Human calcium/calmodulin-dependent protein kinase (CaM kinase) II beta + inhibitor

Target ID:

CAMK2BA

Aliases:

CAM2CAMK2CAMK2BCAMKBMGC29528

Deposition Date:

Wednesday 28th November 2007

Families:

Protein Kinase

Structure type:

protein-ligand

Download Coordinates:

3BHH

Authors:

Filippakopoulos, P., Rellos, P., Niesen, F., Burgess, N., Bullock, A., Berridge, G., Pike, A.C.W., Ugochukwu, E., Pilka, E., von Delft, F., Arrowsmith, C.H., Edwards, A., Weigelt, J., Knapp, S.

Site:

Oxford

3BHH

tabs

Structure Details

The protein kinase CAMK2β is highly expressed in endocrine tissues such as adrenals, pituitary and beta cells and moderately expressed in the liver. Low levels of expression have also been reported in lymphocytes, adipose tissue, muscle, primary fibroblasts and in primary osteoblasts. The full length protein is organized in an oligomeric, ring like structures composed of 6-12 subunits. There are four isoforms present in mammalian cells (alpha, beta, gamma, and delta).

By functioning as a local calcium sensor, CAMK2β exerts a critical influence on the architecture of the developing brain. The enzyme plays an important role in long-term potentiation and neurotransmitter release, in regulating unitary synaptic strength as well as mEPSC frequency and it may play a role in maturation and plasticity of synapses. In addition, CAMK2β serves as a structural protein that organizes F-actin required for the maintenance of the postsynaptic architecture of the dendritic spine. A role of CAMK2β has also been suggested as a platelet-activating factor, regulating sperm motility and insulin secretion from pancreatic beta-cells.

Low expression levels of CAMK2β have been described in a variety of tumor cells but new tumor specific transcripts have been described. An important function of CAMK2β has been discussed in pathways that lead to evasion of apoptosis under androgen ablation in prostate cancer cells and in facilitating the progression to an androgen independent state. In addition, expression of CAMK2β is elevated in brain tissue of patients suffering form schizophrenia or depression and may contribute to these diseases by influencing the expression of many neuroreceptors, neural outgrowth and pruning. Here we present the structure of the kinase domain of CAMK2β in complex with an ATP
competitive inhibitor.

Materials and Methods

CAMK2B + inhibitor

PDB:3BHH

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|26051204
Entry Clone Source:MGC
SGC Clone Accession:IMAGE:5014712
Tag:Cleavable N-terminal His6 tag
Host:BL21(DE3)-R3-pRARE2-Phage resistant

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfq*SMTDEYQLYEDIGKGAFSVVRRCVKLCTGHEYAAKIINTKKLSARDHQKLEREARICRLLKHSNIVRLHDSISEEGFHYLVFDLVTGGELFEDIVAREYYSEADASHCIQQILEAVLHCHQMGVVHRDLKPENLLLASKCKGAAVKLADFGLAIEVQGDQQAWFGFAGTPGYLSPEVLRKEAYGKPVDIWACGVILYILLVGYPPFWDEDQHKLYQQIKAGAYDFPSPEWDTVTPEAKNLINQMLTINPAKRITAHEALKHPWVCQRSTVASMMHRQETVECLKKFNARRKLKGA
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:Transformed 50 µl competent BL-21 (DE3) phage resistant cells with 10 µl of the plasmid DNA and plated out onto LB plate plus 50 µg/ml kanamycin and 35 µg/ml chloramphenicol. The next day colonies were picked into fresh deep well blocks containing 1 ml LB + 50 µg/ml kanamycin and 35 µg/ml chloramphenicol. Cultures were grown overnight and glycerol stocks were prepared by adding 333 ml of 60 % glycerol to 1 ml of cell suspension, which were stored at -80°C and used for future scale up preparations.The glycerol stock was used to inoculate 10 ml of LB supplemented with 50 µg/ml kanamycin and 35 µg/ml chloramphenicol. This starter culture was grown overnight at 37°C and used to inoculate a 1 liter culture in the same medium. The culture was grown at 37°C until the OD600 reached ~0.5. After that the temperature was lowered to 18°C. Protein production was induced with 1mM IPTG and recombinant CAMK2B was expressed at that temperature over night. The next day cells were harvested by centrifugation at 4000 rpm for 15 minutes. The cell pellet was stored at -80°C degrees.

