Human calcium/calmodulin-dependent protein kinase (CaM kinase) II gamma

Target ID:

CAMK2GA

Aliases:

CAMKCAMK-IICAMK2GCAMKGFLJ16043MGC26678

Deposition Date:

Tuesday 31st July 2007

Families:

Protein Kinase

Related Diseases:

MetabolismCancerSignallingNeurobiology

Structure type:

protein-ligand

Download Coordinates:

2V7O

Authors:

A.C.W.Pike, P.Rellos, O.Fedorov, N.Burgess-Brown, L.Shrestha, E.Ugochukwu, E.S.Pilka, F.von Delft, A.Edwards, J.Weigelt, C.H.Arrowsmith, M.Sundstrom, S.Knapp

Site:

Oxford

ICM Datapack:

ICM Datapack

2V7O

tabs

Structure Details

The protein kinase CAMK2γ is ubiquitously expressed and forms oligomeric, ring like structures composed of 6-12 subunits. There are four isoforms present in mammalian cells (alpha, beta, gamma, and delta).

CAMK2γ is one of the most important transducers of Ca2+ signals and has been linked to a multitude of regulatory pathways. For example, regulatory functions for CAMK2γ have been suggested in cardiomyocyte apoptosis, memory, influencing immature T cell lifespan and T cell memory function and modulation of TCR signalling strength. A scaffolding function for the assembly of a postsynaptic signalosome, a role as mediator of synaptic plasticity and in bipolar spindle formation, as well as regulation of osteoclastogenesis has also been proposed.

The overall organization of each of the four CAMK2γ isoforms is similar: an N-terminal catalytic domain is followed by a regulatory domain that contains an autoinhibitory region with a calmodulin (CaM) binding site and a C-terminal association domain, through which the subunits interact to assemble into holoenzymes. A variable domain is located between the CaM-binding domain and the association domain. Most splice variant differences map to this region. CAMK2γ is activated by a two step activation process: The first activation event involves the dissociation of the autoinhibitory regulatory segment. This process involves binding of Ca2+/ CaM which leads to the exposure of a regulatory threonine residue. The now accessible threonine is phophorylated by a neighboring kinase domain within the oligomeric dodecameric assembly. Once activated phophorylation prevents rebinding of the regulatory segment to the kinase domain and binding of Ca2+/CaM . Thus, at high Ca2+/CaM concentration the phosphorylation at the regulatory threonine propagates rapidly through the holoenzyme resulting in active Ca2+/CaM -independent CAMK2γ .

Surprisingly, the structure of the association domain of mouse CAMK2α revealed a stacked arrangement of two 7-fold symmetric rings which has been attributed to the lacking kinase domain in the complex. However this association behavior might be specific for the alpha isoform or splice variant used. Here we present the structure of the association domain of in CAMK2γ in its dodecameric association state as well as the structure of the kinase domain in complex with the ATP competitive inhibitor BIM9.

Materials and Methods

CAMK2G

PDB:2V7O

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|26667203
Entry Clone Source:MGC
SGC Clone Accession:
Tag:mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*) N-terminal his6 tag.
Host:BL21(DE3)-R3-pRARE2

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfq*smATCTRFTDDYQLFEELGKGAFSVVRRCVKKTPTQEYAAKIINTKKLSARDHQKLEREARICRLLKHPNIVRLHDSISEEGFHYLVFDLVTGGELFEDIVAREYYSEADASHCIHQILESVNHIHQHDIVHRDLKPENLLLASKCKGAAVKLADFGLAIEVQGEQQAWFGFAGTPGYLSPEVLRKDPYGKPVDIWACGVILYILLVGYPPFWDEDQHKLYQQIKAGAYDFPSPEWDTVTPEAKNLINQMLTINPAKRITADQALKHPWVCQRSTVASMMHRQETVECLRKFNARRKLKGAILTTMLVSRNFSVG
Vector:pNIC28-Bsa4.

Growth

Medium:LB
Antibiotics:
Procedure:3ml from a 50 ml overnight culture was used to inoculate each of two flasks containing 1 litre of LB media containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol. Cultures were grown at 37°C to an OD600 of ~0.4 and then cooled to 18°C. Expression was induced for 4 hours using 0.5mM IPTG at an OD600 of 0.8. The cells were collected by centrifugation and the pellet resuspended in binding buffer and frozen.

Purification

Procedure
Column 1: Ni-affinity chromatography.
Column 2: Size exclusion chromatography (Sephacryl S200)

5 ml of 50% Ni-sepharose slurry (Amersham Biosciences) was applied to a 1.5 x 10 cm gravity column. The column was equilibrated with 50 ml binding buffer. The lysate was applied to the column which was subsequently washed with 50 ml wash buffer. CaMK2G was eluted in 5 ml step elutions containing 50 mM, 100 mM, 150 mM and 250 mM imidazole. The eluted protein was collected and analyzed by SDS-PAGE. DTT was added to the protein sample to a final concentration of 10 mM. TEV-protease was added to the protein over night. However, the N-terminal his 6 -tag was not cleaved due to phosphorylation of a serine residue within the protease cleavage site.

The cleaved protein was concentrated to 3 ml using Centricon concentrators (10kDa cut off) and applied to a Sephacryl S200 column equilibrated in SEC buffer at a flow rate of 1.0 ml/min. CaMK2G eluted at 65 minutes corresponding to a retention time of a monomeric protein of that size. Eluted fractions were 95% pure as judged by SDS-PAGE. 10 mM DTT was added to the final eluted sample.

Extraction

Procedure
Cell pellets in binding buffer plus 1 mM PMSF, 0.5 mM TCEP were lysed using a high pressure cell disrupter. The lysate was centrifuged at 17,000 rpm for 30 minutes and the supernatant collected for purification. Prior to purification the lysate was passed through a DE52 column (10g/L resin) to remove DNA.
Concentration:Centricon with a 10kDa cut off in SEC-buffer
Ligand
MassSpec:
Crystallization:The inhibitor BIM9 (AXXORA) was added to the protein in 3-fold molar excess and the complex concentrated to 13 mg/ml. Crystals were obtained in sitting drops using the vapor diffusion method by mixing 50 nl of the concentrated protein with 100 nl of a well solution containing 0.10M MgCl2, 0.1M HEPES pH 7.0, 20% PEG 6K, 10% ethylene glycol. A single crystal appeared after several days at 4°C and reached maximum size after two months.
NMR Spectroscopy:
Data Collection:The crystal was cryo-protected using the well solution supplemented with 25% ethylene glycol and flash frozen in liquid nitrogen. Diffraction data were collected at the SLS beam line X10SA to a maximum resolution of 2.25Å.
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.
White-Grindley, E., and Si, K. (2006). RISC-y Memories. Cell 124 , 23-26.
Yang, Y., Zhu, W. Z., Joiner, M. L., Zhang, R., Oddis, C. V., Hou, Y., Yang, J., Price, E. E., Gleaves, L., Eren, M. , et al. (2006). Calmodulin kinase II inhibition protects against myocardial cell apoptosis in vivo. Am J Physiol Heart Circ Physiol 291, H3065-3075.
Zhu, W., Woo, A. Y., Yang, D., Cheng, H., Crow, M. T., and Xiao, R. P. (2007). Activation of CaMKII-delta C is a common intermediate of diverse death stimuli-induced heart muscle cell apoptosis. J Biol Chem. in press.