Cryptosporidium Parvum CDPK1, cgd3_920, in complex with AMPPNP

Target ID:

PP-CDPK4

Deposition Date:

Tuesday 28th July 2009

Families:

Malaria

Related Diseases:

Parasitic disease

Structure type:

protein-ligand

Download Coordinates:

3IGO

Authors:

Wernimont AK, Artz JD, Finnerty P, Lin YH, Amani M, Hassani AA, Vedadi M, Tempel W, MacKenzie F, Bochkarev A, Arrowsmith CH, Edwards AM, Bountra C, Weigelt J, Hui R

Site:

Toronto

3IGO

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. Cryptosporidium parvum - the parasite responsible for transmission of cryptosporidiosis - has a number of CDPKs in its genome[1]. We have previously solved the structures of the kinase domain (KD) of CpCDPK1 (3DFA), CpCDPK2 (2QG5) and CpCDPK4 (3HKO).

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 CpCDPK1 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.

Materials and Methods

CDPK1

PDB:3IGO

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:
SGC Clone Accession:cgd3_920:MAC024-B07:C29724
Tag:N-terminal tag: MHHHHHHSSGRENLYFQG
Host:BL21(DE3)V2RpACYC-LIC+LamP-phosphatase

Construct

Prelude:M1-G70 truncated
Sequence:TFAERYNIVCMLGKGSFGEVLKCKDRITQQEYAVKVINKASAKNKDTSTILREVELLKKLDHPNIMKLFEILEDSSSFYIVGELYTGGELFDEIIKRKRFSEHDAARIIKQVFSGITYMHKHNIVHRDLKPENILLESKEKDCDIKIIDFGLSTCFQQNTKMKDRIGTAYYIAPEVLRGTYDEKCDVWSAGVILYILLSGTPPFYGKNEYDILKRVETGKYAFDLPQWRTISDDAKDLIRKMLTFHPSLRITATQCLEHPWIQKYSSETPTISDLPSLESAMTNIRQFQAEKKLAQAALLYMASKLTTLDETKQLTEIFRKLDTNNDGMLDRDELVRGYHEFMRLKGVDSNSLIQNEGSTIEDQIDSLMPLLDMDGSGSIEYSEFIASAIDRTILLSRERMERAFKMFDKDGSGKISTKELFKLFSQADSSIQMEELESIIEQVDNNKDGEVDFNEFVEMLQNFVRNE
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 50 mL of TB with carbenicillin(100mg/ml)+chloramphenicol (34 mg/mL) in a 250 mL shaking flask and incubated at 37 °C for overnight. Then the culture was transfer into 1.8 L of TB with carbenicillin(100mg/ml)+chloramphenicol (34 mg/mL) and 0.3 mL of antifoam (Sigma) in a 2 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. 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 then added to 1 - 5 mM.

Gel filtration:The sample was loaded onto a Sephadex S200 26/60 column equilibrated with Crystal 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 mechanically lysed with a microfluidizer (Microfluidizer Processor, M-110EH) at approximately 18000 psi; 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:29mg/ml
Ligand
MassSpec:
Crystallization:The protein was crystallized at 20 ºC in 20%peg3350, 0.2M di-NH4tart with ligand 2mM AMPPNP,2mM CaCl2,4mM MgCl2 and 6.25mM TCEP using the Sitting drop method.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Billker O, Lourido S, Sibley LD (2009) Calcium-dependent signaling and kinases in apicomplexan parasites. Cell Host Microbe 5: 612-622.