Purification

Procedure
Column 1: Ni-affinity chromatographyThe cell pellet (38 g) was re-suspended in one volume (38 ml) of binding buffer. The re-suspended cells were lysed by sonication. The lysate was cleared DNA was by a centrifugation at 17,000 rpm (4°C).5 ml of 50% Ni-NTA slurry (Qiagen) was applied to a 1.5 x 10 cm gravity column. The column was equilibrated with 100 ml binding buffer. The lysate was applied to the column and was subsequently washed with 50 ml wash buffer 1 and 2. CAMK2B was eluted with 25 mls of elution buffer. The eluted protein was collected and analyzed by SDS-PAGE. DTT was added to the protein sample to a final concentration of 10mM. The N-terminal his6-tag was cleaved by incubating the protein overnight with TEV protease.Column 2: Size exclusion chromatography (Superdex S75, 60 x 1cm)The fractions eluted of the Ni-affinity chromatography were concentrated to about 4 mls using Centricon concentrators (10kDa cut off). The concentrated protein was applied to a Superdex S75 column equilibrated in SEC buffer at a flow rate of 0.8 ml/min. Eluted fractions were 95% pure as judged by SDS-PAGE.

Extraction

Procedure

Concentration:10.6 mg/ml in SEC buffer using a centricon with a 10kDa cut off
Ligand
{4-[4-(5-Cyclopropyl-1H-pyrazol-3-ylamino_-6-methylamino-pyrimidin-2-ylamino]-phenyl}-acetonotrileMassSpec:ESI-MS revealed that the protein had the expected mass of 33515 Da for the TEV cleaved protein
Crystallization:CAMK2B was crystallized at 4°C using the sitting-drop vapor diffusion method at 10.6 mg/ml in the presence of 1 mM {4-[4-(5-Cyclopropyl-1H-pyrazol-3-ylamino_-6-methylamino-pyrimidin-2-ylamino]-phenyl}-acetonotrile. Diffraction quality crystals were obtained by mixing 150 nl of protein solution with 50 nl of 20% PEG 3350; 0.20M (NH4)Cl.
NMR Spectroscopy:
Data Collection:Crystals were flash frozen in liquid nitrogen. Diffraction data were collected to 2.1 Å at the Swiss light source beam-line X10SA at a single wavelength of 0.9999 Å.
Data Processing:

References

Hoelz, A., Nairn, A. C., and Kuriyan, J. (2003). Crystal structure of a tetradecameric assembly of the association domain of Ca2+/calmodulin-dependent kinase II. Mol Cell 11, 1241-1251.

Laabich, A., Li, G., and Cooper, N. G. (2001). Characterization of apoptosis-genes associated with NMDA mediated cell death in the adult rat retina. Brain Res Mol Brain Res 91, 34-42.

Rosenberg, O. S., Deindl, S., Comolli, L. R., Hoelz, A., Downing, K. H., Nairn, A. C., and Kuriyan, J. (2006). Oligomerization states of the association domain and the holoenyzme of Ca2+/CaM kinase II. Febs J 273, 682-694.

Rosenberg, O. S., Deindl, S., Sung, R. J., Nairn, A. C., and Kuriyan, J. (2005). Structure of the autoinhibited kinase domain of CaMKII and SAXS analysis of the holoenzyme. Cell 123, 849-860.

Blanquet, P.R., and Lamour, Y. (1997). Brain-derived neurotrophic factor increases Ca2+/calmodulin-dependent protein kinase 2 activity in hippocampus. The Journal of biological chemistry 272, 24133-24136.

Maler, L., and Hincke, M.T. (1999). Distribution of calcium/calmodulin-dependent kinase 2 in the brain of Apteronotus leptorhynchus. The Journal of comparative neurology 408, 177-203.