Human choline kinase beta in complex with phosphorylated hemicholinium-3 and adenosine nucleotide

Target ID:

CHKB

Aliases:

CHETKCHKL

Deposition Date:

Sunday 30th November 2008

Families:

Non-protein Kinase

Related Diseases:

Metabolism

Structure type:

protein-ligand

Download Coordinates:

3FEG

Authors:

Hong B, Tempel W, Rabeh WM, MacKenzie F, Arrowsmith CH, Edwards AM, Bountra C, Weigelt J, Bochkarev A, Park H

Site:

Toronto

3FEG

tabs

Structure Details

Phospholipids play a role as a source of lipid molecules and a structural role in membranes by creating the milieu in which membrane proteins function. Eukaryotic cell membranes contain many different phospholipid species with the most abundant one being phosphatidylcholine (PC), which is synthesized through the CDP-choline pathway, also known as the Kennedy pathway. Initially, choline is imported into the cell and is rapidly converted to phosphocholine by choline kinase[1].

In humans, two choline kinase genes have been found: choline kinase alpha (CHKA) and choline kinase beta (CHKB). CHKA is the most abundant in testis and liver whereas CHKB is ubiquitous. CHK is a homodimer (A/A or B/B) as well as a heterodimer (A/B)[1]. Alterations in phospholipids metabolism are linked to programmed cell death and oncogenic transformation. Increased CHK activity and elevated phosphocholine levels have been found in human cancer cells, suggesting that the upregulation of CHK participates in the generation of human tumors. Therefore, CHK isoforms may be potential targets for anticancer drug discovery[2, 3].

We have determined the crystal structures of CHK isoforms in complex with hemicholinium-3 (HC-3), which is well-known as a competitive inhibitor of CHK with a structural homology to a substrate choline[4]. These crystallographic data should provide useful clues for the design of more selective inhibitors against the cancer-specific CHK.

Materials and Methods

CHKB

PDB:3FEG

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi:6978649
Entry Clone Source:GenScript
SGC Clone Accession:
Tag:N-terminal histag with thrombin cleavage site: mgsshhhhhhssglvprgs
Host:BL21 DE3

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsRRRASSLSRDAERRAYQWCREYLGGAWRRVQPEELRVYPVSGGLSNLLFRCSLPDHLPSVGEEPREVLLRLYGAILQGVDSLVLESVMFAILAERSLGPQLYGVFPEGRLEQYIPSRPLKTQELREPVLSAAIATKMAQFHGMEMPFTKEPHWLFGTMERYLKQIQDLPPTGLPEMNLLEMYSLKDEMGNLRKLLESTPSPVVFCHNDIQEGNILLLSEPENADSLMLVDFEYSSYNYRGFDIGNHFCEWVYDYTHEEWPFYKARPTDYPTQEQQLHFIRHYLAEAKKGETLSQEEQRKLEEDLLVEVSRYALASHFFWGLWSILQASMSTIEFGYLDYAQSRFQFYFQQKGQLTSVHSSS
Vector:p28a-LIC

Growth

Medium:
Antibiotics:
Procedure:We prepared the seeds by inoculating freshly transforming E. coli cells (BL21 DE3) into 80 mL of Luria-Bertani medium. After overnight, all of the seeds were inoculated into 1.8 L of Terrific Broth medium in the presence of 50 µg/mL of kanamycin at 37°C and grown to an OD600 of 4.0. Cells were then induced by isopropyl-1-thio-D-galactopyranoside at the final concentration of 1.5 mM and grown overnight at 20°C in a LEX bubbling system.

Purification

Procedure
The supernatant was passed through DE52 (Whatman) column equilibrated with the binding buffer and then loaded onto 3 mL Ni-NTA column (Qiagen) equilibrated with the same binding buffer at 4 ºC. The Ni-NTA column was washed with 150 mL of the wash buffer and the protein was eluted with 15 mL of the elution buffer. The proteins were further purified and desalted using gel filtration column, Superdex 200 (26/60), which was pre-equilibrated with Gel filtration buffer. The protein was concentrated using an Amicon Ultra centrifugal filter at the final concentration of 30 mg/mL. About 20 mg of protein was obtained from 1.8 L of cell culture.

Extraction

Procedure
Cultures were centrifuged and the cell pellets were suspended in 100 mL of the binding buffer with a protease inhibitor cocktail (0.1 mM M benzamidine-HCl and 0.1 mM phenylmethyl sulfonyl fluoride) and flash frozen. The thawed cell pellet was lysed by a combination of 0.5% CHAPS (Sigma) and sonication. The lysate was centrifuged at 15000 rpm for 30 min and the supernatant was used for subsequent steps of purification.
Concentration:30 mg/mL
Ligand
HC-3, ADPMassSpec:
Crystallization:The protein was mixed with 3-5 fold molar excess of the inhibitor and ADP, respectively, and incubated overnight at room temperature. Crystals were obtained by the sitting drop vapor diffusion method. 1ul of the protein was mixed with 1 ul of unbuffered reservoir solution consisting of 0.1M sodium cacodylate (pH6.5), 30% polyethylene glycol-4000 and 0.2M ammonium sulfate. Thin plate-like crystals were grown within 3 days. For data collection a single crystal was separated from the cluster and cryoprotected in a 50:50 mixture of Paratone-N and mineral oil before flash cooling in liquid nitrogen.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Aoyama C., Liao H., and Ishidate K. 2004. Structure and function of choline kinase isoforms in mammalian cells. Progress in Lipid Research. 43, 266-281
Eliyahu G., Kreizman T., and Degani H. 2007. Phosphocholine as a biomarker of breast cancer : Molecular and biochemical studies. International Journal of Cancer. 120, 1721-1730
Hernandez-Alcoceba R., Fernandez F., and Lacal JC. 1999. In vivo antitumor activity of choline kinase inhibitors : a novel target for anticancer drug discovery. Cancer Research. 59, 3112-3118.
Hernandez-Alcoceba R., Saniger L., Campos J., Nunez M. C., Khales, F., Gallo M. A., Espinosa A., and Lacal JC. 1997. Choline kinase inhibitors as a novel approach for antiproliferative drug design. Oncogene. 15, 2289-2301.