and the protein forms a tight dimer in solution. An engineered monomer mutant lost all cytotoxic activity against Raji cells, but retained some haemagglutination activity, showing that the quaternary structure of the protein is important for its cellular effects.
- Published
- 2016
29. Fission Yeast Swi5-Sfr1 Protein Complex, an Activator of Rad51 Recombinase, Forms an Extremely Elongated Dogleg-shaped Structure
- Author
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Yasuhiro Tsutsui, Mamoru Sato, Naohito Nozaki, Yasuto Murayama, Toshiyuki Shimizu, Yuichi Kokabu, Hiroshi Iwasaki, Satoko Akashi, Satoru Unzai, Hiroshi Hashimoto, Mitsunori Ikeguchi, Naoyuki Kuwabara, and Tomotaka Oroguchi
- Subjects
Spectrometry, Mass, Electrospray Ionization ,DNA Repair ,RAD51 ,Swi5-Sfr1 ,Fission Yeast ,Plasma protein binding ,Biochemistry ,Protein filament ,chemistry.chemical_compound ,Mass Spectrometry (MS) ,Schizosaccharomyces ,Recombinase ,Homologous Recombination ,Molecular Biology ,biology ,Cell Biology ,biology.organism_classification ,Yeast ,Protein Structure, Tertiary ,Crystallography ,chemistry ,Protein Structure and Folding ,Schizosaccharomyces pombe ,Rad51 Recombinase ,Schizosaccharomyces pombe Proteins ,Homologous recombination ,Ultracentrifugation ,X-ray Scattering ,DNA ,Protein Binding - Abstract
Background: The Swi5-Sfr1 protein complex is an activator of Rad51 recombinase, which mediates DNA strand exchange in homologous recombination. Results: Swi5 and Sfr1 form a 1:1 complex, which exhibits an extremely elongated dogleg-shaped structure in solution. Conclusion: The Swi5-Sfr1 structure is suitable for binding within the helical groove of the Rad51 filament. Significance: A structural model will advance our understanding of the molecular mechanism of homologous recombination., In eukaryotes, DNA strand exchange is the central reaction of homologous recombination, which is promoted by Rad51 recombinases forming a right-handed nucleoprotein filament on single-stranded DNA, also known as a presynaptic filament. Accessory proteins known as recombination mediators are required for the formation of the active presynaptic filament. One such mediator in the fission yeast Schizosaccharomyces pombe is the Swi5-Sfr1 complex, which has been identified as an activator of Rad51 that assists in presynaptic filament formation and stimulates its strand exchange reaction. Here, we determined the 1:1 binding stoichiometry between the two subunits of the Swi5-Sfr1 complex using analytical ultracentrifugation and electrospray ionization mass spectrometry. Small-angle x-ray scattering experiments revealed that the Swi5-Sfr1 complex displays an extremely elongated dogleg-shaped structure in solution, which is consistent with its exceptionally high frictional ratio (f/f0) of 2.0 ± 0.2 obtained by analytical ultracentrifugation. Furthermore, we determined a rough topology of the complex by comparing the small-angle x-ray scattering-based structures of the Swi5-Sfr1 complex and four Swi5-Sfr1-Fab complexes, in which the Fab fragments of monoclonal antibodies were specifically bound to experimentally determined sites of Sfr1. We propose a model for how the Swi5-Sfr1 complex binds to the Rad51 filament, in which the Swi5-Sfr1 complex fits into the groove of the Rad51 filament, leading to an active and stable presynaptic filament.
- Published
- 2011
30. Crystal structure of basic 7S globulin, a xyloglucan-specific endo-β-1,4-glucanase inhibitor protein-like protein from soybean lacking inhibitory activity against endo-β-glucanase
- Author
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Mamoru Sato, Yuji Nishiuchi, Satoru Unzai, Naoki Shichijo, Toshiyuki Shimizu, Takuya Yoshizawa, Misako Taichi, Hiroshi Hashimoto, Hisashi Hirano, and Mayuki Yamabe
- Subjects
chemistry.chemical_classification ,Cell Biology ,Inhibitor protein ,Biology ,Glucanase ,Biochemistry ,Xyloglucan ,Cell wall ,chemistry.chemical_compound ,Enzyme ,chemistry ,Plant protein ,Xylanase ,Glycoside hydrolase ,Molecular Biology - Abstract
β-Linked glucans such as cellulose and xyloglucan are important components of the cell walls of most dicotyledonous plants. These β-linked glucans are constantly exposed to degradation by various endo-β-glucanases from pathogenic bacteria and fungi. To protect the cell wall from degradation by such enzymes, plants secrete proteinaceous endo-β-glucanases inhibitors, such as xyloglucan-specific endo-β-1,4-glucanase inhibitor protein (XEGIP) in tomato. XEGIPs typically inhibit xyloglucanase, a member of the glycoside hydrolase (GH)12 family. XEGIPs are also found in legumes, including soybean and lupin. To date, tomato XEGIP has been well studied, whereas XEGIPs from legumes are less well understood. Here, we determined the crystal structure of basic 7S globulin (Bg7S), a XEGIP from soybean, which represents the first three-dimensional structure of XEGIP. Bg7S formed a tetramer with pseudo-222 symmetry. Analytical centrifugation and size exclusion chromatography experiments revealed that the assembly of Bg7S in solution depended on pH. The structure of Bg7S was similar to that of a xylanase inhibitor protein from wheat (Tritinum aestivum xylanase inhibitor) that inhibits GH11 xylanase. Surprisingly, Bg7S lacked inhibitory activity against not only GH11 but also GH12 enzymes. In addition, we found that XEGIPs from azukibean, yardlongbean and mungbean also had no impact on the activity of either GH12 or GH11 enzymes, indicating that legume XEGIPs generally do not inhibit these enzymes. We reveal the structural basis of why legume XEGIPs lack this inhibitory activity. This study will provide significant clues for understanding the physiological role of Bg7S. Database Coordinates and structure factors have been deposited in the Protein Data Bank Japan (PDBj) (http://www.pdbj.org/) under the accession number 3AUP. Structured digital abstract • Bg7S binds to Bg7S by x-ray crystallography (View interaction) • Bg7S binds to Bg7S by cosedimentation in solution (View Interaction 1, 2) • Bg7S binds to Bg7S by molecular sieving (View Interaction 1, 2)
- Published
- 2011
