Plasmodium vivax 2-cys peroxiredoxin - reduced conformation

Target ID:

Pv-PF14_0368

Deposition Date:

Thursday 31st August 2006

Families:

Malaria

Related Diseases:

Parasitic disease

Structure type:

apo

Download Coordinates:

2I81

Authors:

J.D.ARTZ,W.QIU,A.DONG,J.LEW,H.REN,Y.ZHAO,I.KOZIERADSKI,A.M.EDWARDS,C.H.ARROWSMITH,J.WEIGELT,M.SUNDSTROM,A.BOCHKAREV,R.HUI,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2I81

tabs

Structure Details

Two Plasmodium vivax 2-cys peroxiredoxin (Pv-2CP) structures have been solved: (1) oxidized Pv-2CP (2H66); and (2) reduced Pv-2CP (2I81).
As a typical 2-CysPrx, Pv-2CP is an obligate homodimer with domain swapped C-termini, such that the active site peroxidatic cysteine (Cys50-SP-) and the resolving cysteine (Cys170-SR-H) are from different subunits.
The 2-CysPrx enzyme also forms higher order oligomers (i.e. octamers, decamers and even higher).
Pv-2CP is a decamer, while
Plasmodium yoelii 2-Cys-Prx
is an octamer. The significance of these higher order oligomers is not well understood.

Peroxiredoxins catalyze the reduction of hydrogen peroxide in a two-step reaction mechanism: (1) the peroxidatic cysteine (probably as the thiolate, Cys-SP-) attacks the peroxide substrate (i.e. a hydroperoxide, peroxide, or peroxynitrite) and is oxidized to cysteinyl sulfenic acid, Cys-SP-OH, while the RO- leaving group is likely protonated; and (2) the Cys-SP-OH is subsequently attacked by the resolving cysteine (CysR) to form a stable disulfide by a condensation reaction. Then, a cellular oxidoreductase reduces the disulfide, completing the catalytic cycle. Structural information about the reduced and oxidized forms of the 2-CysPrx will aid in the understanding of this mechanism.

Reduction of a hydroperoxide to alcohol by a typical 2-CysPrx

Pv-2CP was purified in the oxidized form with at least one disulfide formed between two subunits within the homodimer.
Oxidized Pv-2CP could be reduced with 20 mM dithiothreitol or 5 mM tris(2-carboxyethyl)phosphine.
Oxidation and reduction of the enzyme was confirmed by mass spectroscopy.

The above figure shows the overall structural differences between the reduced Pv-2CP homodimer (red/pink) and the oxidized Pv-2CP homodimer (dark blue/light blue). The reduced Pv-2CP is well structured to the end of the C-terminus (residue 195), while the oxidized Pv-2CP is disordered beginning at residue 178, so the electron density could only be refined to residue 177. The figure shows this difference in the structure and length of the C-terminus. The resolving cysteine (Cys170) located at the C-terminus is in a different orientation in the reduced and oxidized forms (see below). In the oxidized structure there is a disulfide bridge formed between the two homodimers (light blue/dark blue). In the reduced structure, the C-terminal tail has an extra loop; and the two cysteines are not within disulfide forming distance or orientation.

The above electron density map shows the disulfide bridge for the oxidized form while the figure below shows no corresponding disulfide bridge for the reduced Pv-2CP.

Materials and Methods

Pv-2CysPrx

PDB:2I81

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:Pv118545
Entry Clone Source:Plasmodium vivax Salvador I genomic DNA
SGC Clone Accession:
Tag:N-terminal: His6-tag with integrated thrombin protease site: mgsshhhhhhssglvpr*gs
Host:E. coli BL21-(DE3)-Rosetta Oxford (customized strain)

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsPTYVGKEAPFFKAEAVFGDNSFGEVNLTQFIGKKYVLLYFYPLDFTFVCPSEIIALDKALDAFHERNVELLGCSVDSKYTHLAWKKTPLAKGGIGNIKHTLLSDITKSISKDYNVLFDDSVSLRAFVLIDMNGIVQHLLVNNLAIGRSVDEILRIIDAIQHHEKYGDVCPANWQKGKVSMKPSEEGVAQYLSTL
Vector:p28a-thrombin-LIC

Growth

Medium:
Antibiotics:
Procedure:Pv-2CysPrx was expressed in E. coli BL21-(DE3)-Rosetta Oxford in Terrific Broth (TB) in the presence of kanamycin/chloramphenicol (50 microgram/mL and 25 microgram/mL respectively). A single colony was inoculated into 10 mL of LB with of kanamycin/chloramphenicol (50 microgram/mL and 25 microgram/mL respectively) in a 50 mL Falcon tube and incubated with shaking at 250 rpm overnight at 37 ºC. The culture was transferred into 50 mL of TB with 50 microgram/mL kanamycin in a 250 mL shaking flask and incubated at 37 ºC for 3 hours. Then the culture was transfer into 1.8 L of TB with 50 microgram/mL kanamycin and 0.3 mL of antifoam (Sigma) in a 2 L bottle and cultured using the LEX system to an OD600 of ~5, cooled to 15 ºC, and induced with 0.5 mM isopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15 ºC.

Purification

Procedure

Extraction

Procedure
The culture was harvested by centrifugation. Pellets from 4 L of culture were resuspended to approximately 40 mL/L of cell culture in Binding Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM imidazole, and 5 % glycerol) with the addition of protease inhibitors (1 mM benzamidine and 1 mM phenylmethyl sulfonyl fluoride (PMSF)). Resuspended pellets stored at -80 oC were thawed overnight at 4 oC on the day before purification. Prior to mechanical lysis, each pellet from 1 L of culture was pretreated with 0.5 % CHAPS and 500 units of benzonase for 40 minutes at room temperature. Cells were mechanically lysed with a microfluidizer (Microfluidizer Processor, M-110EH) at approximately 18000 psi; and the cell lysate was centrifuged using a Beckman JA-25.50 rotor at ~75000 x g (24000 rpms) for 20 minutes at 10 oC.
Concentration:8 mg/mL for Pv-2CysPrx with His6-tag.
Ligand
MassSpec:
Crystallization:Purified Pv-2CysPrx was crystallized using the hanging drop vapor diffusion method in a VDXm plate with 350 µL of mother liquor at 18 ºC. 1.5 µL of the protein solution treated with 5 mM TCEP was mixed with 1.5 µL of the reservoir solution containing 19% PEG 3350, 150 mM Li3Citrate. Crystals appeared overnight.
NMR Spectroscopy:
Data Collection:
Data Processing: