skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Crystallization and preliminary X-ray crystallographic studies of the axin DIX domain

Abstract

The DIX domain of rat axin has been purified and crystallized. Crystals diffracted to 2.9 Å resolution using synchrotron radiation. Axin is a negative regulator of the canonical Wnt signalling pathway that mediates the phosphorylation of β-catenin by glycogen synthase kinase 3β. The DIX domain of rat axin, which is important for its homooligomerization and interactions with other regulators in the Wnt pathway, was purified and crystallized by the sitting-drop vapour-diffusion technique using polyethylene glycol 6000 and lithium sulfate as crystallization agents. Crystals belong to space group P6{sub 1} or P6{sub 5}, with unit-cell parameters a = b = 91.49, c = 84.92 Å. An X-ray diffraction data set has been collected to a nominal resolution of 2.9 Å.

Authors:
 [1];  [2];  [3]; ; ; ; ; ;  [1]; ;  [1];  [2]; ;  [4];  [5]; ;  [6];  [1];  [2]
  1. Department of Life Science, Graduate School of Science, University of Hyogo, 3-2-1 Koto, Kamigori-cho, Ako-gun, Hyogo 678-1297 (Japan)
  2. (Japan)
  3. Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo, Kyoto 606-8502 (Japan)
  4. Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo, Kyoto 606-8502 (Japan)
  5. Mitsubishi Pharma Corporation, 1000 Kamoshida-cho, Aoba-ku, Yokohama, Kanagawa 227-0033 (Japan)
  6. Department of Biochemistry, Graduate School of Biomedical Sciences, Hiroshima University, Minami-ku, Hiroshima 734-8551 (Japan)
Publication Date:
OSTI Identifier:
22360349
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 63; Journal Issue: Pt 6; Other Information: PMCID: PMC2335085; PMID: 17554179; PUBLISHER-ID: nj5002; OAI: oai:pubmedcentral.nih.gov:2335085; Copyright (c) International Union of Crystallography 2007; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United Kingdom
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CRYSTALLIZATION; CRYSTALS; DIFFUSION; INTERACTIONS; RATS; RESOLUTION; SPACE GROUPS; SYNCHROTRON RADIATION; X-RAY DIFFRACTION