31. Relationship between heat-induced fibrillogenicity and hemolytic activity of thermostable direct hemolysin and a related hemolysin of Vibrio parahaemolyticus
- Author
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Kentaro Shiraki, Kumiko Nakahira, Takeshi Honda, Kouta Mayanagi, Hiroshi Hashimoto, Itaru Yanagihara, Kiyouhisa Ohnishi, and Satoru Unzai
- Subjects
chemistry.chemical_classification ,Circular dichroism ,Vibrio parahaemolyticus ,food and beverages ,Virulence ,Hemolysin ,Biology ,biology.organism_classification ,Microbiology ,Molecular biology ,Amino acid ,chemistry.chemical_compound ,Tetramer ,Biochemistry ,chemistry ,Vibrionaceae ,Genetics ,Thioflavin ,Molecular Biology - Abstract
The formation of nonspecific ion channels by small oligomeric amyloid intermediates is toxic to the host's cellular membranes. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are major virulence factors of Vibrio parahaemolyticus. We have previously reported the crystal structure of TDH tetramer with the central channel. We have also identified the molecular mechanism underlying the paradoxical responses to heat treatment of TDH, known as the Arrhenius effect, which is the reversible amyloidogenic property. In the present report, we describe the biophysical properties of TRH, which displays 67% amino acid similarity with TDH. Molecular modeling provided a good fit of the overall structure of TDH and TRH. Size-exclusion chromatography, ultracentrifugation, and transmission electron microscopy revealed that TRH formed tetramer in solution. These toxins showed similar hemolytic activity on red blood cells. However, TRH had less amyloid-like structure than TDH analyzed by thioflavin T-binding assay and far-UV circular dichroism spectra. These data indicated that amyloidogenicity upon heating is not essential for the membrane disruption of erythrocytes, but the maintenance of tetrameric structure is indispensable for the hemolytic activity of the TDH and TRH.
- Published
- 2011
32. Structural basis for the dual RNA-recognition modes of human Tra2-β RRM
- Author
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Kanako Kuwasako, Peter Güntert, Kengo Tsuda, Takushi Harada, M. Takahashi, Takaho Terada, Takanori Kigawa, Satoru Unzai, Yutaka Muto, Makoto Inoue, Naohiro Kobayashi, Shigeyuki Yokoyama, Satoru Watanabe, Fahu He, Akiko Tanaka, Tatsuhiko Someya, Sumio Sugano, and Mikako Shirouzu
- Subjects
Models, Molecular ,Guanine ,Protein family ,Amino Acid Motifs ,Molecular Sequence Data ,Nerve Tissue Proteins ,RNA-binding protein ,Biology ,chemistry.chemical_compound ,Structural Biology ,Genetics ,Aromatic amino acids ,Humans ,Amino Acid Sequence ,Nuclear Magnetic Resonance, Biomolecular ,Peptide sequence ,chemistry.chemical_classification ,Base Sequence ,Serine-Arginine Splicing Factors ,RNA recognition motif ,fungi ,Alternative splicing ,RNA-Binding Proteins ,RNA ,Amino acid ,Biochemistry ,chemistry ,Protein Binding - Abstract
Human Transformer2-β (hTra2-β) 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-β specifically binds to two types of RNA sequences [the CAA and (GAA)(2) sequences]. We determined the solution structure of the hTra2-β 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 β-sheet surface. We then solved the complex structure of the hTra2-β 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 β-sheet surface. Further NMR experiments revealed that the hTra2-β RRM recognizes the CAA sequence when it is integrated in the stem-loop structure. This study indicates that the hTra2-β RRM recognizes two types of RNA sequences in different RNA binding modes.
- Published
- 2010
33. Purification, crystallization and initial X-ray diffraction study of human REV7 in complex with a REV3 fragment
- Author
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Mamoru Sato, Satoko Akashi, Hiroshi Hashimoto, Toshiyuki Shimizu, Kodai Hara, and Satoru Unzai
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DNA polymerase ,Protein subunit ,Biophysics ,DNA-Directed DNA Polymerase ,Crystallography, X-Ray ,Biochemistry ,DNA-binding protein ,law.invention ,Structural Biology ,law ,Genetics ,Humans ,Protein Interaction Domains and Motifs ,Crystallization ,biology ,DNA synthesis ,DNA replication ,Proteins ,Space group ,Condensed Matter Physics ,Peptide Fragments ,Recombinant Proteins ,DNA-Binding Proteins ,Crystallography ,Amino Acid Substitution ,Crystallization Communications ,Multiprotein Complexes ,Mad2 Proteins ,Mutagenesis, Site-Directed ,Recombinant DNA ,biology.protein - Abstract
REV7 is involved in various cellular functions including DNA replication, signal transduction and cell-cycle regulation. In DNA replication, REV7 interacts with REV3 and forms DNA polymerase zeta, which plays a central role in error-prone DNA synthesis. REV3 is a catalytic subunit and its activity is stimulated by REV7. To clarify the structural basis of the interaction between REV7 and REV3, human REV7 was crystallized in complex with a REV3 fragment. Two crystal forms were obtained. Crystal forms I and II belonged to space groups P2(1), with unit-cell parameters a = 43.8, b = 50.0, c = 107.3 A, beta = 96.9 degrees , and P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 76.6, c = 118.4 A, respectively.
- Published
- 2009
34. The nature of the TRAP–Anti-TRAP complex
- Author
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Sam-Yong Park, Satoko Akashi, Jonathan G. Heddle, Jeremy R. H. Tame, Ken-ichi Kikuchi, Masahiro Watanabe, and Satoru Unzai
- Subjects
Models, Molecular ,Spectrometry, Mass, Electrospray Ionization ,Protein Conformation ,Population ,Molecular Conformation ,Bacillus ,Plasma protein binding ,Crystallography, X-Ray ,Mass spectrometry ,Models, Biological ,Mass Spectrometry ,trp operon ,Trap (computing) ,Protein structure ,Bacterial Proteins ,Binding site ,education ,education.field_of_study ,Binding Sites ,Multidisciplinary ,Chemistry ,Tryptophan ,RNA-Binding Proteins ,RNA ,Hydrogen Bonding ,Gene Expression Regulation, Bacterial ,Biological Sciences ,Crystallography ,Solvents ,Biophysics ,Protein Binding ,Transcription Factors - Abstract
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-Å 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.
