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Yoshikawa H.Y.,Saitama University | Yoshikawa H.Y.,Osaka University | Murai R.,Kyoto University | Adachi H.,Osaka University | And 15 more authors.
Chemical Society Reviews | Year: 2014

With the recent development in pulsed lasers with ultrashort pulse widths or wavelengths, spatially precise, low-damage processing by femtosecond or deep-UV laser ablation has shown promise for the production of protein single crystals suitable for X-ray crystallography. Femtosecond laser processing of supersaturated solutions can shorten the protein nucleation period or can induce nucleation at low supersaturation, which improves the crystal quality of various proteins including membrane proteins and supra-complexes. In addition to nucleation, processing of protein crystals by femtosecond or deep-UV laser ablation can produce single crystalline micro- or macro-seeds without deterioration of crystal quality. This tutorial review gives an overview of the successful application of laser ablation techniques to nucleation and seeding for the production of protein single crystals, and also describes the advantages from a physico-chemical perspective. This journal is © the Partner Organisations 2014.


Watanabe Y.S.,Sanwa Kagaku Kenkyusho Co. | Yasuda Y.,Sanwa Kagaku Kenkyusho Co. | Kojima Y.,Mitsubishi Group | Kojima Y.,Synergy Inc. | And 4 more authors.
Journal of Enzyme Inhibition and Medicinal Chemistry | Year: 2015

The single-crystal structure of anagliptin, N-[2-({2-[(2S)-2-cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-2-methylpropyl]-2-methylpyrazolo[1,5-a]pyrimidine-6-carboxamide, was determined. Two independent molecules were held together by intermolecular hydrogen bonds, and the absolute configuration of the 2-cyanopyrrolidine ring delivered from l-prolinamide was confirmed to be S. The interactions of anagliptin with DPP-4 were clarified by the co-crystal structure solved at 2.85 Å resolution. Based on the structure determined by X-ray crystallography, the potency and selectivity of anagliptin were discussed, and an SAR study using anagliptin derivatives was performed. © 2015 Informa UK Ltd.


PubMed | Chiba University, Tokyo Institute of Technology, Osaka University, Chiba Institute of Science and 2 more.
Type: | Journal: The international journal of biochemistry & cell biology | Year: 2016

Spermidine acetyltransferase (SAT) from Escherichia coli, which catalyses the transfer of acetyl groups from acetyl-CoA to spermidine, is a key enzyme in controlling polyamine levels in prokaryotic cells. In this study, we determined the crystal structure of SAT in complex with spermidine (SPD) and CoA at 2.5 resolution. SAT is a dodecamer organized as a hexamer of dimers. The secondary structural element and folding topology of the SAT dimer resemble those of spermidine/spermine N(1)-acetyltransferase (SSAT), suggesting an evolutionary link between SAT and SSAT. However, the polyamine specificity of SAT is distinct from that of SSAT and is promiscuous. The SPD molecule is also located at the inter-dimer interface. The distance between SPD and CoA molecules is 13. A deep, highly acidic, water-filled cavity encompasses the SPD and CoA binding sites. Structure-based mutagenesis and in-vitro assays identified SPD-bound residues, and the acidic residues lining the walls of the cavity are mostly essential for enzymatic activities. Based on mutagenesis and structural data, we propose an acetylation mechanism underlying promiscuous polyamine recognition for SAT.


Nomura Y.,Chiba Institute of Technology | Nomura Y.,Japan Science and Technology Agency | Sugiyama S.,Osaka University | Sugiyama S.,Japan Science and Technology Agency | And 24 more authors.
Nucleic Acids Research | Year: 2010

Aptamers are short single-stranded nucleic acids with high affinity to target molecules and are applicable to therapeutics and diagnostics. Regardless of an increasing number of reported aptamers, the structural basis of the interaction of RNA aptamer with proteins is poorly understood. Here, we determined the 2.15 crystal structure of the Fc fragment of human IgG1 (hFc1) complexed with an anti-Fc RNA aptamer. The aptamer adopts a characteristic structure fit to hFc1 that is stabilized by a calcium ion, and the binding activity of the aptamer can be controlled many times by calcium chelation and addition. Importantly, the aptamer-hFc1 interaction involves mainly van der Waals contacts and hydrogen bonds rather than electrostatic forces, in contrast to other known aptamer-protein complexes. Moreover, the aptamer-hFc1 interaction involves human IgG-specific amino acids, rendering the aptamer specific to human IgGs, and not crossreactive to other species IgGs. Hence, the aptamer is a potent alternative for protein A affinity purification of Fc-fusion proteins and therapeutic antibodies. These results demonstrate, from a structural viewpoint, that conformational plasticity and selectivity of an RNA aptamer is achieved by multiple interactions other than electrostatic forces, which is applicable to many protein targets of low or no affinity to nucleic acids. © 2010 The Author(s).


