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Title: Crystallization and Preliminary X-ray Crystallographic Studies of the Oligomeric Death-Domain Complex Between PIDD and RAIDD

Abstract

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. 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}.

Authors:
;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
929889
Report Number(s):
BNL-80471-2008-JA
TRN: US200822%%1071
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica Section F: Structural Biology and Crystallization Communications; Journal Volume: 63
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; APOPTOSIS; COMPLEXES; CRYSTALLIZATION; CRYSTALS; ESCHERICHIA COLI; INTERACTIONS; LIGHT SCATTERING; PROTEINS; SPACE GROUPS; X-RAY DIFFRACTION; national synchrotron light source

Citation Formats

Park,H., and Wu, H. Crystallization and Preliminary X-ray Crystallographic Studies of the Oligomeric Death-Domain Complex Between PIDD and RAIDD. United States: N. p., 2007. Web. doi:10.1107/S1744309107007889.
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Park,H., & Wu, H. Crystallization and Preliminary X-ray Crystallographic Studies of the Oligomeric Death-Domain Complex Between PIDD and RAIDD. United States. doi:10.1107/S1744309107007889.
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Park,H., and Wu, H. Mon . "Crystallization and Preliminary X-ray Crystallographic Studies of the Oligomeric Death-Domain Complex Between PIDD and RAIDD". United States. doi:10.1107/S1744309107007889.
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@article{osti_929889,
title = {Crystallization and Preliminary X-ray Crystallographic Studies of the Oligomeric Death-Domain Complex Between PIDD and RAIDD},
author = {Park,H. and Wu, H.},
abstractNote = {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. 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}.},
doi = {10.1107/S1744309107007889},
journal = {Acta Crystallographica Section F: Structural Biology and Crystallization Communications},
number = ,
volume = 63,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
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Journal Article:
DOI:  10.1107/S1744309107007889
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  • ; - Acta Crystallographica. Section F
    The PIDD DD–RAIDD DD complex has been crystallized. The crystals are hexagonal and belong to space group P6{sub 5}, with unit-cell parameters a = b = 138.4, c = 207.6 Å. The crystals were obtained at room temperature; a native crystal diffracted to 3.2 Å resolution and a Hg-derivatized crystal to 4.0 Å resolution. 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 andmore » 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. 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 Å 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 Å.« less

  • ; - International Journal of Molecular Sciences
    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

  • ; ; - Acta Crystallogr. F
    Smc5/6, a protein complex that belongs to the structural maintenance of chromosome (SMC) family, plays a key role in DNA replication, sister chromatid recombination and DNA damage repair. The complex contains eight subunits, including a SUMO E3 ligase Mms21 (Nse2). The activity of Mms21 is important for regulation of Smc5/6 in the response to DNA damage. Mms21 and the Mms21-binding region of Smc5 were overexpressed and purified individually in Escherichia coli with a C-terminal LEHHHHHH tag. The Mms21-Smc5 protein complex was crystallized. The diffraction of the crystals was improved greatly by glutaraldehyde treatment. X-ray diffraction data sets were collected tomore » resolutions of 2.3 and 3.9 {angstrom} from native and selenomethionine-derivative protein crystals, respectively. The crystals belonged to space group C2221, with unit-cell parameters a = 47.465, b = 97.574, c = 249.215 {angstrom} for the native crystals.« less

  • ; ; ; ... - Acta Crystallographica. Section F
    Crystals of a complex of the E. coli proteins BtuB (outer membrane cobalamin transporter) and TonB (carboxy-terminal domain) diffracting to 2.1 Å resolution have been obtained. The energy-dependent uptake of organometallic compounds and other micronutrients across the outer membranes of Gram-negative bacteria is carried out by outer membrane active-transport proteins that utilize the proton-motive force of the inner membrane via coupling to the TonB protein. The Escherichia coli outer membrane cobalamin transporter BtuB and a carboxy-terminal domain of the TonB protein, residues 147–239 of the wild-type protein, were expressed and purified individually. A complex of BtuB and TonB{sup 147–239} wasmore » formed in the presence of the substrate cyanocobalamin (CN-Cbl; vitamin B{sub 12}) and calcium and was crystallized. BtuB was purified in the detergent LDAO (n-dodecyl-N,N-dimethylamine-N-oxide) and the complex was formed in a detergent mixture of LDAO and C{sub 8}E{sub 4} (tetraethylene glycol monooctylether). Crystals were obtained by sitting-drop vapor diffusion, with the reservoir containing 30%(v/v) polyethylene glycol (PEG 300) and 100 mM sodium acetate pH 5.2. The crystals belong to space group P2{sub 1}2{sub 1}2{sub 1} (unit-cell parameters a = 74.3, b = 82.4, c = 122.6 Å). The asymmetric unit consists of a single BtuB–TonB complex. Data sets have been collected to 2.1 Å resolution at a synchrotron beamline (APS SER-CAT 22-ID)« less

  • ; ; ; ... - Acta Crystallographica. Section F, 66(12):1610-1613
    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