- Published
- 2009
35. Cloning, expression and purification of cytochromec6from the brown algaHizikia fusiformisand complete X-ray diffraction analysis of the structure
- Author
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Sam-Yong Park, Toshiyuki Nishio, Yuichirou Matsumoto, Satoru Unzai, Mao Hirayama, Ken Hoshikawa, Hirotaka Chida, Hideharu Akazaki, Fumihiro Kawai, Tadatake Oku, and Wataru Hakamata
- Subjects
Protein Conformation ,Stereochemistry ,Biophysics ,Crystal structure ,Crystallography, X-Ray ,Phaeophyta ,Biochemistry ,Cytochromes c6 ,Structural Biology ,Genetics ,Protein Structure Communications ,Cloning, Molecular ,DNA Primers ,Cloning ,Base Sequence ,Endosymbiosis ,biology ,Cytochrome c ,fungi ,Condensed Matter Physics ,biology.organism_classification ,Electron transport chain ,Chloroplast ,Brown algae ,Crystallography ,biology.protein ,Eukaryote - Abstract
The primary sequence of cytochrome c(6) from the brown alga Hizikia fusiformis has been determined by cDNA cloning and the crystal structure has been solved at 1.6 A resolution. The crystal belonged to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 84.58, c = 232.91 A and six molecules per asymmetric unit. The genome code, amino-acid sequence and crystal structure of H. fusiformis cytochrome c(6) were most similar to those of red algal cytochrome c(6). These results support the hypothesis that brown algae acquired their chloroplasts via secondary endosymbiosis involving a red algal endosymbiont and a eukaryote host.
- Published
- 2008
36. Interaction and Stoichiometry of the Peripheral Stalk Subunits NtpE and NtpF and the N-terminal Hydrophilic Domain of NtpI of Enterococcus hirae V-ATPase
- Author
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Takuya Kobayashi, Shinya Saijo, Yukako Yabuki-Miyata, So Iwata, Ichiro Yamato, Satoru Unzai, Chiyo Suno-Ikeda, Mitsutoshi Toyama, Misaki Yamamoto, Kazuki Ito, Takaho Terada, Yoshimi Kakinuma, Mikako Shirouzu, Takeshi Murata, Shigeyuki Yokoyama, and Kenji Mizutani
- Subjects
Vacuolar Proton-Translocating ATPases ,Circular dichroism ,biology ,Chemistry ,Protein subunit ,Cell Biology ,Plasma protein binding ,biology.organism_classification ,Biochemistry ,Protein Structure, Tertiary ,Protein Subunits ,Crystallography ,Bacterial Proteins ,Stalk ,Enterococcus hirae ,Biophysics ,V-ATPase ,Surface plasmon resonance ,Protein Structure, Quaternary ,Hydrophobic and Hydrophilic Interactions ,Molecular Biology ,Ternary complex ,Enterococcus ,Protein Binding - Abstract
The vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain and an integral membrane domain connected by a central stalk and a few peripheral stalks. The number and arrangement of the peripheral stalk subunits remain controversial. The peripheral stalk of Na+-translocating V-ATPase from Enterococcus hirae is likely to be composed of NtpE and NtpF (corresponding to subunit G of eukaryotic V-ATPase) subunits together with the N-terminal hydrophilic domain of NtpI (corresponding to subunit a of eukaryotic V-ATPase). Here we purified NtpE, NtpF, and the N-terminal hydrophilic domain of NtpI (NtpI(Nterm)) as separate recombinant His-tagged proteins and examined interactions between these three subunits by pulldown assay using one tagged subunit, CD spectroscopy, surface plasmon resonance, and analytical ultracentrifugation. NtpI(Nterm) directly bound NtpF, but not NtpE. NtpE bound NtpF tightly. NtpI(Nterm) bound the NtpE-F complex stronger than NtpF only, suggesting that NtpE increases the binding affinity between NtpI(Nterm) and NtpF. Purified NtpE-F-I(Nterm) complex appeared to be monodisperse, and the molecular masses estimated from analytical ultracentrifugation and small-angle x-ray scattering (SAXS) indicated that the ternary complex is formed with a 1:1:1 stoichiometry. A low resolution structure model of the complex produced from the SAXS data showed an elongated "L" shape.
- Published
- 2008
37. Self-Assembling Nano-Architectures Created from a Protein Nano-Building Block Using an Intermolecularly Folded Dimeric de Novo Protein
- Author
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Satoru Unzai, Keiichi Yanase, Naoya Kobayashi, Ryoichi Arai, Takaaki Sato, and Michael H. Hecht
- Subjects
Chemistry ,Small-angle X-ray scattering ,Size-exclusion chromatography ,Protein Data Bank (RCSB PDB) ,Multiangle light scattering ,Supramolecular chemistry ,General Chemistry ,medicine.disease_cause ,Biochemistry ,Fusion protein ,Catalysis ,Synthetic biology ,Crystallography ,Colloid and Surface Chemistry ,medicine ,Escherichia coli - Abstract
The design of novel proteins that self-assemble into supramolecular complexes is an important step in the development of synthetic biology and nanotechnology. Recently, we described the three-dimensional structure of WA20, a de novo protein that forms an intermolecularly folded dimeric 4-helix bundle (PDB code 3VJF ). To harness the unusual intertwined structure of WA20 for the self-assembly of supramolecular nanostructures, we created a protein nanobuilding block (PN-Block), called WA20-foldon, by fusing the dimeric structure of WA20 to the trimeric foldon domain of fibritin from bacteriophage T4. The WA20-foldon fusion protein was expressed in the soluble fraction in Escherichia coli, purified, and shown to form several homooligomeric forms. The stable oligomeric forms were further purified and characterized by a range of biophysical techniques. Size exclusion chromatography, multiangle light scattering, analytical ultracentrifugation, and small-angle X-ray scattering (SAXS) analyses indicate that the small (S form), middle (M form), and large (L form) forms of the WA20-foldon oligomers exist as hexamer (6-mer), dodecamer (12-mer), and octadecamer (18-mer), respectively. These findings suggest that the oligomers in multiples of 6-mer are stably formed by fusing the interdigitated dimer of WA20 with the trimer of foldon domain. Pair-distance distribution functions obtained from the Fourier inversion of the SAXS data suggest that the S and M forms have barrel- and tetrahedron-like shapes, respectively. These results demonstrate that the de novo WA20-foldon is an effective building block for the creation of self-assembling artificial nanoarchitectures.
- Published
- 2015
38. A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation
- Author
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Tomas Obsil, Helmut Cölfen, Anna Modrak-Wójcik, José García de la Torre, Caitlin I. Stoddard, Ute Curth, Thomas M. Laue, Geeta J. Narlikar, Szu Huan Wang, Carlos Alfonso, Carlos R. Escalante, Jia Ma, Gali Prag, Masanori Noda, Katherine Stott, Klaus Richter, Carrie A. May, Peter Schuck, Fumio Arisaka, Edward Eisenstein, Stephen J. Perkins, Ilan Attali, John P. Sumida, Catherine T. Chaton, Erby E. Perdue, Davis Jose, David Staunton, Matthew A. Perugini, Jin Ku Park, Amanda Nourse, Peter E. Prevelige, Craig L. Peterson, Norbert Mücke, Satoru Unzai, Peter D. Pawelek, Jayesh Gor, Arne C. Rufer, Rose Rosenberg, Hauke Lilie, Martin G. Peverelli, Yee-Foong Mok, Martin Wolff, Jacinta M. Wubben, Luitgard Nagel-Steger, Peter H. von Hippel, Karolin Luger, Edward Wright, Holger M. Strauss, Lenka Rezabkova, Agnieszka Bzowska, Aline Le Roy, Steven E. Weitzel, Jonathan B. Chaires, Elena Krayukhina, Donald F. Becker, Alexandra S. Solovyova, Alison E. Ringel, Chad A. Brautigam, Dalibor Kosek, David J. Scott, Kimberly A. Crowley, Cynthia Wolberger, Soon-Jong Kim, Adam G. Larson, Leslie E. Eisele, Susumu Uchiyama, Gregory J. Bedwell, Javier Seravalli, Bertrand Raynal, Ron M. Finn, Hyewon Kwon, Tabot M. D. Besong, Marina Bakhtina, Ahmet Bekdemir, Shu-Chuan Jao, Bashkim Kokona, Catherine Birck, Roman H. Szczepanowski, Andrew B. Herr, Christine Wandrey, Stephen E. Harding, Daniel D Krzizike, David L. Bain, José G. Hernández Cifre, Grzegorz Piszczek, Renjie Song, Stephan Uebel, Tina Daviter, Damien Hall, Debra M. Eckert, Patrick England, Ingrid Tessmer, Eric Kusznir, Ernest L. Maynard, William L. Dean, Werner Streicher, Christine Ebel, Anna Vitlin Gruber, Beata Wielgus-Kutrowska, Rodolfo Ghirlando, Yu Sung Wu, Ana I. Díez, Olwyn Byron, Robert Fairman, William Brennerman, Henning Gustafsson, Richard Stefan Isaac, Wolfgang Fischle, Elizabeth E. Howell, Ronald T. Toth, Sarah G. Swygert, Ashutosh Tripathy, Arthur J. Rowe, Chad K. Park, Jeffrey A. Fagan, Keith D. Connaghan, Huaying Zhao, Jack A. Kornblatt, Andrew P. Leech, Juan Román Luque-Ortega, BGI Shenzhen, Laboratoire d'Informatique pour l'Entreprise et les Systèmes de Production (LIESP), Université Lumière - Lyon 2 (UL2)-École Centrale de Lyon (ECL), Université de Lyon-Université de Lyon-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA), Innovative Computing Laboratory [Knoxville] (ICL), The University of Tennessee [Knoxville], Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), EDF R&D (EDF R&D), EDF (EDF), Physikalische Chemie, Universität Konstanz, Photonic Crystal Fibres group, University of Bath [Bath], Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Observatoire Astronomique de l'Université de Genève (ObsGE), Université de Genève (UNIGE), Biophysique des macromolécules et leurs interactions, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Laboratory of Chromatin Biochemistry [Göttingen], Max Planck Institute for Biophysical Chemistry (MPI-BPC), Max-Planck-Gesellschaft-Max-Planck-Gesellschaft, Stellenbosch University, Department of Biochemistry, The Centre for Ornithology, School of Biosciences, College of Life and Environmental Sciences, University of Alabama at Birmingham [ Birmingham] (UAB), Département de Chimie Moléculaire - Chimie Inorganique Redox Biomimétique (DCM - CIRE), Université Joseph Fourier - Grenoble 1 (UJF)-Institut de Chimie Moléculaire de Grenoble (ICMG)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Hormone Research Center, Chonnam National University, Anglophonie : Communautés, Ecritures (ACE ), Université de Rennes 2 (UR2), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES), Institut de Chimie des Milieux et Matériaux de Poitiers (IC2MP), Université de Poitiers-Centre National de la Recherche Scientifique (CNRS), Department of Animal Science, Iowa State University (ISU), Université Laval, School of Mathematics [Bristol], University of Bristol [Bristol], Chimie et Biocatalyse, Laboratoire de Physique des Solides (LPS), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Department of marine Ecology, University of Gothenburg (GU), Laboratoire de physique de la matière condensée (LPMC), École polytechnique (X)-Centre National de la Recherche Scientifique (CNRS), Pharma Research Discovery Chemistry, F. Hoffmann-La Roche AG, University of KwaZulu-Natal (UKZN), Institute for Cell and Molecular Biosciences, Newcastle University [Newcastle], Changzhou University, Institut für Geologie und Paläontologie, Westfälische Wilhelms-Universität Münster (WWU), Eindhoven University of Technology [Eindhoven] (TU/e), Department of Biochemistry and Biophysics, UNC Macromolecular Interactions Facility, University of North Carolina [Chapel Hill] (UNC), University of North Carolina System (UNC)-University of North Carolina System (UNC), Institute of Botany, University of Innsbruck, NSF Center for EUV Science and Technology, NSF, Sustainable Energy Economics, Department of mathematics and MOE-LSC, Shanghai Jiao Tong University [Shanghai], Science et Technologie du Lait et de l'Oeuf (STLO), Institut National de la Recherche Agronomique (INRA)-AGROCAMPUS OUEST, Platforms of the Grenoble Instruct centre (ISBG, CNRS-CEA-UJF-EMBL), ANR-10-INBS-0005-02,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-10- LABX-49-01,Labex GRAL,Labex GRAL, Beijing Genomics Institute [Shenzhen] (BGI), Institut National des Sciences Appliquées de Lyon (INSA Lyon), Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université Lumière - Lyon 2 (UL2)-École Centrale de Lyon (ECL), Université de Lyon, Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Département de Chimie Moléculaire (DCM), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Joseph Fourier - Grenoble 1 (UJF), Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Poitiers-Institut de Chimie du CNRS (INC), Université Laval [Québec] (ULaval), Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), Stott, Katherine [0000-0002-4014-1188], Apollo - University of Cambridge Repository, Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Université de Genève = University of Geneva (UNIGE), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Département de Chimie Moléculaire - Chimie Inorganique Redox (DCM - CIRE), Université Joseph Fourier - Grenoble 1 (UJF)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Université de Poitiers-Institut national des sciences de l'Univers (INSU - CNRS)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), F. Hoffmann-La Roche [Basel], University of KwaZulu-Natal [Durban, Afrique du Sud] (UKZN), Westfälische Wilhelms-Universität Münster = University of Münster (WWU), and Leopold Franzens Universität Innsbruck - University of Innsbruck
- Subjects
Accuracy and precision ,Analytical chemistry ,lcsh:Medicine ,Magnification ,02 engineering and technology ,bcs ,Standard deviation ,Analytical Ultracentrifugation ,03 medical and health sciences ,Range (statistics) ,Calibration ,ddc:610 ,lcsh:Science ,030304 developmental biology ,Mathematics ,0303 health sciences ,Field flow fractionation ,Multidisciplinary ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,lcsh:R ,Reproducibility of Results ,021001 nanoscience & nanotechnology ,3. Good health ,ddc ,Data set ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM] ,ddc:540 ,lcsh:Q ,0210 nano-technology ,Ultracentrifugation ,Research Article - Abstract
30 p.-14 fig. Zhao, Huaying, et alt., Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.
- Published
- 2015
39. Fine-tuning of protein domain boundary by minimizing potential coiled coil regions
- Author
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Natsuko Goda, Kentaro Tomii, Masahiro Shirakawa, Naoko Iwaya, Kenichiro Fujiwara, Satoru Unzai, Hidehito Tochio, Hidekazu Hiroaki, and Toshiki Tanaka
- Subjects
Adenosine Triphosphatases ,Coiled coil ,Chemistry ,Circular Dichroism ,Molecular Sequence Data ,Protein domain ,Sequence alignment ,Biochemistry ,Protein Structure, Secondary ,Protein Structure, Tertiary ,Structural genomics ,Domain (software engineering) ,Mice ,Crystallography ,Spectrometry, Fluorescence ,Protein structure ,Biophysics ,Animals ,Humans ,Amino Acid Sequence ,Nuclear Magnetic Resonance, Biomolecular ,Sequence Alignment ,Peptide sequence ,Spectroscopy ,Heteronuclear single quantum coherence spectroscopy - Abstract
Structural determination of individual protein domains isolated from multidomain proteins is a common approach in the post-genomic era. Novel and thus uncharacterized domains liberated from intact proteins often self-associate due to incorrectly defined domain boundaries. Self-association results in missing signals, poor signal dispersion and a low signal-to-noise ratio in (1)H-(15)N HSQC spectra. We have found that a putative, non-canonical coiled coil region close to a domain boundary can cause transient hydrophobic self-association and monomer-dimer equilibrium in solution. Here we propose a rational method to predict putative coiled coil regions adjacent to the globular core domain using the program COILS. Except for the amino acid sequence, no preexisting knowledge concerning the domain is required. A small number of mutant proteins with a minimized coiled coil region have been rationally designed and tested. The engineered domains exhibit decreased self-association as assessed by (1)H-(15)N HSQC spectra with improved peak dispersion and sharper cross peaks. Two successful examples of isolating novel N-terminal domains from AAA-ATPases are demonstrated. Our method is useful for the experimental determination of domain boundaries suited for structural genomics studies.
- Published
- 2006
40. The Swi5–Sfr1 complex stimulates Rhp51/Rad51 - and Dmc1-mediated DNA strand exchange in vitro
- Author
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Yasuto Murayama, Satoru Unzai, Hiroshi Iwasaki, Nami Haruta, Yufuko Akamatsu, Yumiko Kurokawa, and Yasuhiro Tsutsui
- Subjects
RAD51 ,Swi5-Sfr1 complex ,Biology ,biology.organism_classification ,Molecular biology ,Nucleoprotein ,Cell biology ,DNA-Binding Proteins ,Recombinases ,enzymes and coenzymes (carbohydrates) ,chemistry.chemical_compound ,chemistry ,Structural Biology ,Schizosaccharomyces ,Schizosaccharomyces pombe ,Recombinase ,Nucleic Acid Conformation ,DMC1 ,Rad51 Recombinase ,Schizosaccharomyces pombe Proteins ,DNA, Fungal ,Molecular Biology ,Replication protein A ,DNA ,Protein Binding - Abstract
Nucleoprotein filaments made up of Rad51 or Dmc1 recombinases, the core structures of recombination, engage in ATP-dependent DNA-strand exchange. The ability of recombinases to form filaments is enhanced by recombination factors termed 'mediators'. Here, we show that the Schizosaccharomyces pombe Swi5-Sfr1 complex, a conserved eukaryotic protein complex, at substoichiometric concentrations stimulates strand exchange mediated by Rhp51 (the S. pombe Rad51 homolog) and Dmc1 on long DNA substrates. Reactions mediated by both recombinases are completely dependent on Swi5-Sfr1, replication protein A (RPA) and ATP, although RPA inhibits the reaction when it is incubated with single-stranded DNA (ssDNA) before the recombinase. The Swi5-Sfr1 complex overcomes, at least partly, the inhibitory effect of RPA, representing a novel class of mediator. Notably, the Swi5-Sfr1 complex preferentially stimulates the ssDNA-dependent ATPase activity of Rhp51, and it increases the amounts of Dmc1 bound to ssDNA.
