Putative Plasmodium yoelii cyclophilin

Target ID:

PY00693

Deposition Date:

Monday 3rd October 2005

Families:

Malaria

Related Diseases:

Parasitic disease

Structure type:

apo

Download Coordinates:

2B71

Authors:

A.DONG,P.FINERTY,G.WASNEY,M.VEDADI,J.LEW,Y.ZHAO,I.KOZIERADZKI,M.MELONE,Z.ALAM,A.M.EDWARDS,C.H.ARROWSMITH,J.WEIGELT,M.SUNDSTROM,A.BOCHKAREV,R.HUI,T.HILLS,STRUCTURALGENOMICS CONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2B71

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 family of PPIâ€™s is comprised of parvulins, FK506 binding proteins, and cyclophilins.

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 bind to the
calcium calmodulin dependent serine/threonine phosphatase, calcineurin,
thereby inhibiting its dephosphorylation activity.
This binding, in turn, inhibits activation of T-cells,
preventing the formation of IL-2 and resulting in suppression of 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 sometimes used in anti-malarial combination therapy;
however, their mode of action is still unknown.

We have solved the structure cyclophilin-like protein from the rodent malarial parasite Plasmodium yoelii,
namely PY00693, to 2.5 Å.
This protein is 85% identical to PFE1430c
from the human malarial parasite Plasmodium falciparum.
It also has 46% identity with human cyclophilin J
(1XYH) which was used for the molecular replacement solution.

The structure of another P. falciparum cyclophilin (PfCyp19A or
PFC0975c)
which is 48 % identical to PY00693, has previously been solved in complex with CsA.2
From this structure it has been shown that thirteen residues are involved in the binding of CsA, nine of which are conserved in PY00693.
Mutagenesis of Trp128 in PfCyp19a has further revealed that this residue is essential for binding of CsA, since this mutant with the addition of CsA was unable to inhibit calcineurin activity.3
This tryptophan residue is not conserved within the P. yoelii cyclophilin PY00693 , therefore, further analysis is required to determine whether PY00693 is capable of binding CsA and inhibiting calcineurin activity.

Py-CyP; Plasmodium yoelii cyclophilin (PY00693)

PDB:2B71

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:cgd5_440
Entry Clone Source:Plasmodium yoelii 17XNL genomic DNA
SGC Clone Accession:PY00693:Y7-D201; plate MAC00E:D8
Tag:N-terminal: N-terminal His-tag with integrated TEV protease site: MGSSHHHHHHSSGRENLYFQG
Host:E. coli BL21-(DE3)-CodonPlus-RIL from Stratagene

Construct

Prelude:
Sequence:gMGSSHHHHHHSSGRENLYFQGYSDDEEEESNAINVVSEKTKSLEEKIAYYKMKGHTERGYITIYTNLGDFEVELYWYHSPKTCLNFYTLCEMGFYDNTIFHRVIPNFVIQGGDPTGTGKGGKSIYGEYFEDEINKELKHTGAGILSMSNNGPNTNSSQFFITLAPLPHLDGKHTIFARVSKNMTCIENIASVQTTATNKPIFDLKILRTSTAVNAD
Vector:p15TvL

Growth

Medium:Terrific Broth (TB)
Antibiotics:50 microG/mL kanamycin and 25 microG/mL chloramphenicol
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 5, cooled to 15 degC and induced with 0.5 mM isopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15 degC.

Purification

Procedure
The cleared cell lysate was loaded onto a DE52 (Whatman) column packed with 10 g of resin (previously activated with 2.5 M NaCl and equilibrated with Binding Buffer), and subsequently onto a 2.5 mL Ni-NTA column at approximately 1.5 mL/min. When all the lysate was loaded, 20 mL of Binding Buffer was added to the DE52 column. Then the Ni-NTA column was washed with 200 mL of Wash Buffer at 2Â2.5 mL/min. After washing, the protein was eluted from the Ni-NTA column with 15 mL of Elution Buffer. EDTA was added immediately to 1 mM. DTT was then added to 1 mM 15 minutes later.

The eluted protein was applied to a Sephadex S75 26/60 gel filtration column pre-equilibrated with Gel Filtration Buffer. The collected fractions corresponding to the eluted protein peak were concentrated using a 15 mL Amicon Ultra centrifugal filter device from Millipore (10 kD cutoff). The concentrated sample was flash frozen in N2(l) and stored at -80 degC.

From the thawed sample, the His-tag was cleaved with Tev protease overnight at 4 degC in the presence of 5mM B-mercaptoethanol. The cleaved sample was applied to a 2.5 mL Ni-NTA column pre-equilibrated with Binding Buffer 2. Imidazole was added to the cleaved sample to 15 mM and applied to the Ni-NTA column. The flow-through was collected and the column was rinsed with 5 mL of Wash Buffer 2. These fractions were pooled and concentrated using a 15 mL Amicon Ultra centrifugal filter device from Millipore (5 kD cutoff). The concentrated protein was flash frozen in N₂(l) in 150 microL aliquots and stored at -80 ºC.

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:7.9 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 cleaved protein and 0.5 microL buffer in each drop, and 100 microL reservoir volume per well. Crystals appeared after 3 days in 3.2M Sodium Chloride 0.1M Bis-Tris Propane pH 7.0 at 18 degC.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

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

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

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