Human peroxiredoxin 4 (thioredoxin peroxidase)

Target ID:

PRDX4A

Aliases:

AOE37-2PRX-4

Deposition Date:

Tuesday 24th April 2007

Families:

Oxidoreductases

Related Diseases:

CancerMetabolism

Structure type:

apo

Download Coordinates:

2PN8

Authors:

E.S.PILKA,K.L.KAVANAGH,C.COOPER,F.VON DELFT,M.SUNDSTROM,C.A.ARROWSMITH,J.WEIGELT,A.EDWARDS,U.OPPERMANN,STRUCTURALGENOMICS CONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2PN8

tabs

Structure Details

Oxidative stress is an inevitable process coupled to respiration and is defined as an imbalance between pro-oxidative and anti-oxidative states. This imbalance leads to an overall increase in cellular levels of reactive oxygen species such as hydrogen peroxide, superoxide anion or hydroxyl radicals that can progress to further lipid or macromolecule oxidation. Oxidative stress is accompanied with an altered gene transcription profile, carried out by redox-sensitive transcription factors, e.g., NFkB, AP1, or MAP kinases.

Various antioxidative mechanisms have been identified, such as small molecule antioxidants (glutathione, lipoic acid, ascorbic acid, tocopherol) or enzymatic mechanisms such as thioredoxin dependent peroxiredoxins (PRDXs) or glutathione dependent glutaredoxins (GLRXs). These enzymes regulate the intracellular redox state and inactivate hydrogen peroxide or lipid peroxides.

Peroxiredoxin 4 (Prdx4) belongs to a family of highly conserved peroxiredoxins, which have been identified in yeast, plant and animal cells, and most, if not all, eubacteria and archaea. Six members are expressed in mammalian cells (Prdx I to VI), which are classified into three subgroups based on the number and position of active site Cys residues that participate in catalysis. PRDX4 (Prx III) together with Prx 1, 2 and 3 belongs to the first subgroup.

The enzyme is widely expressed in human tissues with highest levels found in pancreas, liver, heart spleen and thymus. In sperm cells Prdx4 serves in the acrosome formation and vesicular reorganization during spermiogenesis.

Materials and Methods

PRDX4

PDB:2PN8

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|5453549
Entry Clone Source:MGC
SGC Clone Accession:PRDX4A-c004
Tag:N-terminal, TEV cleavable hexahistidine tag. Tag sequence: mhhhhhhssgvdlgtenlyfq(*)sm
Host:BL21(DE3)-R3-pRARE2

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfq*smPAPYWEGTAVIDGEFKELKLTDYRGKYLVFFFYPLDFTFVCPTEIIAFGDRLEEFRSINTEVVACSVDSQFTHLAWINTPRRQGGLGPIRIPLLSDLTHQISKDYGVYLEDSGHTLRGLFIIDDKGILRQITLNDLPVGRSVDETLRLVQAFQYTDKHGEVCPAGWKPGSETIIPDPAGKLKYFDKLN
Vector:pNIC28-Bsa4.

Growth

Medium:TB
Antibiotics:
Procedure:10µl of BL21(DE3)-R3-pRARE2 glycerol stock were inoculated into 60ml of TB with 50µg/ml of kanamycin and 34µg/ml chloramphenicol and grown overnight at 37°C, 200rpm. 10ml of overnight culture were added to 1L of TB with 50µg/ml kanamycin and incubated at 37°C, 160rpm. After the OD600 reached 1.0, the temperature was dropped to 18 °C and 500ul of 1M IPTG was added to the final concentration of ~0.5mM. The culture was then incubated with shaking overnight at 18 °C, 160rpm. The following morning the 4L culture was harvested and centrifuged for 15min at 4000rpm. Supernatant was discarded and cell pellets were resuspended in 80ml of a lysis buffer and frozen at -80 °C.

Purification

Procedure
The cell extract was loaded on the AKTA Express system The extinction at 280nm was monitored and fractions were collected and analyzed by SDS-PAGE. Positive fractions were pooled and concentrated.

Extraction

Procedure
The thawed cells were broken by 5 passes at 16.000 psi through a high pressure homogeniser followed by centrifugation for 45 min at 15.000rpm.
Concentration:Using VivaSpin-15 concentrators with 10kDa cutoff, the sample was concentrated to 8mg/ml. Concentrations were determined from the absorbance at 280nm using NanoDrop.
Ligand
MassSpec:Calculated mass of the construct was 24001. The mass of 24000 was confirmed by the mass spec.
Crystallization:Crystals were grown by vapor diffusion at 20°C in 3ul hanging drops. The drops were prepared by mixing 2ul of protein solution and 1ul of precipitant consisting of 0.1M HEPES pH 7.5, 10% PEG 10K and 8% Ethylene Glycol. Crystals were transferred to a cryo-protectant consisting of 10% Ethylene Glycol and 90% well solution before flash-cooling in liquid nitrogen.
NMR Spectroscopy:
Data Collection:Resolution: 1.8Å; X-ray source: SLS beam X10SA.
Data Processing: