Plasmodium yoelii 1-Cys Peroxidoxin

Target ID:

PY04285

Deposition Date:

Wednesday 1st September 2004

Families:

Malaria

Related Diseases:

Parasitic disease

Structure type:

apo

Download Coordinates:

1XCC

Authors:

M.VEDADI,S.SHARMA,S.HOUSTON,J.LEW,G.WASNEY,M.AMANI,X.XU,J.BRAY,M.SUNDSTROM,C.ARROWSMITH,A.EDWARDS,R.HUI,A.BOCHKAREV,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Toronto

1XCC

tabs

Structure Details

All aerobic mechanisms have well developed detoxification mechanisms to deal with oxidative stress.
Specifically, failure to maintain tolerable reactive oxygen species (ROS) such as superoxide anions
(O2â€¢-), hydrogen peroxide and hydroxyl radicals would lead to cellular damage.
Malaria parasites are particularly dependent on redox control because digestion of host hemoglogin
produces free haem which in turn generates O2â€¢-.
Three systems are involved in elimination of ROS at different stages:
the superoxide dismutase system,
the glutathione system and the thioredoxin system.1, 2
Catalases are also involved in many organisms but not protozoans.

The thioredoxin system includes the peroxiredoxins, also known as thioredoxin peroxidases
or thiol-dependent peroxidases.
Unlike other peroxidases, peroxiredoxins act without the benefit of redox co-factors.
So far, both 1-cys-peroxiredoxin (1-cys-Prx) and 2-cys-preoxiredoxin (2-cys-Prx)
have been identified and characterized in both P. falciparum3 and P. yoelii.4
As their names imply, these two classes of peroxiredoxins
are distinguished by the number of redox-active cysteines.

All peroxiredoxins have in common a N-terminal peroxidatic cysteine which is oxidized to cysteine sulfenic acid
upon attacking peroxide substrates.
This is often the 47th residue in 1-cys-Prx and the 50th residue in 2-cys-Prx.
2-cys-Prx has a second catalytic cysteine,
dubbed the resolving cysteine and typically from the other subunit of a homodimer in a domain swapping arrangement,
to reduce the peroxidatic cysteine via formation of a disulfide bond.
In contrast, 1-cys-Prx depends on a thiol-containing redox partner.

Our structure of Py-1-cys-Prx
(PY04285,
ortholog of P. falciparum protein
PF08_0131)
is a homodimer, the common conformation of all active peroxiredoxins.
Similar to the human 1-cys-Prx structure (1PRX), each monomer features a thioredoxin domain
and a C-terminal domain connected by a helix and a loop.
The homodimer is held in place by interaction between the thioredoxin domain of one subunit
and the C-terminal domain of the other.

See SGC's Py-2-cys-Prx structure.

Materials and Methods

Py-1cys-Prx: Plasmodium yoelii 1-cys-peroxidoxin (PY04285 - ortholog of PF08_0131)

PDB:1XCC

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:PY04285
Entry Clone Source:Plasmodium yoelii 17XNL genomic DNA
SGC Clone Accession:PY04285:; plate 2002:B10
Tag:N-terminal: His-tag with integrated TEV protease site: mgsshhhhhhssgrenlyfqghm. C-terminal: gs
Host:BL21 (DE3) Rosetta-2 from Novagen

Construct

Prelude:
Sequence:mgsshhhhhhssgrenlyfqghmGYHLGATFPNFTAKASGIDGDFELYKYIENSWAILFSHPNDFTPVCTTELAELGKMHEDFLKLNCKLIGFSCNSKESHDKWIEDIKYYGKLNKWEIPIVCDESRELANKLKIMDEQEKDITGLPLTCRCLFFISPEKKIKATVLYPATTGRNAHEILRVLKSLQLTYTTPVATPVNWNEGDKCCVIPTLQDDEISKHFKNEITKVEMPSKKKYLRFVNLgs
Vector:p11

Growth

Medium:Studier auto-induction media
Antibiotics:
Procedure:A single colony was inoculated into 10 mL of LB with of carbenicillin (50 microG/mL) and incubated with shaking at 250 rpm overnight at 37 degC. The culture was transferred into 50 mL of above-specified medium with carbenicillin (50 microG/mL) in a 250 mL shaking flask and incubated at 37 degC for 3 hours. The culture was then transferred into 1.8 L of above-specified growth medium with carbenicillin (50 microG/mL)in a 2L baffled flask and grown at 37 degC overnight in an Innova shaker from New Brunswick Scientific (150 rpm).

Purification

Procedure
The cleared lysate was loaded onto a column of 3 mL Ni-NTA from Qiagen at 4 degC. The column was washed with 150 mL Wash Buffer and the protein was eluted with 15 mL Elution Buffer. About 25 mg of pure protein was obtained from 1L of cell culture. The purified protein was dialyzed overnight into Crystal Buffer at 4 degC and concentrated using a Amicon Ultra centrifugal filter device (15 kD cutoff).

Extraction

Procedure
Cultures were centrifuged and the cell pellets were resuspended in binding buffer with protease inhibitor (1 mM benzamidine-HCl and 1 mM phenylmethyl sulfonyl fluoride, PMSF)and flash frozen. The thawed cell pellet was lysed by a combination of 0.5% CHAPS (Sigma) and sonication (1x 30 sec). Lysate was cleared by centrifugation and passed through DE52 from Whatman in 0.5 M NaCl.
Concentration:15 mg/mL
Ligand
MassSpec:
Crystallization:The protein was crystallized by means by hanging drop vapor diffusion in a VDXm plate. The plate was set with 1.5 microL uncleaved protein (15 mg/mL) and 1.5 microL buffer in each drop, and 350 microL reservoir volume per well. Crystals emerged in 23% Peg 3350, 0.1M Bis-Tris pH 5.5, 0.2M ammonium sulfate and 5% ethylene glycol at 20 degC.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

1. Z. A. Wood, E. Schröder, J. R. Harris, and L. B. Poole. Structure, mechanism, and regulation of peroxiredoxins. Trends Biochem. Sci. 28 (2003) 32-40.

2. Sylke Müller. Redox and antioxidant systems of the malaria parasite Plasmodium falciparum. Molecular Microbiology (2004) 53 (5), 1291-1305

3. Z. Krnajski, R. D. Walter, S. Muller. Isolation and functional analysis of two thioredoxin peroxidases
(peroxiredoxins) from Plasmodium falciparum. Molecular & Biochemical Parasitology 113 (2001) 303-308.

4. Kawazu S, Nozaki T, Tsuboi T, Nakano Y, Komaki-Yasuda K, Ikenoue N, Torii M, Kano S. Expression profiles of peroxiredoxin proteins of the rodent malaria parasite Plasmodium yoelii. Int J Parasitol. (2003) Nov; 33(13):1455-61.

5. Choi HJ, Kang SW, Yang CH, Rhee SG, Ryu SE. Crystal structure of a novel human peroxidase enzyme at 2.0 A resolution. Nat Struct Biol. 1998 May;5(5):400-6.