Toxoplasma gondii CDPK1, TGME49_101440, inactivated form

Target ID:

PP-CDPK14

Deposition Date:

Thursday 26th November 2009

Families:

Protein Kinase

Related Diseases:

Parasitic disease

Structure type:

protein-ligand

Download Coordinates:

3KU2

Authors:

Wernimont, A.K., Artz, J.D., Finnerty, P., Xiao, T., He, H., Mackenzie, F., Sinestera, G., Hassani, A.A., Wasney, G., Vedadi, M., Lourido, S., Bochkarev, A., Arrowsmith, C.H., Edwards, A.M., Bountra, C., Weigelt, J., Sibley, D.L., Hui, R., Lin, Y.H.

Site:

Toronto

3KU2

tabs

Structure Details

Calcium controls various essential pathways in apicomplexan parasites including protein secretion, motility, host invasion and egress. To mediate calcium pathways, these organisms employ calcium-dependent protein kinases (CDPK), which are also found in plants and ciliates but not in animals or fungi. Toxoplasma gondii - the parasite responsible for transmission of toxoplasmosis - has a number of CDPKs in its genome, with both TgCDPK1 and TgCDPK3 characterized in previous studies[1,2].

Canonical CDPKs are comprised of a kinase domain (KD) that is highly homologous to calmodulin-dependent kinases (CaMK), followed by 4 EF-hands, which bind Ca2+ and play the role of intramolecular regulation. We call this regulatory domain the CDPK activation domain (CAD).

We have previously solved the structure of the calcium-bound state of TgCDPK1, with both the KD and the CAD intact. Here, we present the structure of the same protein with calcium absent. This structure represents the general autoinhibited form of a canonical CDPK, and takes the same overall fold as the autoinhibited form of TgCDPK3 (3HZT).

The CAD begins with a long helix, which we have dubbed CH1 (shown in cyan) and starts a few residues downstream of the conserved HXW motif (X is proline in TgCDPK3) in the C-terminal lobe of the KD and spans the autoinhibitory region and culminates in the E-helix of the first EF-hand. On this helix, A lysine (Lys338 shown in the figure) extends a lysine to interact with Glu135 and Asp138 located on α-helix D as part of autoinhibitory mechanism also found in CaMKs. The F-helix of the second EF-hand is also the start of the second helix, which we have dubbed CH2 and extends into the helix of the third EF-hand.

Materials and Methods

TgCDPK1

PDB:3KU2

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:
SGC Clone Accession:TgCDPK4:MAC03F-A01:C208088
Tag:
Host:BL21(DE3)V2RpACYC-LIC+LamP-phosphatase

Construct

Prelude:Full length; His tag on.
Sequence:MHHHHHHSSGRENLYFQGMGQQESTLGGAAGEPRSRGHAAGTSGGPGDHLHATPGMFVQHSTAIFSDRYKGQRVLGKGSFGEVILCKDKITGQECAVKVISKRQVKQKTDKESLLREVQLLKQLDHPNIMKLYEFFEDKGYFYLVGEVYTGGELFDEIISRKRFSEVDAARIIRQVLSGITYMHKNKIVHRDLKPENLLLESKSKDANIRIIDFGLSTHFEASKKMKDKIGTAYYIAPEVLHGTYDEKCDVWSTGVILYILLSGCPPFNGANEYDILKKVEKGKYTFELPQWKKVSESAKDLIRKMLTYVPSMRISARDALDHEWIQTYTKEQISVDVPSLDNAILNIRQFQGTQKLAQAALLYMGSKLTSQDETKELTAIFHKMDKNGDGQLDRAELIEGYKELMRMKGQDASMLDASAVEHEVDQVLDAVDFDKNGYIEYSEFVTVAMDRKTLLSRERLERAFRMFDSDNSGKISSTELATIFGVSDVDSETWKSVLSEVDKNNDGEVDFDEFQQMLLKLCGN
Vector:pET15-MHL

Growth

Medium:TB
Antibiotics:
Procedure:Express plasmid in E. coli BL21(DE3)V2RpACYC-LIC+LamP-phosphatase on LB(Lauria broth) plate in the presence of carbenicillin(100mg/ml)+chloramphenicol (34 mg/mL). A single colony was inoculated into 25 mL of TB with carbenicillin(100mg/ml)+chloramphenicol (34 mg/mL) in a 50 mL falcon tube and incubated with shaking at 250 rpm overnight at 37 °C. Then the culture was transfer into 1L of TB with carbenicillin(100mg/ml)+chloramphenicol (34 mg/mL) and 0.3 mL of antifoam (Sigma) in a 1 L bottle and cultured using the LEX system to an OD600 of ~5, cooled to 15 °C, and induced with 0.5 mM isopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15 °C.

Purification

Procedure
Affinity column: 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. 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. TCEP was then added to 1 - 5 mM.

Gel filtration: The sample was loaded onto a Sephadex S200 26/60 column equilibrated with Gel Filtration Buffer. The fractions from the peak corresponding to monomer protein were collected.

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 degC were thawed overnight at 4 °C on the day before purification. Prior to mechanical lysis, each pellet from 1 L of culture was pretreated with protease inhibitors, 0.5% CHAPS and 500 units of benzonase for 40 minutes at room temperature. Cells were sonicated for effective time 10 minutes(about 120 watts, pulsed 10s on, 10s off) and the cell lysate was centrifuged using a Beckman JA-25.25 rotor at 24,000 rpms for 20 minutes at 10 ºC
Concentration:protein concentration: 12mg/ml
Ligand
MassSpec:
Crystallization:The protein was crystallized at 20 ºC in 20% peg3350 and 0.2M KF with ligand 2mM AMPPNP,4mM MgCl2,6.25mM TCEP using the Sitting drop method.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Kieschnick H, Wakefield T, Narducci CA, Beckers C (2001) Toxoplasma gondii attachment to host cells is regulated by a calmodulin-like domain protein kinase. J Biol Chem 276: 12369-12377.
Nagamune K, Sibley LD (2006) Comparative genomic and phylogenetic analyses of calcium ATPases and calcium-regulated proteins in the apicomplexa. Mol Biol Evol 23: 1613-1627.