Citation Formats

Shibata, Naoki, E-mail: shibach@sci.u-hyogo.ac.jp, RIKEN SPring-8 Center, 1-1-1 Koto, Sayo-cho, Sayo-gun, Hyogo 679-5248, Tomimoto, Yusuke, Hanamura, Toru, Yamamoto, Ryo, Ueda, Mai, Ueda, Yasufumi, Mizuno, Nobuhiro, Ogata, Hideaki, Komori, Hirofumi, Shomura, Yasuhito, RIKEN SPring-8 Center, 1-1-1 Koto, Sayo-cho, Sayo-gun, Hyogo 679-5248, Kataoka, Michihiko, Shimizu, Sakayu, Kondo, Jun, Yamamoto, Hideki, Kikuchi, Akira, Higuchi, Yoshiki, E-mail: shibach@sci.u-hyogo.ac.jp, and RIKEN SPring-8 Center, 1-1-1 Koto, Sayo-cho, Sayo-gun, Hyogo 679-5248. Crystallization and preliminary X-ray crystallographic studies of the axin DIX domain. United Kingdom: N. p., 2007. Web. doi:10.1107/S1744309107022579.
Shibata, Naoki, E-mail: shibach@sci.u-hyogo.ac.jp, RIKEN SPring-8 Center, 1-1-1 Koto, Sayo-cho, Sayo-gun, Hyogo 679-5248, Tomimoto, Yusuke, Hanamura, Toru, Yamamoto, Ryo, Ueda, Mai, Ueda, Yasufumi, Mizuno, Nobuhiro, Ogata, Hideaki, Komori, Hirofumi, Shomura, Yasuhito, RIKEN SPring-8 Center, 1-1-1 Koto, Sayo-cho, Sayo-gun, Hyogo 679-5248, Kataoka, Michihiko, Shimizu, Sakayu, Kondo, Jun, Yamamoto, Hideki, Kikuchi, Akira, Higuchi, Yoshiki, E-mail: shibach@sci.u-hyogo.ac.jp, & RIKEN SPring-8 Center, 1-1-1 Koto, Sayo-cho, Sayo-gun, Hyogo 679-5248. Crystallization and preliminary X-ray crystallographic studies of the axin DIX domain. United Kingdom. doi:10.1107/S1744309107022579.
Shibata, Naoki, E-mail: shibach@sci.u-hyogo.ac.jp, RIKEN SPring-8 Center, 1-1-1 Koto, Sayo-cho, Sayo-gun, Hyogo 679-5248, Tomimoto, Yusuke, Hanamura, Toru, Yamamoto, Ryo, Ueda, Mai, Ueda, Yasufumi, Mizuno, Nobuhiro, Ogata, Hideaki, Komori, Hirofumi, Shomura, Yasuhito, RIKEN SPring-8 Center, 1-1-1 Koto, Sayo-cho, Sayo-gun, Hyogo 679-5248, Kataoka, Michihiko, Shimizu, Sakayu, Kondo, Jun, Yamamoto, Hideki, Kikuchi, Akira, Higuchi, Yoshiki, E-mail: shibach@sci.u-hyogo.ac.jp, and RIKEN SPring-8 Center, 1-1-1 Koto, Sayo-cho, Sayo-gun, Hyogo 679-5248. Fri . "Crystallization and preliminary X-ray crystallographic studies of the axin DIX domain". United Kingdom. doi:10.1107/S1744309107022579.
@article{osti_22360349,
title = {Crystallization and preliminary X-ray crystallographic studies of the axin DIX domain},
author = {Shibata, Naoki, E-mail: shibach@sci.u-hyogo.ac.jp and RIKEN SPring-8 Center, 1-1-1 Koto, Sayo-cho, Sayo-gun, Hyogo 679-5248 and Tomimoto, Yusuke and Hanamura, Toru and Yamamoto, Ryo and Ueda, Mai and Ueda, Yasufumi and Mizuno, Nobuhiro and Ogata, Hideaki and Komori, Hirofumi and Shomura, Yasuhito and RIKEN SPring-8 Center, 1-1-1 Koto, Sayo-cho, Sayo-gun, Hyogo 679-5248 and Kataoka, Michihiko and Shimizu, Sakayu and Kondo, Jun and Yamamoto, Hideki and Kikuchi, Akira and Higuchi, Yoshiki, E-mail: shibach@sci.u-hyogo.ac.jp and RIKEN SPring-8 Center, 1-1-1 Koto, Sayo-cho, Sayo-gun, Hyogo 679-5248},
abstractNote = {The DIX domain of rat axin has been purified and crystallized. Crystals diffracted to 2.9 Å resolution using synchrotron radiation. Axin is a negative regulator of the canonical Wnt signalling pathway that mediates the phosphorylation of β-catenin by glycogen synthase kinase 3β. The DIX domain of rat axin, which is important for its homooligomerization and interactions with other regulators in the Wnt pathway, was purified and crystallized by the sitting-drop vapour-diffusion technique using polyethylene glycol 6000 and lithium sulfate as crystallization agents. Crystals belong to space group P6{sub 1} or P6{sub 5}, with unit-cell parameters a = b = 91.49, c = 84.92 Å. An X-ray diffraction data set has been collected to a nominal resolution of 2.9 Å.},
doi = {10.1107/S1744309107022579},
journal = {Acta Crystallographica. Section F},
number = Pt 6,
volume = 63,
place = {United Kingdom},
year = {Fri Jun 01 00:00:00 EDT 2007},
month = {Fri Jun 01 00:00:00 EDT 2007}
}