- Published
- 2006
41. Crystal Structure of Penicillin Binding Protein 4 (dacB) from Escherichia coli, both in the Native Form and Covalently Linked to Various Antibiotics
- Author
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Sam-Yong Park, Satoru Unzai, David I. Roper, H. Kishida, Adrian J. Lloyd, and Jeremy R. H. Tame
- Subjects
Models, Molecular ,Penicillin binding proteins ,biology ,Chemistry ,Escherichia coli Proteins ,Molecular Sequence Data ,Active site ,Crystallography, X-Ray ,Serine-Type D-Ala-D-Ala Carboxypeptidase ,medicine.disease_cause ,Biochemistry ,Anti-Bacterial Agents ,Cell wall ,Hydrolase ,Escherichia coli ,biology.protein ,medicine ,Penicillin-Binding Proteins ,Penicillin binding ,Amino Acid Sequence ,Peptide sequence - Abstract
The crystal structure of penicillin binding protein 4 (PBP4) from Escherichia coli, which has both DD-endopeptidase and DD-carboxypeptidase activity, is presented. PBP4 is one of 12 penicillin binding proteins in E. coli involved in the synthesis and maintenance of the cell wall. The model contains a penicillin binding domain similar to known structures, but includes a large insertion which folds into domains with unique folds. The structures of the protein covalently attached to five different antibiotics presented here show the active site residues are unmoved compared to the apoprotein, but nearby surface loops and helices are displaced in some cases. The altered geometry of conserved active site residues compared with those of other PBPs suggests a possible cause for the slow deacylation rate of PBP4.
- Published
- 2005
42. Investigation of molecular size of transcription factor TFIIE in solution
- Author
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Satoko Akashi, Satoru Unzai, Yoshiyuki Itoh, Mamoru Sato, Aritaka Nagadoi, Masahiko Okuda, and Yoshifumi Nishimura
- Subjects
Spectrometry, Mass, Electrospray Ionization ,Recombinant Fusion Proteins ,Electrospray ionization ,Amino Acid Motifs ,Molecular Sequence Data ,RNA polymerase II ,Biochemistry ,Transcription Factors, TFII ,Structural Biology ,Transcription (biology) ,Humans ,Scattering, Radiation ,Molecular Biology ,Chromatography ,biology ,Molecular mass ,General transcription factor ,Chemistry ,Heterotetramer ,Molecular Weight ,Solutions ,Transcription preinitiation complex ,Chromatography, Gel ,biology.protein ,Biophysics ,RNA Polymerase II ,Transcription factor II E ,Ultracentrifugation - Abstract
Human general transcription factor IIE (TFIIE), a component of a transcription preinitiation complex associated with RNA polymerase II, was characterized by size-exclusion chromatography, mass spectrometry, analytical ultracentrifugation, and small-angle X-ray scattering (SAXS). Recombinant human TFIIE was purified to homogeneity and shown to contain equimolar amounts of TFIIEα (50 kDa) and TFIIEβ (35 kDa) by SDS-PAGE. In the analysis of size-exclusion chromatography of the purified sample, as already reported, TFIIE was shown to be a 170-kDa α2β2 heterotetramer. However, by using electrospray ionization mass spectrometry the purified sample gave the molecular mass of 84,152 ± 5, indicating that TFIIE is an αβ heterodimer but not a heterotetramer. Analytical ultracentrifugation experiment of TFIIE provided that only a single component with the molecular mass of ca. 80,000 existed in solution, also suggesting an αβ heterodimer. In addition, its extraordinarily rod-like molecular shape was confirmed by SAXS. It is likely that the rod-like molecular shape of TFIIE has misled larger molecular size in size-exclusion chromatography, which was calibrated by globular proteins. It is demonstrated that TFIIE exists as a heterodimer under our present conditions in solution, although two molecules of heterodimer might be required for the formation of the preinitiation complex with RNA polymerase II for starting the transcription process. Proteins 2005. © 2005 Wiley-Liss, Inc.
- Published
- 2005
43. Novel Mechanisms of pH Sensitivity in Tuna Hemoglobin
- Author
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Daniel T.-b. Shih, Khoon Tee Chong, Satoru Unzai, Gentaro Miyazaki, Takeshi Yokoyama, Jeremy R. H. Tame, Sam-Yong Park, and Hideki Morimoto
- Subjects
Chemistry ,Mutagenesis ,Root effect ,Hemoglobin variants ,Cell Biology ,Ligand (biochemistry) ,Biochemistry ,chemistry.chemical_compound ,Hemoglobin A ,Hemoglobin ,Molecular Biology ,Peptide sequence ,Carbon monoxide - Abstract
The crystal structure of hemoglobin has been known for several decades, yet various features of the molecule remain unexplained or controversial. Several animal hemoglobins have properties that cannot be readily explained in terms of their amino acid sequence and known atomic models of hemoglobin. Among these, fish hemoglobins are well known for their widely varying interactions with heterotropic effector molecules and pH sensitivity. Some fish hemoglobins are almost completely insensitive to pH (within physiological limits), whereas others show extremely low oxygen affinity under acid conditions, a phenomenon called the Root effect. X-ray crystal structures of Root effect hemoglobins have not, to date, provided convincing explanations of this effect. Sequence alignments have signally failed to pinpoint the residues involved, and site-directed mutagenesis has not yielded a human hemoglobin variant with this property. We have solved the crystal structure of tuna hemoglobin in the deoxy form at low and moderate pH and in the presence of carbon monoxide at high pH. A comparison of these models shows clear evidence for novel mechanisms of pH-dependent control of ligand affinity.
