Human glutathione peroxidase 5

Target ID:

GPX5A

Deposition Date:

Monday 21st August 2006

Families:

Oxidoreductases

Related Diseases:

Metabolism

Structure type:

apo

Download Coordinates:

2I3Y

Authors:

K.L.KAVANAGH,C.JOHANSSON,A.ROJKOVA,C.UMEANO,G.BUNKOCZI,O.GILEADI,F.VON DELFT,J.WEIGELT,C.ARROWSMITH,M.SUNDSTROM,A.EDWARDS,U.OPPERMANN,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2I3Y

tabs

Structure Details

Glutathione peroxidase 5 (GPX5) belongs to the glutathione peroxidase family, members of which are involved in scavenging and inactivating hydrogen and lipid peroxides to water or lipid hydroxyls in a glutathione-dependent reductive reaction.
The GPX family therefore constitutes an important mechanism in the protection against oxidative stress.
At this point, seven members of the GPX family were identified in humans with distinct catalytic properties and tissue-specific expression profiles.

The hallmark of the active site in GPX enzymes is the presence of a catalytically competent selenocysteine residue, rendering it highly active towards peroxides.
GPX5 differs from most other members of the GPX family by the replacement of the selenocysteine residue by cysteine, and possibly as a result of this substitution GPX5 has lower peroxidase activity.

GPX5 is specifically expressed and secreted in the male reproductive tract.
It is postulated that GPX5, which is bound to the acrosome of sperm, may act to protect sperm cells from a premature acrosome reaction in the epididymis and thus protect against peroxidative damage.

Differential splicing of exon 3 of the human GPX5 gene, which is located on chromosome 6p22.1, leads to two variant forms, one of which is lacking the active site residues.
The majority of transcripts found in human reproductive tissue appears to have this deletion of exon 3.

Materials and Methods

GPX5: Human glutathione peroxidase 5

PDB:2I3Y

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:Codon optimised, protein sequence derivied from NP_001500
SGC Clone Accession:
Tag:Tag sequence: mhhhhhhssgvdlgtenlyfqs*(m), TEV-cleavable (*), N-terminal his6 tag.
Host:Rosetta-R3

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfqsmKMDCHKDEKGTIYDYEAIALNKNEYVSFKQYVGKHILFVNVATYCGLTAQYPELNALQEELKPYGLVVLGFPCNQFGKQEPGDNKEILPGLKYVRPGGGFVPSFQLFEKGDVNGEKEQKVFSFLKHSCPHPSEILGTFKSISWDPVKVHDIRWNFEKFLVGPDGIPVMRWSHRATVSSVKTDILAYLKQFKT
Vector:pNIC-Bsa4

Growth

Medium:
Antibiotics:
Procedure:Medium: TB + 50 µg/ml Kanamycin + 34 µg/ml chloramp .
2 x 1 liter TB in 2.5-L baffled flasks were inoculated with 2 x 10 ml overnight culture and grown at 37°C. The protein expression was induced with 1 mM IPTG at OD600 = 5.5 at 18°C over night. The cells were collected by centrifugation and frozen at -80°C.

Purification

Procedure
Column 1 : Ni-affinity, HisTrap, 1 ml (GE/Amersham Biosciences )
The cell extract was loaded on the column at 0.8 ml/minute on an AKTA-express system (GE/Amersham). The column was then washed with 10 volumes of lysis buffer, 10 volumes of wash buffer, and then eluted with elution buffer at 0.8 ml/min. The eluted peak of A280nm was automatically collected.

Column 2: Hiload 16/60 Superdex 200 prep grade 120 ml (GE/Amersham Biosciences)
The eluted fractions from the Ni-affinity Histrap columns were loaded on the gel filtration column in GF buffer at 0.80 ml/min. Eluted proteins were collected in 2 ml fractions

Concentration : The protein was concentrated in Amicon (5 K) to 8 mg/ml. The protein concentration was determined spectrophotometrically using the predicted molar extinction coefficient 36900(M-1 cm-1).

Extraction

Procedure
Frozen cell pellets from 2 liter were thawed at 37°C and resuspended in a total volume of 100 ml lysis buffer. The cells were disrupted by high pressure (20 kpsi) and nucleic acids and cell debris removed by adding 0.15 % PEI , followed by centrifugation for 30 minutes at 40 000 x g. The supernatant was further clarified by filtration (0.20 m m).
Concentration:
Ligand
MassSpec:The mass determined for GPX 5Ap004 was 24550.2 Da, in agreement with the predicted mass of 24550 for the his-tagged protein.
Crystallization:Crystals were grown by vapor diffusion at 20°C. A sitting drop consisting of 200 nl protein and 100 nl well solution was equilibrated against well solution containing 20% PEG 3350, 200 mM sodium formate, 5 % ethylene glycol. The crystal was transferred to a cryoprotectant composed of 19 % glycerol before flash-cooling in liquid nitrogen.
NMR Spectroscopy:
Data Collection:Resolution: 1.8 Å; X-ray source: Synchrotron SLS -X10, single wavelength.
Data Processing: