Plasmodium falciparum CDPK3 (PFC0420w), CAD domain

Target ID:

PP-CDPK3

Deposition Date:

Tuesday 29th September 2009

Families:

Protein Kinase

Related Diseases:

Parasitic disease

Structure type:

apo

Download Coordinates:

3K21

Authors:

Wernimont AK, Hutchinson A, Artz JD, MacKenzie F, Cossar D, Kozieradzki I, Arrowsmith CH, Edwards AM, Bountra C, Weigelt J, Bochkarev A, Hui R, Amani M

Site:

Toronto

ICM Datapack:

ICM Datapack

3K21

tabs

Structure Details

Calcium dependent protein kinases, absent in animals but found in apicomplexan parasites and specific plants, typically consist of a kinase domain with a calcium-binding CDPK Activation Domain (CAD) in the C-terminus. Often, the CAD is made up of a long flexible helix (CH1) followed by two pairs of EF-hands linked by another flexible helix (CH2). In Plasmodium falciparum, the parasite responsible for infecting humans with a lethal strain of malaria, there are five known CDPKs in the genome, including PFC0420w.

Using the activated and inactivated structures of CDPKs from Toxoplasma and Cryptosporidium(3HZT, 3HX4, 3IGO), we have previously proposed that a model for the activation of CDPKs based on calcium-triggered refolding of the CAD from the state exemplified in 3HXT to the conformation in 3IGO, concurrent with the translocation of this domain from the substrate-blocking position to the "opposite" side of the kinase domain. Here, we present the structure of the CAD domain alone from PfCDPK3, which provides a deeper understanding of the mechanism of refolding.

The CAD domain forms a dimer in solution in the absence of the kinase domain. For the most part, this domain is folded in the same manner as it is in the activated full length CDPK structure (3HX4, 3IGO). The major difference here is that the CH1 helix, in the absence o the kinase domain, is unbent, unlike in the full length structures where it is bent and wrapped around the EF hands.

Materials and Methods

CDPK3

PDB:3K21

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:PFC0420w
Entry Clone Source:
SGC Clone Accession:PFC0420w:I388-L560:G2
Tag:N-terminal tag: mhhhhhhssgrenlyfqg
Host:E. coli BL21(DE3)-V2R-pRARE2

Construct

Prelude:
Sequence:IHVLENFKNYGLLLKFQKLAMTIIAQQSNDYDVEKLKSTFLVLDEDGKGYITKEQLKKGLEKDGLKLPYNFDLLLDQIDSDGSGKIDYTEFIAAALDRKQLSKKLIYCAFRVFDVDNDGEITTAELAHILYNGNKKGNITQRDVNRVKRMIRDVDKNNDGKIDFHEFSEMMKL
Vector:p15-mhl

Growth

Medium:TB
Antibiotics:
Procedure:PFC0420w:I388-L560:G2 was expressed in E. coli BL21(DE3)-V2R-pRARE2 cells in Terrific Broth (TB) in the presence of ampicillin/chloramphenicol (50 microg/mL and 25 microg/mL respectively). A single colony was inoculated into 10 mL of LB with of ampicillin/chloramphenicol (50 microg/mL and 25 microg/mL respectively) in a 50 mL Falcon tube and incubated with shaking at 250 rpm overnight at 37 degC. The culture was transferred into 50 mL of TB with 50 microg/mL ampicillin in a 250 mL shaking flask and incubated at 37 degC for 3 hours. Then the culture was transfered into 1.8 L of TB with 50 microg/mL kanamycin and 0.3 mL of antifoam (Sigma) in a 1 L bottle and cultured using the LEX system to an OD 600 of ~5, cooled to 15 degC, and induced with 0.5 mM isopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15 degC.

Purification

Procedure
STEP1: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 3 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,and 1mMTCEP was added to protein.

STEP2: The sample was loaded onto a Sephadex S200 26/60 gel filtration column pre-equilibrated with 10 mM HEPES, pH 7.5 and 500 mM NaCl. The collected fractions corresponding to the correct eluted protein peak were concentrated using a 15 mL Amicon Ultra centrifugal filter device (Millipore). The protein sample identity were evalulated by mass spectroscopy. The concentrated sample (14.5 mg/ml) was stored at -80 degC.

Extraction

Procedure
The culture was harvested by centrifugation. Pellets from 2 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 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 30 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 JA-25.50 rotor at ~75000 x g (24000 rpms) for 20 minutes at 10 degC.
Concentration:14.5 mg/ml
Ligand
MassSpec:
Crystallization:The protein was crystallized at 20 degC in 21.8% w/v PEG 3350, 0.1M Acetate pH 4.2 using the Sitting drop vapor diffusion method. 10mM CaCl2 and 1mM TECEP added to protein before setting up the plate
NMR Spectroscopy:
Data Collection:
Data Processing: