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.
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.
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.
@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}
}
-
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 »
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Purification, Crystallization and Preliminary X-ray Crystallographic Studies of RAIDD Death-Domain (DD)
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High-level expression, purification, crystallization and preliminary X-ray crystallographic studies of the receptor binding domain of botulinum neurotoxin serotype D
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 »