Matsumura H.,Osaka University | Matsumura H.,SoSho Inc | Kai A.,Osaka University | Maeda T.,Osaka University | And 10 more authors.
Structure | Year: 2011

The reversible formation of a glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-CP12-phosphoribulokinase (PRK) supramolecular complex, identified in oxygenic photosynthetic organisms, provides light-dependent Calvin cycle regulation in a coordinated manner. An intrinsically disordered protein (IDP) CP12 acts as a linker to sequentially bind GAPDH and PRK to downregulate both enzymes. Here, we report the crystal structures of the ternary GAPDH-CP12-NAD and binary GAPDH-NAD complexes from Synechococcus elongates. The GAPDH-CP12 complex structure reveals that the oxidized CP12 becomes partially structured upon GAPDH binding. The C-terminus of CP12 is inserted into the active-site region of GAPDH, resulting in competitive inhibition of GAPDH. This study also provides insight into how the GAPDH-CP12 complex is dissociated by a high NADP(H)/NAD(H) ratio. An unexpected increase in negative charge potential that emerged upon CP12 binding highlights the biological function of CP12 in the sequential assembly of the supramolecular complex. © 2011 Elsevier Ltd All rights reserved.


PubMed | Osaka University, SoSho Inc and Kyoto University
Type: Journal Article | Journal: Acta crystallographica. Section F, Structural biology communications | Year: 2015

-Conglycinin is a major seed storage protein in soybeans, which are an important source of protein. The major subunits (, and ) of -conglycinin are sorted to protein-storage vacuoles in seed cells. Vacuolar sorting receptor (VSR) is an integral membrane protein that recognizes the sorting determinant of vacuolar proteins, including -conglycinin, and regulates their sorting process. Vacuolar sorting determinants of the and subunits of -conglycinin exist in their C-terminal peptides. Here, the preliminary X-ray diffraction analysis of the binding domain of soybean VSR crystallized with the peptide responsible for the sorting determinant in -conglycinin is reported. X-ray diffraction data were collected to a resolution of 3.5 . The crystals belonged to space group P3121, with unit-cell parameters a = b = 116.4, c = 86.1 .


Sugiyama S.,Osaka University | Sugiyama S.,Japan Science and Technology Agency | Maruyama M.,Osaka University | Maruyama M.,Japan Science and Technology Agency | And 23 more authors.
Journal of the American Chemical Society | Year: 2012

High-throughput protein X-ray crystallography offers a significant opportunity to facilitate drug discovery. The most reliable approach is to determine the three-dimensional structure of the protein-ligand complex by soaking the ligand in apo crystals. However, protein apo crystals produced by conventional crystallization in a solution are fatally damaged by osmotic shock during soaking. To overcome this difficulty, we present a novel technique for growing protein crystals in a high-concentration hydrogel that is completely gellified and exhibits high strength. This technique allowed us essentially to increase the mechanical stability of the crystals, preventing serious damage to the crystals caused by osmotic shock. Thus, this method may accelerate structure-based drug discoveries. © 2012 American Chemical Society.


Sogabe Y.,Osaka Prefecture University | Kitatani T.,Osaka Prefecture University | Yamaguchi A.,Osaka Prefecture University | Kinoshita T.,Osaka Prefecture University | And 17 more authors.
Acta Crystallographica Section D: Biological Crystallography | Year: 2011

Arabinanase Abnx from Penicillium chrysogenum 31B, which belongs to the GH93 family, releases arabinobiose from the nonreducing terminus of α-1,5-L-arabinan, which is distributed in the primary cell walls of higher plants. Crystal structures of Abnx and of its complex with arabinobiose were determined at the high resolutions of 1.14 Å to an Rwork of 10.7% (Rfree = 12.8%) and 1.04 Å to an Rwork of 10.4% (Rfree = 12.5%). Abnx has a six-bladed β-propeller fold with a typical ring-closure mode called Velcro, in which the last four-stranded β-sheet is completed by the incorporation of a strand from the N - terminus. Catalytic residues which act as a nucleophile and an acid/base were proposed from the structures and confirmed by site-directed mutagenesis. The substrate-binding groove is enclosed at one end by two residues, Glu64 and Tyr66, which contribute to the recognition of the nonreducing chain end of the polysaccharide. A comparison with the related enzyme Arb93A which has a quite similar overall structure suggested that Abnx has different mechanisms to funnel substrates to the active site and/or to stabilize the transition state. © 2011 International Union of Crystallography.


Terai T.,University of Tokyo | Terai T.,Chiyoda Corporation | Maki E.,University of Tokyo | Maki E.,Chiyoda Corporation | And 12 more authors.
Chemistry and Biology | Year: 2011

Biotin-(strept)avidin complex is widely used in biotechnology because of its extremely high binding constant, but there is no report describing spatiotemporally controlled formation of the complex in live cells. Here, based on X-ray crystal structure analysis and calorimetric data, we designed and synthesized photoreleasable biotins, which show greatly reduced affinity for (strept)avidin, but recover native affinity after UV irradiation. For application at the cell surface, we introduced an amine-reactive moiety into these "caged" biotin molecules. Specific fluorescence imaging of live cells that had been labeled with these agents and then UV-irradiated, was accomplished by addition of streptavidin conjugated with a fluorophore. We also demonstrated the applicability of these compounds for UV-irradiated-cell- specific drug delivery by using caged-biotin-labeled cells, a prodrug, and streptavidin conjugated with a prodrug-activating enzyme. © 2011 Elsevier Ltd All rights reserved.

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