Toxoplasma gondii CDPK1, TGME49_101440, in presence of calcium

Target ID:

PP-CDPK4

Deposition Date:

Friday 19th June 2009

Families:

Malaria

Related Diseases:

Parasitic disease

Structure type:

apo

Download Coordinates:

3HX4

Authors:

Wernimont AK, Artz JD, Finnerty P, Xiao T, He H, MacKenzie F, Sinestera G, Hassani AA, Wasney G, Vedadi M, Lourido S, Bochkarev A, Arrowsmith CH, Edwards AM, Bountra C, Weigelt J, Sibley DL, Hui R, Yuhui LL

Site:

Toronto

ICM Datapack:

ICM Datapack

3HX4

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).

Here, we present the structure of TgCDPK1 with the KD (in gray) and the CAD intact. This structure represents the general activated form of a canonical CDPK. Each EF-hand in the CAD has a Ca2+ bound. As found with calmodulin, binding of calcium opens up each EF-hand, exposing hydrophobic surfaces and resulting in a refolding of CAD. The extent of refolding can be seen by comparing this structure against that of TgCDPK3 (3HZT), which is in the inhibited conformation. Furthermore, we can also see that the CAD has translocated to a new location relative to the KD, away from the substrate binding site and therefore allowing it to become active.

In the refolded CAD, the CH1 helix (teal), which is a single long helix in the inhibited conformation (3HZT), is bent into three segments in this activated conformation. The same is also true of the CH2 helix (magenta). The two are intertwined around each other, in addition to interaction with the two EF-lobes.

In its new position, the refolded CAD interacts with the KD via various salt bridges. Most interestingly, a stretch of residues in the N-terminus of the KD (shown in green in the figure below) latches into a cleft in the CAD.

Materials and Methods

CDPK1

PDB:3HX4

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:
SGC Clone Accession:TgCDPK4:MAC03F-A01:C208088
Tag:C-terminal His6-tag
Host:BL21(DE3)V2RpACYC-LIC+LamP-phosphatase

Construct

Prelude:
Sequence:MMGQQESTLGGAAGEPRSRGHAAGTSGGPGDHLHATPGMFVQHSTAIFSDRYKGQRVLGKGSFGEVILCKDKITGQECAVKVISKRQVKQKTDKESLLREVQLLKQLDHPNIMKLYEFFEDKGYFYLVGEVYTGGELFDEIISRKRFSEVDAARIIRQVLSGITYMHKNKIVHRDLKPENLLLESKSKDANIRIIDFGLSTHFEASKKMKDKIGTAYYIAPEVLHGTYDEKCDVWSTGVILYILLSGCPPFNGANEYDILKKVEKGKYTFELPQWKKVSESAKDLIRKMLTYVPSMRISARDALDHEWIQTYTKEQISVDVPSLDNAILNIRQFQGTQKLAQAALLYMGSKLTSQDETKELTAIFHKMDKNGDGQLDRAELIEGYKELMRMKGQDASMLDASAVEHEVDQVLDAVDFDKNGYIEYSEFVTVAMDRKTLLSRERLERAFRMFDSDNSGKISSTELATIFGVSDVDSETWKSVLSEVDKNNDGEVDFDEFQQMLLKLCGNLEHHHHHH
Vector:PET22

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:15mg/ml
Ligand
MassSpec:
Crystallization:The protein was crystallized at 20 ºC in 26.36% peg3350, tris8.0 and 0.2M LiSO4 with ligand 2mMAMPPNP,2mMCaCl2,4mM MgCl2 and 6.25mMTCEP 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.