Human choline kinase alpha

Target ID:

CHKA

Aliases:

CHKCKI

Deposition Date:

Thursday 31st August 2006

Families:

Non-protein Kinase

Related Diseases:

Cancer

Structure type:

apo

Download Coordinates:

2I7Q

Authors:

W.M.RABEH,W.TEMPEL,L.NEDYALKOVA,G.WASNEY,R.LANDRY,C.H.ARROWSMITH,A.M.EDWARDS,M.SUNDSTROM,J.WEIGELT,A.BOCHKAREV,H.PARK,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2I7Q

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.

In humans, two choline kinase genes have been found: CHKA and 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).
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.

We have solved the structure of the apo-choline kinase CHKA and
CHKB, both of which consist of a mixed α/β fold.
The overall fold of choline kinase A and B is similar to the actin fold.
Choline kinase A and B were both found to be homodimeric in solution and in the crystal lattice.
Each monomer contains small and large domains (CHKA, CHKB) and the putative active site is located between the two domains, and is is highly conserved in CHKA and CHKB.
The small domain of CHKA contains five-stranded antiparallel β-sheet surrounded by two α-helices from each side.
The large domain CHKA contains mainly α-helices and four β-sheets: seven long α-helices and four single turn α-helices construct the large domain along with two double-stranded β-sheets. The linker regionof the two domains is a three-stranded antiparallel β-sheet.

Materials and Methods

Human choline kinase A

PDB:2I7Q

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi:34192558
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal histag
Host:E.coli BL21 (DE3) codon plus RIL.

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsPQPPADEQPEPRTRRRAYLWCKEFLPGAWRGLREDEFHISVIRGGLSNMLFQCSLPDTTATLGDEPRKVLLRLYGAILQMRSCNKEGSEQAQKENEFQGAEAMVLESVMFAILAERSLGPKLYGIFPQGRLEQFIPSRRLDTEELSLPDISAEIAEKMATFHGMKMPFNKEPKWLFGTMEKYLKEVLRIKFTEESRIKKLHKLLSYNLPLELENLRSLLESTPSPVVFCHNDCQEGNILLLEGRENSEKQKLMLIDFEYSSYNYRGFDIGNHFCEWMYDYSYEKYPFFRANIRKYPTKKQQLHFISSYLPAFQNDFENLSTEEKSIIKEEMLLEVNRFALASHFLWGLWSIVQAKISSIEFGYMDYAQARFDAYFHQKRKLGV
Vector:p28a-LIC

Growth

Medium:
Antibiotics:
Procedure:

Purification

Procedure
Column 1: DE52 column
Column 2: 3 mL Ni-NTA column (Qiagen)
Column 3: Superdex 200 column (26x60, Amersham Biosciences)

The lysate was centrifuged at 15000 rpm for 30 min and 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°C. 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 protein were further purified and desalted using gel filtration column, Superdex 200 (26/60), which was pre-equilibrated with Gel filtration buffer.

Concentration: All proteins were concentrated using an Amicon Ultra centrifugal filter to a final concentration of 30 mg/mL after the addition of 5mM GDP. Protein concentrations were measured using Bradford assay with purity >95% based on SDS-PAGE analysis.

Extraction

Procedure

Concentration:
Ligand
MassSpec:
Crystallization:Crystallization trials were set up using the sitting drop vapor diffusion method. The protein drop was equilibrated against a reservoir solution (1:1 volume ratio) containing 25% PEG3350, 0.1M Ammonium sulfate, 0.1M bis-Tris, pH 6.5, vapor diffusion, sitting drop, temperature 291K.
NMR Spectroscopy:
Data Collection:Resolution: 1.90Å, X-ray source: The Industrial Macromolecular Crystallography Association 17-ID beamline at the Advanced Photon Source.
Data Processing:

References

Malito E, Sekulic N, Too WC, Konrad M, Lavie A. Elucidation of Human Choline Kinase Crystal Structures in Complex with the Products ADP or Phosphocholine. J Mol Biol. 2006 Sep 3.