- Published
- 2004
44. Crystal Structures of the Catalytic Domains of Pseudouridine Synthases RluC and RluD from Escherichia coli
- Author
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Satoru Unzai, Jeremy R. H. Tame, Kenji Mizutani, Yoshitaka Machida, and Sam-Yong Park
- Subjects
Models, Molecular ,Molecular Sequence Data ,Static Electricity ,Biology ,Crystallography, X-Ray ,medicine.disease_cause ,Biochemistry ,Pseudouridine ,chemistry.chemical_compound ,Catalytic Domain ,medicine ,Amino Acid Sequence ,Escherichia coli ,Hydro-Lyases ,Sequence Homology, Amino Acid ,Escherichia coli Proteins ,RNA-Binding Proteins ,RNA ,Ribosomal RNA ,Lyase ,Peptide Fragments ,Uridine ,chemistry ,Transfer RNA ,Mutagenesis, Site-Directed ,Crystallization ,Sequence Alignment ,Binding domain - Abstract
The most frequent modification of RNA, the conversion of uridine bases to pseudouridines, is found in all living organisms and often in highly conserved locations in ribosomal and transfer RNA. RluC and RluD are homologous enzymes which each convert three specific uridine bases in Escherichia coli ribosomal 23S RNA to pseudouridine: bases 955, 2504, and 2580 in the case of RluC and 1911, 1915, and 1917 in the case of RluD. Both have an N-terminal S4 RNA binding domain. While the loss of RluC has little phenotypic effect, loss of RluD results in a much reduced growth rate. We have determined the crystal structures of the catalytic domain of RluC, and full-length RluD. The S4 domain of RluD appears to be highly flexible or unfolded and is completely invisible in the electron density map. Despite the conserved topology shared by the two proteins, the surface shape and charge distribution are very different. The models suggest significant differences in substrate binding by different pseudouridine synthases.
- Published
- 2004
45. Structure and catalytic mechanism of 2-C-methyl-<scp>D</scp>-erythritol 2,4-cyclodiphosphate (MECDP) synthase, an enzyme in the non-mevalonate pathway of isoprenoid synthesis
- Author
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Takaho Terada, Tomohisa Kuzuyama, Mikako Shirouzu, Jeremy R. H. Tame, Motoki Takagi, H. Kishida, Takashi Wada, Satoru Unzai, Shigeyuki Yokoyama, and Sam-Yong Park
- Subjects
Models, Molecular ,Protein Conformation ,Stereochemistry ,Molecular Sequence Data ,Mevalonic Acid ,Crystallography, X-Ray ,Catalysis ,Substrate Specificity ,Bacterial Proteins ,Polyisoprenyl Phosphates ,Structural Biology ,Cytidine Monophosphate ,Magnesium ,Amino Acid Sequence ,Non-mevalonate pathway ,Magnesium ion ,chemistry.chemical_classification ,Binding Sites ,Sequence Homology, Amino Acid ,biology ,Escherichia coli Proteins ,Lysine ,Thermus thermophilus ,Active site ,Substrate (chemistry) ,General Medicine ,biology.organism_classification ,Lyase ,Recombinant Proteins ,Enzyme ,chemistry ,Biochemistry ,biology.protein ,Mevalonate pathway ,Phosphorus-Oxygen Lyases - Abstract
Precursors for isoprenoid synthesis are essential in all organisms. These compounds are synthesized by one of two known routes: the well characterized mevalonate pathway or a recently discovered non-mevalonate route which is used in many bacteria and human pathogens. Since the second pathway is both vital and unlike any found in humans, enzymes catalysing reactions along this synthetic route are possible drug targets. The structure of one such enzyme from the thermophilic bacterium Thermus thermophilus has been solved to high resolution in the presence of substrate and with a substrate analogue. Enzyme co-crystallized with substrate shows only one product, cytosine monophosphate (CMP), in the active site. At the high resolution of the refinement (1.6 A) the positions and coordination of the magnesium ions in the active site are clearly seen.
- Published
- 2002
46. Decreased amyloidogenicity caused by mutational modulation of surface properties of the immunoglobulin light chain BRE variable domain
- Author
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Hidekazu Hiroaki, Yuta Kobayashi, Hironari Kamikubo, Tetsuyuki Abe, Hirotaka Tsutsumi, Daizo Hamada, Satoru Unzai, Yuki Tashiro, Mikio Kataoka, and Kyohei Ikeda
- Subjects
Models, Molecular ,Amyloid ,Protein Conformation ,Surface Properties ,Molecular Sequence Data ,Immunoglobulin Variable Region ,Immunoglobulin light chain ,medicine.disease_cause ,Biochemistry ,Protein structure ,medicine ,Native state ,Humans ,Amino Acid Sequence ,Peptide sequence ,Mutation ,Sequence Homology, Amino Acid ,Chemistry ,Protein Stability ,Amyloidosis ,Point mutation ,medicine.disease ,Recombinant Proteins ,Spectrometry, Fluorescence ,Amino Acid Substitution ,Biophysics ,Mutagenesis, Site-Directed ,Immunoglobulin Light Chains ,Mutant Proteins - Abstract
Amyloid formation by immunoglobulin light chain (LC) proteins is associated with amyloid light chain (AL) amyloidosis. Destabilization of the native state of the variable domain of the LC (VL) is known to be one of the critical factors in promoting the formation of amyloid fibrils. However, determining the key residues involved in this destabilization remains challenging, because of the existence of a number of intrinsic sequence variations within VL. In this study, we identified the key residues for destabilization of the native state of amyloidogenic VL in the LC of BRE by analyzing the stability of chimeric mutants of BRE and REI VL; the latter immunoglobulin is not associated with AL amyloidosis. The results suggest that the surface-exposed residues N45 and D50 are the key residues in the destabilization of the native state of BRE VL. Point mutations at the corresponding residues in REI VL (K45N, E50D, and K45N/E50D) destabilized the native state and increased amyloidogenicity. However, the reverse mutations in BRE VL (N45K, D50E, and N45K/D50E) re-established the native state and decreased amyloidogenicity. Thus, analyses using chimeras and point mutants successfully elucidated the key residues involved in BRE VL destabilization and increased amyloidogenic propensity. These results also suggest that the modulation of surface properties of wild-type VL may improve their stability and prevent the formation of amyloid fibrils.
