Human Ca2+/calmodulin-dependent protein kinase I-gamma

Target ID:

CAMK1GA

Aliases:

CLICKIIIdJ272L16.1VWS1

Deposition Date:

Wednesday 29th November 2006

Families:

Protein Kinase

Related Diseases:

NeurobiologySignalling

Structure type:

apo

Download Coordinates:

2JAM

Authors:

J.E.DEBRECZENI,A.BULLOCK,T.KEATES,F.H.NIESEN,E.SALAH,L.SHRESTHA,C.SMEE,F.SOBOTT,A.C.W.PIKE,G.BUNKOCZI,F.VON DELFT,A.TURNBULL,J.WEIGELT,C.H.ARROWSMITH,A.EDWARDS,M.SUNDSTROM,S.KNAPP

Site:

Oxford

ICM Datapack:

ICM Datapack

2JAM

tabs

Structure Details

The Ser/Thr kinases Ca2+/calmodulin-dependent protein kinase I (CaMKI) constitutes a family of closely related isoforms (alpha, beta and gamma). CaMK isoforms play a critical role in establishing distinct types of memory in mammals. The activation of CAMK1G requires binding of Ca2+/CaM and phosphorylation by other kinases, presumably by a CaMKK.

CAMK1G expression is first detectable at embryonic day 11 (E11) and it is predominantly expressed in neurons but not glia. Within the CNS, highest expression is found in the forebrain neocortex (cerebral cortex, occipital pole, frontal lobe, and temporal lobe), the striatum (putamen and caudate nucleus), and the limbic system (amygdale and hippocampus). CAMK1G expression is altered in response to brain-derived neurotrophic factor (BDNF).

Here we present the structure of CaMK1G in complex with the ATP competitive inhibitor 5-[(Z)- (5-Chloro -2-oxo-1,2- dihydro- 3H-indol- 3-ylidene) methyl]-N- [2-(diethylamino) ethyl]-2,4- dimethyl-1H- pyrrole-3- carboxamide. This inhibitor has been initially described as a tyrosine kinase receptor and angiogenic inhibitor that exhibits selectivity for PDGFRb (IC50 = 3 ηM), VEGFR2 (IC50 = 27 ηM), FGFR1 (IC50 = 170 ηM), and Kit family members (IC50 ~ 10-500 ηM) over EGFR (IC50 >20 μM). This inhibitor has been reported to display anti-proliferative and pro-apoptotic properties in tumor cells.

Materials and Methods

CAMK1GA: Human Ca2+/calmodulin-dependent protein kinase I-gamma

PDB:2JAM

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|14196445
Entry Clone Source:MGC
SGC Clone Accession:
Tag:Tag sequence: mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*) N-terminal his6 tag.
Host:BL21 (DE3)

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfq^sMQTTNI RKTFIFMEVLGSGAFSEVFLVKQRLTGKL FALKCIKKSPAFRDSSLENEIAVLKKIKH ENIVTLEDIYESTTHYYLVMQLVSGGELF DRILERGVYTEKDASLVIQQVLSAVKYLH ENGIVHRDLKPENLLYLTPEENSKIMITD FGLSKMEQNGIMSTACGTPGYVAPEVLAQ KPYSKAVDCWSIGVITYILLCGYPPFYEE TESKLFEKIKEGYYEFESPFWDDISESAK DFICHLLEKDPNERYTCEKALSHPWIDGN TALHRDIYPSVSLQIQKNFAKSKWRQAFN AAAVVHH
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:1ml from a 10 ml overnight culture containing 50 µg/ml kanamycine was used to inoculate 1 liter of LB media containing 100 µg/ml ampiciline. Cultures were grown at 37°C until the OD 600 reached ~0.3. After that the temperature was adjusted to 18°C. Expression was induced for 4 hours using 1mM IPTG at an OD600 of 0.8. The cells were collected by centrifugation and the pellet resuspended in binding buffer and frozen. Binding buffer: 50mM HEPES pH 7.5; 300 mM NaCl; 20 mM imidazole.

Purification

Procedure

Extraction

Procedure
Cell pellets were lysed using a high pressure cell disrupter. The lysate was centrifuged at 19,000 rpm for 60 minutes and the supernatant collected for purification.
Concentration:
Ligand
MassSpec:
Crystallization:Crystals were obtained using the vapor diffusion method and a protein concentration of 10 mg/ml containing 1 mM 5-[(Z)- (5-Chloro- 2-oxo-1,2- dihydro-3H- indol-3- ylidene) methyl]- N-[2- (diethylamino) ethyl]-2,4- dimethyl-1H- pyrrole-3- carboxamide by mixing 100nl of the concentrated protein with 100nl of a well solution containing 0.20M Na(malonate); 0.1M BTProp pH 7.5; 20.0% PEG 3350; 10.0% EtGly. Crystals appeared after several days at 4°C.
NMR Spectroscopy:
Data Collection:Crystals were cryo-protected using the well solution and 25% ethylene glycole and flash frozen in liquid nitrogen. Resolution: 1.8 Å. X-ray source: Diffraction data were collected at the SLS beam line X10 at a single wavelength (0.99 nm).
Data Processing:

References

Glorioso, C., Sabatini, M., Unger, T., Hashimoto, T., Monteggia, L. M., Lewis, D. A., and Mirnics, K. (2006). Specificity and timing of neocortical transcriptome changes in response to BDNF gene ablation during embryogenesis or adulthood. Mol Psychiatry 11 , 633-648.

Takemoto-Kimura, S., Terai, H., Takamoto, M., Ohmae, S., Kikumura, S., Segi, E., Arakawa, Y., Furuyashiki, T., Narumiya, S., and Bito, H. (2003). Molecular cloning and characterization of CLICK-III/CaMKIgamma, a novel membrane-anchored neuronal Ca2+/calmodulin-dependent protein kinase (CaMK). J Biol Chem 278 , 18597-18605.