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Toxoplasma gondii CDPK3, TGME49_105860

PDB Code 3HZT Target Class Malaria

Target CDPK3
Alias n/a
Disease Area/Function Parasitic Disease
Date Deposited 2009-06-24
Authors Wernimont, AK, Artz, JD, Finnerty, P, Wasney G, Allali-Hassani A, Vedadi M, Bochkarev A, Arrowsmith CH, Edwards AM, Bountra C, Weigelt J, Hui R, Amani M
Related Structure 3DXN, 3KHE, 3L19, 3LIJ, 3Q5I

Struc Details 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 KD of TgCDPK3 (3DXN). Here, we present the structure of the KD (in gray) with the CAD intact. This structure represents the general autoinhibited form of a canonical CDPK, with the CAD resembling the closed form of calmodulin (3CLN) but with an additional long helix in the N-terminus. This helix, which we have dubbed CH1 (shown in cyan), 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.

References

  1. 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.
  2. 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.
Materials & Methods
StructureCDPK3
PDB Code3HZT
Entry clone accession 541.m00134
SGC clone accession 541.m00134:L72-H537:B4
Tag N-terminal tag: mhhhhhhssgrenlyfqg
Construct sequenceLSDRYQRVKKLGSGAYGEVLLCKDKLTGAERAIKIIKKSSVTTTSNSGALLDEVAVLKQLDHPNIMKLYEFFEDKRNYYL
VMEVYRGGELFDEIILRQKFSEVDAAVIMKQVLSGTTYLHKHNIVHRDLKPENLLLESKSRDALIKIVDFGLSAHFEVGG
KMKERLGTAYYIAPEVLRKKYDEKCDVWSCGVILYILLCGYPPFGGQTDQEILKRVEKGKFSFDPPDWTQVSDEAKQLVK
LMLTYEPSKRISAEEALNHPWIVKFCSQKHTDVGKHALTGALGNMKKFQSSQKLAQAAMLFMGSKLTTLEETKELTQIFR
QLDNNGDGQLDRKELIEGYRKLMQWKGDTVSDLDSSQIEAEVDHILQSVDFDRNGYIEYSEFVTVCMDKQLLLSRERLLA
AFQQFDSDGSGKITNEELGRLFGVTEVDDETWHQVLQECDKNNDGEVDFEEFVEMMQKICDVKVKH
Vector p15-mhl
Expression host BL21(DE3)R3pACYC-LIC+LamP-phosphatase
Growth medium TB
Growth method 541.m00134:L72-H537:B4 was expressed in E. coli BL21(DE3)R3pACYC-LIC+LamP-phosphatase 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 ºC. The culture was transferred into 50 mL of TB with 50 microg/mL ampicillin in a 250 mL shaking flask and incubated at 37 ºC 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 2 L bottle and cultured using the LEX system to an OD 600 of ~5, cooled to 15 ºC, and induced with 0.5 mM isopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15 ºC.
Extraction buffersBinding Buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM imidazole, and 5 % glycerol
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 ºC were thawed overnight at 4 ºC 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 ºC.
Purification buffersWash Buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 30 mM imidazole, and 5 % glycerol
Elution Buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 250 mM imidazole, and 5 % glycerol
Gel Filtration Buffer: 10 mM HEPES, pH 7.5, 500 mM NaCl, 5mMDTT
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. TCEP was added to 1 mM, and MgCl2 to 1mM.

STEP2:(Cut His Tag) Added TEV (with activity 1:50 and concentration of 12mg/mL) to the protein and dialysis in 10mM HEPES, 500mM NACL, 5mM Imidazole, and 5mM DTT overnight. The day after running mass spectroscopy to make sure His Tag completely cut and then pass through Ni-NTA column, and filterated with syringe driven filter unite(0.22um) for running Gel filtration.

STEP3: 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, 5mMDTT. 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 (20 mg/mL) was stored at -80 ºC.
Crystallization The protein was crystallized at 20 ºC in 19% w/v PEG 3350, 0.2 M KF using the Hanging drop vapor diffusion method. 3mM SU11652 (Calbiochem) added to protein before setting up plate.