Cryptosporidium parvum calcium dependent protein kinase cgd7_1840 in complex with indirubin E804

Target ID:

PP-CDPK8

Deposition Date:

Friday 31st October 2008

Families:

Malaria

Related Diseases:

Parasitic disease

Structure type:

protein-ligand

Download Coordinates:

3F3Z

Authors:

Wernimont AK, Lew J, Wasney G, Kozieradzki I, Cossar D, Bochkarev A, Arrowsmith CH, Bountra C, Weigelt J, Edwards AM, Hui R, Artz JD, Armani M

Site:

Toronto

ICM Datapack:

ICM Datapack

3F3Z

tabs

Structure Details

Calcium dependent protein kinases (CDPK) are found only in some plants - e.g. Arabidopsis thanalia - and some protozoan organisms, including Plasmodium, Cryptosporidium and Toxoplasma parasites.
We have previously determined the apo structure of kinase domain of
(encoded by the gene cgd7_1840.

From Stargazer screening, we identified a number of kinase inhibitors which stabilized CpCDPK8, including staurosporine and indirubin derivative E804.
In addition, we have determined determined that indirubin E804 effectively inhibited the enzyme's phosphorylation activity with a IC50 value of 1.2 μM.

Indirubins (IRD) are a class of natural chemicals extracted from the indigo plant as well as gastropods of the Muricidae and Thaididae families (Polychronopoulos et al., 2004). They are also the active ingredients in the Chinese herbal mixture Dang Gui Long Hui Wan, which was found to have antileukemic activity back in 1980's. The mechanism of action is based on inhibition of both S/T and tyrosine protein kinases including cyclin-dependent kinases (CDK), glycogen synthase kinase (GSK) and Stat3 (Meijer et al., 2003; Polychronopoulos et al., 2004).
We were able to crystallize CpCDPK8's kinase domain with indirubin 804 bound - the first ever complex of a CDPK with a ligand. As noted in most kinases, the presence of a ligand in the nucleotide cleft bends the N lobe towards the C-lobe, particularly the flexible glycine-rich region.
The backbone interaction between the hinge and ligand is conserved in known kinases/indirubin complexes, as shown in the table below.
Different indirubin derivatives are distinguished by different moieties at the 3'and 5-positions on the bisindole.

Structure Ligands Interacting residues CpCDPK8 (3F3Z) Indirubin E804 Glu103 Cys105 hCaMK2A (2VZ6) Indirubin E804 Asp90 Val92 hCDK5 (1UNH) Indirubin 3'-monoxime Glu81 Cys83 hCDK2 (1E9H) Indirubin 3'-monoxime-5-sulphonic acid Glu81 Leu83 hGSK3 (1Q41) Indirubin 3'-monoxime Asp133 Val135 CpGSK (3EB0) Indirubin E804 Glu118 Val119

Indirubin 804 is distinguished by its side chain on the bisindole's 3'position.
This is an important moiety because other indirubin compounds in our screening library, all with the same bisindole structure but different decorations at the 3'or 5 positions, failed to stabilize CpCPDK8.
The figure above shows how this side chain forms polar interaction with Glu109, Glu152 and, via a water molecule, Leu110, Phe111 and Pro151.
These hydrogen bonds result partly from the polar side chain being forced by the hydrophobic side chain of Ile168 (located just before the DFG triad) to bend in a direction perpendicular to the plane of the bisindole structure.
In the absence of the hydrophobic residue, the IRD molecule would adopt the conformation found in the hCaMK2A structure.
The presence of this nonpolar residue and the resulting change in the confomation of IRD804 are unique to CpCDPK8 and may be potentially exploited for development of a selective inhibitor.

Based on our biochemical assays, indirubin compounds are potentially promising anticryptosporidial drug candidates. In particular, IRD804 is able to inhibit CpCPDK8 at low micromolar concentrations. Our structural complex of CpCDPK8 and IRD804 provides evidence of a unique mechanism of inhibition that can be used to develop a more selective and more potent inhibitor. Certainly, it gives us motivation to test other indirubin derivatives with similar side chain decoration.

