Plasmodium falciparum thioredoxin

Target ID:

PFI0790w

Deposition Date:

Thursday 24th April 2008

Families:

Oxidoreductases

Related Diseases:

Parasitic disease

Structure type:

apo

Download Coordinates:

3CXG

Authors:

Wernimont AK, Lew J, Kozieradzki I, Cossar D, Schapira M, Bochkarev A, Arrowsmith CH, Bountra C, Wilkstrom M, Edwards AM, Hui R, Hills T, Pizarro J

Site:

Toronto

3CXG

tabs

Structure Details

Thioredoxins (Trx) are proteins that act as antioxidants by facilitating the reduction of other proteins by cysteine thiol-disulfide exchange. Thioredoxins are found in nearly all known organisms and are essential for life in mammals. Multiple in vitro substrates for thioredoxin have been identified, including ribonuclease, choriogonadotropins, coagulation factors, glucocorticoid receptor, and insulin. The thioredoxins are kept in the reduced state by the flavoenzyme thioredoxin reductase, in a NADPH-dependent reaction. Thioredoxins act as electron donors to peroxidases and ribonucleotide reductase.

The thioredoxin system of Plasmodium falciparum has already been partially characterized, revealing that it supplies reducing equivalents for a wide variety of acceptors, most notably ribonucleotide reductase, and it was also exposed as efficient in the nonspecific reduction of protein disulfide bonds. The Plasmodium genome encodes for at least four Thioredoxins whose transcriptions profiles appear to be sequential and the level of expression similar to those genes involved in basic metabolic functions like glycolysis or nucleotide biosynthesis.

The structure determination of Pf-Trx3 (PFI0790w), a natural substrate of P. falciparum thioredoxin reductase, enlarges our understanding of the redox metabolism of this parasite. This is also of practical importance since the proteins of the antioxidant defense system in Plasmodium represent promising targets for antimalarial drug design because like many intracellular parasites, they are particularly susceptible to oxidative challenge.

Materials and Methods

Crystal structure of Plasmodium falciparum thioredoxin, PFI0790w

PDB:3CXG

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:PFI0790w
Entry Clone Source:
SGC Clone Accession:PFI0790w:Q18-K132:E1
Tag:mgsshhhhhhssgrenlyfq
Host:Ros-Ox

Construct

Prelude:
Sequence:mhhhhhhssgrenlyfqgQSIYIELKNTGSLNQVFSSTQNSSIVIKFGAVWCKPCNKIKEYFKNQLNYYYVTLVDIDVDIHPKLNDQHNIKALPTFEFYFNLNNEWVLVHTVEGANQNDIEKAFQKYCLEKAK
Vector:p15-mhl

Growth

Medium:TB
Antibiotics:
Procedure:The protein was expressed in E. coli BL21-(DE3)-Rosetta-Oxford cells inTerrific Broth (TB) in the presence of ampicillin/chloramphenicol (50microg/mL and 25 microg/mL respectively). A single colony was inoculatedinto 10 mL of LB with the same antibiotic mixture added in a 50 mL Falcontube and incubated with shaking at 250 rpm overnight at 37 °C. The culturewas transferred into 50 mL of TB with 50 ug/mL ampicillin in a 250 mLshaking flask and incubated at 37 °C for 3 hours. Then the culture wastransferred into 1.8 L of TB with the same antibiotic mixture and 0.3 mL ofantifoam (Sigma) added in a 2 L bottle and cultured using the LEX system toan OD₆₀₀ of ~5, cooled to 15 °C, and induced with 0.5 mMisopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15 °C.

Purification

Procedure
The cleared lysate was loaded onto a column prepacked with 10 g DE52 (Whatman) anion exchange resin (previously activated with 2.5 M NaCl and equilibrated with Binding Buffer); and subsequently onto a 2.5 mL Ni-NTA (Qiagen) column pre-equilibrated with Binding Buffer at approximately 1 - 1.5 mL/min. The volume of the Ni-NTA resin was pre-determined by the predicted protein yield from test expression analysis. After the lysate was loaded, the DE52 was further washed with 20 mL of Binding Buffer. The Ni-NTA column was then washed with 200 mL of Wash Buffer at 2 - 2.5 mL/min. After washing, the protein was eluted with 15 mL of Elution Buffer. The protein was further purified by Size exclusion chromatography in a Superdex S75 26/60 column equilibrated with Gel Filtration Buffer. The fractions from the peak eluting at 199 mL corresponding to monomeric protein were pooled and the protein identity was evaluated by SDS-Page and mass spectroscopy.

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 with the addition of protease inhibitors (1 mM benzamidine and 1 mM phenylmethyl sulfonyl fluoride (PMSF)). Resuspended pellets stored at -80 degrees Celsius were thawed overnight at 4 degrees Celsius 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 JLA-16.25 rotor at 15000 rpms for 1 hour at 4 degree Celsius.
Concentration:
Ligand
MassSpec:
Crystallization:MAY5WV:G4-12M AmmSO4 0.2M NaAcetate 0.1M Hepes pH 7.5 5%MPD, 15% Glycerol
NMR Spectroscopy:
Data Collection:
Data Processing: