TPR domain of Plasmodium falciparum FK506-binding protein

Target ID:

PFL2275c

Deposition Date:

Saturday 10th December 2005

Families:

Malaria

Related Diseases:

Parasitic disease

Structure type:

apo

Download Coordinates:

2FBN

Authors:

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

Site:

Toronto

ICM Datapack:

ICM Datapack

2FBN

tabs

Structure Details

FK506 binding proteins, FKBPâ€™s, are enzymes which possess peptidyl-prolyl cis-trans isomerase activity and are considered molecular chaperones involved in protein folding.
FKBPâ€™s also bind to the immunosuppressive drugs, FK506 and rapamycin, commonly used in immune-suppressive therapy following organ transplantation.
FKBP in complex with rapamycin can inhibit the serine/threonine kinase TOR (target of rapamycin).
FKBP /FK506 can form a complex with calcineurin, a calcium calmodulin serine/threonine phosphatase,
thereby inhibiting its dephosphorylation activity.
This inhibits activation of T-cells, preventing the formation of IL-2 and resulting in suppression of immune response.1
In Plasmodium falciparum, FK506 and rapamycin have been shown to have anti-malarial activities.
The mode of action remains unclear; however, it is possible that binding of FK506 or rapamycin to the plasmodium FKBP may inhibit an essential plasmodial protein.
The residues involved in the interaction between human FKBP12 and FK506 are conserved within PFL2275c.
The P. falciparum FKBP binds FK506 and can inhibit calcineurin;
however, interestingly, PFL2275c is also able to inhibit calcineurin activity in the absence of FK506.3
The 35 kDa Plasmodium falciparum FK506 binding protein, PFL2275c, contains an N-terminal FKBP domain and a C-terminal TPR domain.
It is expressed in the erythrocytic stage parasite2 and it appears to be segregated from Plasmodium calcineurin.
During the ring stage of parasite development, FKBP is located in the cytoplasm;
however, FKBP is translocated to the nucleus in the as it enters the trophozoite and shizont stage, while calcineurin remains in the cytoplasm.3

The tetratricopeptide repeat (TPR) is a 34 amino acid repeating motif (exhibiting low sequence similarity),
with each motif comprising 2 antiparallel alpha helices. The PFL2275c C-terminal domain contains 3 TPR motifs and an additional extending alpha helix.
This TPR domain is thought to be a site for protein-protein interactions.
It has been shown that in certain FKBPâ€™s, such as human FKBP51, the TPR domain binds to the molecular chaperone Hsp 90 at the MEEVD motif.
The P. falciparum FKBP has been shown to interact with the Plasmodium falciparum Hsp90,
which also contains the MEEVD motif.
PFL2275c truncation mutants, containing the N-terminal FKBP domain only, were unable to bind Hsp90.
Both the FKBP domain and the TPR containing domain have chaperone activity but only the FKBP domain possesses peptidyl-prolyl cis-trans isomerase activity.3

We have solved the structure of Plasmodium falciparum PFL2275c, C-terminal TPR containing domain, to 1.8 Å.
The full length P. falciparum FL2275c is 30% identical to the large human FKBP-like protein, FKBP51.
The domain solved here, comprises residues 128-304 and is 25% identical to Human FKBP52 C-terminal TPR containing domain.
Due to low identity, the structure was unable to be solved using molecular replacement.
Instead, the structure was solved by Sulfur SAD method with data collected using
Dr. Emil Paiâ€™s Cr anode.

Materials and Methods

Pf-FKBP-TPR: Plasmodium falciparum FK506-binding protein - TPR domain

PDB:2FBN

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:PFL2275c
Entry Clone Source:Plasmodium falciparum 3D7 gDNA
SGC Clone Accession:PFL2275c:A128-N304; plate MAC015:C1
Tag:His-tag with integrated TEV protease site: MGSSHHHHHHSSGRENLYFQ*G
Host:E. coli BL21-(DE3)-CodonPlus-RIL from Stratagene

Construct

Prelude:
Sequence:mgsshhhhhhssgrenlyfqgAKKSIYDYTDEEKVQSAFDIKEEGNEFFKKNEINEAIVKYKEALDFFIHTEEWDDQILLDKKKNIEISCNLNLATCYNKNKDYPKAIDHASKVLKIDKNNVKALYKLGVANMYFGFLEEAKENLYKAASLNPNNLDIRNSYELCVNKLKEARKKDKLTFGGMFDKGPLYEEKKNSAN
Vector:p15-LIC

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 3 M NaCl and equilibrated with Binding Buffer), and subsequently onto a 2.0 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 (Millipore)with a 5 kDa cutoff. Aliquots of the purified protein were flash frozen in N2(l) and stored at -80°C.

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 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 to full size in 5 days in 3.5M Sodium Formate 0.1M Sodium Acetate pH 4.6 at 18 degC.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

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

2. Monaghan P, Bell A. A Plasmodium falciparum FK506-binding protein (FKBP) with peptidyl-prolyl cis-trans isomerase and chaperone acitivities. Molecular & Biochemical Parasitology, 2005;139 : 185-195.

3. Kumar R, Adams B, Musiyenko A, Shulyayeva O, Barik S. The FK506-binding protein of the malaria parasite, Plasmodium falciparum, is a FK506-sensitive chaperone with FK506-independent calcineurin-inhibitory activity. Molecular & Biochemical Parasitology, 2005;141: 163-173.