Materials and Methods

cgd7_1840

PDB:3F3Z

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:cgd7_1840
Entry Clone Source:
SGC Clone Accession:cgd7_1840:S197-L472; plate MAC024:D9
Tag:N-terminal His6-tag with integrated TEV cleavage site (*): mhhhhhhssgrenlyfq*g
Host:E. coli BL21-(DE3)-R3-pRARE2

Construct

Prelude:
Sequence:mhhhhhhssgrenlyfqgSTKGDINQYYTLENTIGRGSWGEVKIAVQKGTRIRRAAKKIPKYFVEDVDRFKQEIEIMKSLDHPNIIRLYETFEDNTDIYLVMELCTGGELFERVVHKRVFRESDAARIMKDVLSAVAYCHKLNVAHRDLKPENFLFLTDSPDSPLKLIDFGLAARFKPGKMMRTKVGTPYYVSPQVLEGLYGPECDEWSAGVMMYVLLCGYPPFSAPTDSEVMLKIREGTFTFPEKDWLNVSPQAESLIRRLLTKSPKQRITSLQALEHEWFEKQLSSSPRNLL
Vector:p15-mhl

Growth

Medium:TB
Antibiotics:100 microG/mL ampicillin and 34 microG/mL chloramphenicol
Procedure:A single colony was inoculated into 10 mL of LB with of Antibiotics and incubated with shaking at 250 rpm overnight at 37 °C. The culture was transferred into 50 mL of TB with Antibiotics in a 250 mL shaking flask and incubated at 37 °C for 3 hours. The culture was then transferred into 1.8 L of above-specified growth medium with Antibiotics and 0.3 mL of antifoam (Sigma) in a 2L 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
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. MgCl2 and CaCl2 was added to the elution fraction to 1 mM; and TCEP was added to 2 mM after elutting from Ni-NTA column.

The sample was loaded onto a Sephadex S200 26/60 gel filtration column pre-equilibrated with Gel Filtration Buffer. The collected fractions corresponding to the correct eluted protein peak were concentrated using a 15 mL Amicon Ultra centrifugal filter device (Millipore). TCEP (2mM), 1mM MgCl₂, 2mM CaCl₂ was added to the concentrated protein. The protein sample identity were evalulated by mass spectroscopyand found to be a mixture of different phosphorylated states.The protein was treated by lambda protein phosphatase from Biolabs.Subsequent evaluation by mass spectroscopy again revealed thatthe phosphatase resulted in a dominant species with a single phosphorylation site.

Extraction

Procedure
Cells were resuspended to approximately 40 mL/L of cell culture in Binding Buffer with protease inhibitor (1 mM benzamidine-HCl 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 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 at ~75000 x g for 20 minutes at 10 ºC.
Concentration:The concentrated sample (20 mg/mL) was stored at 4 °C.
Ligand
MassSpec:
Crystallization:The protein was crystallized using the hanging drop vapor diffusion method at 20 ºC in 18% PEG 3350, 0.1 M NH₄SO₄, 0.1 M NaCacodylate, 5 mM Indirubin E804, pH 5.5.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Meijer, L., Skaltsounis, A.L., Magiatis, P., Polychronopoulos, P., Knockaert, M., Leost, M., Ryan, X.P., Vonica, C.A., Brivanlou, A., Dajani, R., et al. (2003). GSK-3-selective inhibitors derived from Tyrian purple indirubins. Chem Biol 10, 1255-1266.
Polychronopoulos, P., Magiatis, P., Skaltsounis, A.L., Myrianthopoulos, V., Mikros, E., Tarricone, A., Musacchio, A., Roe, S.M., Pearl, L., Leost, M., et al. (2004). Structural basis for the synthesis of indirubins as potent and selective inhibitors of glycogen synthase kinase-3 and cyclin-dependent kinases. J Med Chem 47, 935-946.