- Published
- 2014
47. RBFOX and SUP-12 sandwich a G base to cooperatively regulate tissue-specific splicing
- Author
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Kanako Kuwasako, M. Takahashi, Fahu He, Peter Güntert, Shigeyuki Yokoyama, Naohiro Kobayashi, Takuhiro Ito, Seiko Yoshikawa, Kengo Tsuda, Satoru Unzai, Akiko Tanaka, Masatoshi Hagiwara, Yutaka Muto, Hidehito Kuroyanagi, and Mikako Shirouzu
- Subjects
musculoskeletal diseases ,Models, Molecular ,Base Sequence ,Exonic splicing enhancer ,RNA ,RNA-Binding Proteins ,Biology ,Non-coding RNA ,Crystallography, X-Ray ,Molecular biology ,Cell biology ,Protein Structure, Tertiary ,Alternative Splicing ,Structural Biology ,Fibroblast growth factor receptor ,RNA splicing ,Animals ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Molecular Biology ,Tissue-Specific Splicing - Abstract
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.
- Published
- 2014
48. MS71
- Author
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Roderick E. HUBBARD, Yoshiyuki KAWAKAMI, Satoko AKASHI, Masahiro WATANABE, Jonathan G. HEDDLE, Satoru UNZAI, Sam-Yong PARK, and Jeremy R. H. TAME
- Published
- 2009
49. Crystal structure of the ubiquitin-associated (UBA) domain of p62 and its interaction with ubiquitin
- Author
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Masaaki Komatsu, Yu-shin Sou, Satoru Unzai, Hidehito Tochio, Kyouhei Arita, Shin Isogai, Masahiro Shirakawa, Daichi Morimoto, Keiji Tanaka, Jun Hasegawa, and Takeshi Tenno
- Subjects
Sequestosome-1 Protein ,Magnetic Resonance Spectroscopy ,Ubiquitin binding ,Plasma protein binding ,Protein degradation ,Crystallography, X-Ray ,Biochemistry ,Phase Transition ,Mice ,Protein structure ,Ubiquitin ,Heat shock protein ,Animals ,Protein Interaction Domains and Motifs ,Molecular Biology ,Heat-Shock Proteins ,Adaptor Proteins, Signal Transducing ,biology ,Chemistry ,Signal transducing adaptor protein ,Cell Biology ,Protein Structure and Folding ,Biophysics ,biology.protein ,Protein Multimerization - Abstract
p62/SQSTM1/A170 is a multimodular protein that is found in ubiquitin-positive inclusions associated with neurodegenerative diseases. Recent findings indicate that p62 mediates the interaction between ubiquitinated proteins and autophagosomes, leading these proteins to be degraded via the autophagy-lysosomal pathway. This ubiquitin-mediated selective autophagy is thought to begin with recognition of the ubiquitinated proteins by the C-terminal ubiquitin-associated (UBA) domain of p62. We present here the crystal structure of the UBA domain of mouse p62 and the solution structure of its ubiquitin-bound form. The p62 UBA domain adopts a novel dimeric structure in crystals, which is distinctive from those of other UBA domains. NMR analyses reveal that in solution the domain exists in equilibrium between the dimer and monomer forms, and binding ubiquitin shifts the equilibrium toward the monomer to form a 1:1 complex between the UBA domain and ubiquitin. The dimer-to-monomer transition is associated with a structural change of the very C-terminal end of the p62 UBA domain, although the UBA fold itself is essentially maintained. Our data illustrate that dimerization and ubiquitin binding of the p62 UBA domain are incompatible with each other. These observations reveal an autoinhibitory mechanism in the p62 UBA domain and suggest that autoinhibition plays a role in the function of p62.
- Published
- 2011
50. Crystal structure of basic 7S globulin, a xyloglucan-specific endo-β-1,4-glucanase inhibitor protein-like protein from soybean lacking inhibitory activity against endo-β-glucanase
- Author
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Takuya, Yoshizawa, Toshiyuki, Shimizu, Mayuki, Yamabe, Misako, Taichi, Yuji, Nishiuchi, Naoki, Shichijo, Satoru, Unzai, Hisashi, Hirano, Mamoru, Sato, and Hiroshi, Hashimoto
- Subjects
Models, Molecular ,Glycoside Hydrolases ,Sequence Homology, Amino Acid ,Protein Conformation ,Immunoglobulin G ,Molecular Sequence Data ,Chromatography, Gel ,Amino Acid Sequence ,Soybeans ,Enzyme Inhibitors ,Crystallography, X-Ray - Abstract
β-Linked glucans such as cellulose and xyloglucan are important components of the cell walls of most dicotyledonous plants. These β-linked glucans are constantly exposed to degradation by various endo-β-glucanases from pathogenic bacteria and fungi. To protect the cell wall from degradation by such enzymes, plants secrete proteinaceous endo-β-glucanases inhibitors, such as xyloglucan-specific endo-β-1,4-glucanase inhibitor protein (XEGIP) in tomato. XEGIPs typically inhibit xyloglucanase, a member of the glycoside hydrolase (GH)12 family. XEGIPs are also found in legumes, including soybean and lupin. To date, tomato XEGIP has been well studied, whereas XEGIPs from legumes are less well understood. Here, we determined the crystal structure of basic 7S globulin (Bg7S), a XEGIP from soybean, which represents the first three-dimensional structure of XEGIP. Bg7S formed a tetramer with pseudo-222 symmetry. Analytical centrifugation and size exclusion chromatography experiments revealed that the assembly of Bg7S in solution depended on pH. The structure of Bg7S was similar to that of a xylanase inhibitor protein from wheat (Tritinum aestivum xylanase inhibitor) that inhibits GH11 xylanase. Surprisingly, Bg7S lacked inhibitory activity against not only GH11 but also GH12 enzymes. In addition, we found that XEGIPs from azukibean, yardlongbean and mungbean also had no impact on the activity of either GH12 or GH11 enzymes, indicating that legume XEGIPs generally do not inhibit these enzymes. We reveal the structural basis of why legume XEGIPs lack this inhibitory activity. This study will provide significant clues for understanding the physiological role of Bg7S.
- Published
- 2011
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