  • Three large macromolecular complexes known as the death-inducing signaling complex (DISC), the apoptosome and the PIDDosome mediate caspase activation in apoptosis signaling pathways. The PIDDosome, which activates caspase-2, is composed of three protein components: PIDD, RAIDD and caspase-2. Within the PIDDosome, the interaction between PIDD and RAIDD is mediated by a homotypic interaction between their death domains (DDs). PIDD DD and RAIDD DD were overexpressed in Escherichia coli with engineered C-terminal His tags. The proteins were purified and mixed to allow complex formation. Gel-filtration and multi-angle light scattering (MALS) analyses showed that the complex is around 150 kDa in solution.more » The purified PIDD DD-RAIDD DD complex was crystallized at 293 K. X-ray diffraction data were collected to resolutions of 3.2 and 4.0 {angstrom} from a native and a Hg-derivative crystal, respectively. The crystals belong to space group P6{sub 5}, with unit-cell parameters a = b = 138.4, c = 207.6 {angstrom}.« less
  • Caspase-2 activation by formation of PIDDosome is critical for genotoxic stress induced apoptosis. PIDDosome is composed of three proteins, RAIDD, PIDD, and Caspase-2. RAIDD is an adaptor protein containing an N-terminal Caspase-Recruiting-Domain (CARD) and a C-terminal Death-Domain (DD). Its interactions with Caspase-2 and PIDD through CARD and DD respectively and formation of PIDDosome are important for the activation of Caspase-2. RAIDD DD cloned into pET26b vector was expressed in E. coli cells and purified by nickel affinity chromatography and gel filtration. Although it has been known that the most DDs are not soluble in physiological condition, RAIDD DD was solublemore » and interacts tightly with PIDD DD in physiological condition. The purified RAIDD DD alone has been crystallized. Crystals are trigonal and belong to space group P3121 (or its enantiomorph P3221) with unit-cell parameters a = 56.3, b = 56.3, c = 64.9 and ? = 120 degrees. The crystals were obtained at room temperature and diffracted to 2.0 A resolution.« less
  • Botulinum neurotoxins (BoNTs) are highly toxic proteins for humans and can cause neuroparalytic disease botulism. Due to the limitations of production and manipulation of holoenzymes, expressing non-toxic heavy chain receptor binding domains (HCR) has become a common strategy for vaccine and antibody development. Meanwhile, large quantities and highly purified soluble proteins are required for research areas such as antibody maturation and structural biology. We present high level expression and purification of the BoNT serotype D HCR in E. coli using a codon-optimized cDNA. By varying expression conditions, especially at low temperature, the protein was expressed at a high level withmore » high solubility. About 150-200 mg protein was purified to >90% purity from 1 L cell culture. The recombinant D_HCR was crystallized and the crystals diffracted to 1.65 Å resolution. The crystals belong to space group P212121 with unit cell dimensions a = 60.8 Å, b = 89.7 Å, c = 93.9 Å. Preliminary crystallographic data analysis revealed one molecule in asymmetric unit.« less
  • Botulinum neurotoxins (BoNTs) are highly toxic proteins for humans and animals that are responsible for the deadly neuroparalytic disease botulism. Here, details of the expression and purification of the receptor-binding domain (HCR) of BoNT/D in Escherichia coli are presented. Using a codon-optimized cDNA, BoNT/D{_}HCR was expressed at a high level (150-200 mg per litre of culture) in the soluble fraction. Following a three-step purification protocol, very pure (>98%) BoNT/D{_}HCR was obtained. The recombinant BoNT/D{_}HCR was crystallized and the crystals diffracted to 1.65 {angstrom} resolution. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 60.8,more » b = 89.7, c = 93.9 {angstrom}. Preliminary crystallographic data analysis revealed the presence of one molecule in the asymmetric unit.« less
  • Botulinum neurotoxins (BoNTs) are highly toxic proteins for humans and animals that are responsible for the deadly neuroparalytic disease botulism. Here, details of the expression and purification of the receptor-binding domain (HCR) of BoNT/D in Escherichia coli are presented. Using a codon-optimized cDNA, BoNT/D{_}HCR was expressed at a high level (150-200 mg per litre of culture) in the soluble fraction. Following a three-step purification protocol, very pure (>98%) BoNT/D{_}HCR was obtained. The recombinant BoNT/D{_}HCR was crystallized and the crystals diffracted to 1.65 {angstrom} resolution. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 60.8,more » b = 89.7, c = 93.9 {angstrom}. Preliminary crystallographic data analysis revealed the presence of one molecule in the asymmetric unit.« less