Plasmodium falciparumcyclophilin, putative cyclosporin-binding domain

Target ID:

PFE0505w

Deposition Date:

Wednesday 25th January 2006

Families:

Malaria

Related Diseases:

Parasitic disease

Structure type:

apo

Download Coordinates:

2FU0

Authors:

A.DONG,J.LEW,E.SUNDARARAJAN,Y.ZHAO,G.WASNEY,M.VEDADI,I.KOEIERADZKI,A.M.EDWARDS,C.H.ARROWSMITH,J.WEIGELT,M.SUNDSTROM,A.BOCHKAREV,R.HUI,T.HILLS,STRUCTURAL GENOMICSCONSORTIUM (SGC)

Site:

Toronto

2FU0

tabs

Structure Details

Peptidyl prolyl isomerases (PPIâ€™s) catalyze the cis-trans isomerization of peptidyl-prolyl bonds to aid in the folding of proteins and are often considered to be molecular chaperones.
The PPI family is comprised of parvulins, FK506 binding proteins and cylcophilins.

Cyclophilins are identified by their ability to bind the immune suppressive drug Cyclosporin A (CsA) and are involved in a number of biological functions such as cell cycle regulation.
The resulting cyclophilin /cyclosporin complex can then bind to the calcium calmodulin dependent serine/threonine kinase, calcineurin, thereby inhibiting the calcineurinâ€™s phosphatase activity.
Inactivation of calcineurin inhibits activation of T-cells, preventing the formation of IL-2 and results in suppression of the immune response.1

Cyclosporins are commonly used in immunosuppressive therapy following organ transplantation and as anti-inflammatories.
Cyclosporin A and the non-immunosuppresive Cyclosporin D (Valspodar) are known to have anti-malarial activity;
however, their mechanism of action is unknown and their costs are too high.

We have solved the structure of the PFE0505w putative cyclophilin domain to 1.8 Å using Plasmodium yoelii cyclophilin PY00693 (PDB 2B71) as the molecular replacement model.
PFE0505w is an 87 kDa, putative cyclophilin with a large N-term extension containing a WD40 domain.
Its cyclophilin domain is 63% identical to the same domain in
human PPWD1 (PDB 2A2N).
Therefore, we dub this target Pf-PPWD1.

Comparison with the structure of the human cyclophilin hCYP18 in complex with cyclosporin A and calcineurin (PDB 1M63)2
and the structure of PfCyp19a (another P. falciparum cyclophilin) in complex with CsA (PDB 1QNG)3
reveals that Pf-PPWD1 contains 12 of the 13 residues implicated in binding cyclosporin.
This includes the tryptophan residue corresponding to Trp128 in PfCyp19a, which has previously been shown to be essential for cyclosporin binding through analysis of PfCyp19a CsA binding mutants;4
however, only three of thirteen residues shown to be involved in calcineurin binding are conserved within PFE0505w.

Materials and Methods

Pf-CyP: Plasmodium falciparum cyclophilin PFE0505w

PDB:2FU0

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:PFE0505w
Entry Clone Source:Plasmodium falciparum 3D7 genomic DNA
SGC Clone Accession:PFE0505w:K589-N747; plate MAC015:F3
Tag:N-terminal: His6-tag with integrated TEV protease site: mgsshhhhhhssgrenlyfq*g
Host:E. coli BL21-(DE3)-CodonPlus-RIL from Stratagene

Construct

Prelude:
Sequence:gKNTPKSAIIYTTMGDIHISLFYKECKKTVQNFSVHSINGYYNNCIFHRVIKHFMVQTGDPSGDGTGGESIWGNEFEDEFFDHLNHSKPFMVSMANCGPNTNGSQFFITTVPCPWLDFKHTVFGKVTQGSKIVLDIEKVRTDKRDKPLEDIKILNIKINN
Vector:p15TV-L

Growth

Medium:Terrific Broth (TB)
Antibiotics:
Procedure:A single colony was inoculated into 10 mL of LB with of Antibiotics and incubated with shaking at 250 rpm overnight at 37 degC. The culture was transferred into 50 mL of TB with Antibiotics 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 Antibiotics and 0.3 mL of antifoam (Sigma) in a 2L bottle and cultured using the LEX system to an OD600 of ~10, cooled to 15 degC and induced with 0.5 mM isopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15 degC.

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 the DE52 column was further washed with 20 mL of Binding Buffer. The lysate was subsequently loaded onto a 2.5 mL Ni-NTA (Qiagen) column (pre-equilibrated with Binding Buffer) at approximately 1-1.5 mL/min. The Ni-NTA column was 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. EDTA was immediately added to the elution fraction to 1 mM; and DTT was added to 1-5 mM after approximately 15 more minutes.

The eluted protein was applied to a Sephadex S200 26/60 gel filtration column pre-equilibrated with Gel Filtration Buffer. The fractions corresponding to the eluted protein peak were collected and stored in 4 degC.

The His-tag was cleaved with TEV protease overnight at 4 degC in the presence of 5 mM DTT. The cleaved sample was applied to a 1ml Ni-NTA column pre-equilibrated with Binding buffer 2. Imidazole was added to the cleaved PFE0505w sample to 15 mM and applied to the Ni-NTA column. The flow-through was collected; and the column was rinsed with an additional 5 mL of Binding Buffer 2. These fractions were pooled and concentrated using a 15 mL Amicon Ultra centrifugal filter device (5 kD cutoff)

Extraction

Procedure
Cells were resuspended to approximately 40 mL/L of cell culture in Binding Buffer with protease inhibitor (1 mM benzamidine-HCl and 1 mM phenylmethyl sulfonyl fluoride, PMSF). Resuspended pellets stored at -80 degC were thawed overnight at 4 degC 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 18,000 psi. The cell lysate was centrifuged at ~75,000 x g for 20 minutes at 10 degC.
Concentration:6.5 mg/mL
Ligand
MassSpec:
Crystallization:The protein was crystallized by means of sitting drop vapor diffusion in a 96-well Intelli-Plate. The plate was set with 0.5 microL protein and 0.5 microL buffer in each drop, and 100 microL reservoir volume per well. Crystals grew after 1 day in 20% PEG 2000 MME, 0.2 M Trimethylamine N-oxide, 0.1 M Tris, pH 8.5 at 18 degC.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

1. Wang P, Heitman J. The cyclophilins. Genome Biol. 2005;6(7):226.

2. Huai Q, Kim H-Y, Liu Y, Zhao Y, Mondragon A, Liu J O, Ke H. Crystal structure of calcineurin-cyclophilin-cyclosporin shows common but distinct recognition of immunophilin-drug complexes. PNAS. 2002;99(19):12037-12042.

3. Peterson MR, Hall DR, Berriman M, Nunes JA, Leonard G, Fairlamb AH, Hunter WN. The three-dimensional structure of a Plasmodium falciparum cylcophilin in complex with the potent anti-malarial cyclosporin A. J Mol Biol.2000 Apr 21:298(1)123-33.

4. Kumar R, Musiyenko A,Barik S. Plasmodium falciparum calcineurin and its association with heat shock protein 90: mechanisms for the antimalarial activity of cyclosporine A and synergism with geldanamycin. Mol Biochem Parasitol. 2005 May;141(1):29-37.