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

Target ID:

CAMK2AA

Aliases:

CAMK2ACAMKAKIAA0968

Deposition Date:

Wednesday 30th July 2008

Families:

Protein Kinase

Related Diseases:

SignallingNeurobiology

Structure type:

protein-ligand

Download Coordinates:

2VZ6

Authors:

A.C.W.PIKE,P.RELLOS,O.KING,L.SHRESTHA,N.BURGESS-BROWN,A.ROOS,J.W.MURRAY,F.VON DELFT,A.EDWARDS,C.H.ARROWSMITH,M.WIKSTROEM,C.BOUNTRA,S.KNAPP

Site:

Oxford

2VZ6

tabs

Structure Details

The protein kinase CAMK2α is mainly expressed in brain and forms oligomeric, ring like structures composed of 6-12 subunits. There are four isoforms present in mammalian cells (alpha, beta, gamma, and delta).

CAMK2α is a prominent kinase in the central nervous system which constitutes up to 1% of total brain protein. It plays an important function in long-term potentiation and neurotransmitter release, which has been demonstrated in numerous mouse models. CaMK2α is activated by NMDA receptors and is involved in phosporylation and trafficking of AMPA receptors, both of which are important steps in long term potentiation. Although no mutation of CAMK2α has been identified in the human dysregulated expression of CAMK2α has been described in several neurological disorders like Alzheimers’s disease, depression and bipolar illness. CAMK2α also controls the growth of human osteosarcoma by regulating cell cycle progression.

The overall organization of each of the four CAMK2 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.

CAMK2α 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 CAMK2α.

Here we present the structure of CAMK2α at 2.3 Å resolution in complex with the cell-permeable indirubin derivative IDR E804. This inhibitor blocks the Scr-Stat3 signaling pathway and displays anti-tumor properties and has been shown to be a potent, ATP-competitive inhibitor of the kinase activities of Src (IC50 = 430 nM), Cdk1/cyclin E (IC50 = 210 nM), Cdk2/cyclin A (IC50 = 540 nM), and Cdk1/cyclin B (IC50 = 1.65 µM).

Materials and Methods

Entry Clone Source: Origene

Entry Clone Accession: NM_015981 Variant

SGC Construct ID: CAMK2AA-c026

GenBank GI number: gi|25952114

Vector: pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ]

Tags and additions: Tag sequence: *Cleavable N-terminal His6 tag.

Expressed sequence: (tag sequence in lowercase):
mhhhhhhssgvdlgtenlyfq*SMYQLFE
ELGKGAFSVVRRCVKVLAGQEYAAKIINT
KKLSARDHQKLEREARICRLLKHPNIVRL
HDSISEEGHHYLIFDLVTGGELFEDIVAR
EYYSEADASHCIQQILEAVLHCHQMGVVH
RDLKPENLLLASKLKGAAVKLADFGLAIE
VEGEQQAWFGFAGTPGYLSPEVLRKDPYG
KPVDLWACGVILYILLVGYPPFWDEDQHR
LYQQIKAGAYDFPSPEWDTVTPEAKDLIN
KMLTINPSKRITAAEALKHPWISHRSTVA
SCMHRQETVDCLKKFNARRKLKGA

Host: BL21 (DE3) Rosetta-Phage resistant

Expression protocol: 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 µl 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 OD₆₀₀reached ~0.5. After that the temperature was lowered to 18°C. Protein production was induced with 1mM IPTG and recombinant CAMK2AA 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.

Lysis andNi-affinity chromatography: Buffers: Binding buffer: 50 mM HEPES pH 7.5, 300mM NaCl, 20 mM Imidazole; Wash buffer 1: 50 mM HEPES pH 7.5, 1M NaCl, 20mM Imidazole; Wash buffer 2:as for lysis buffer; Elution buffer: 50mM HEPES pH 7.5, 300mM NaCl, 150 mM Imidazole.

Procedure: The 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. CAMK2AA 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 not cleaved.

Column 2: Size exclusion chromatography (Superdex S75, 60 x 1cm)

SEC-Buffers: 50 mM Hepes, pH 7.5, 300 mM NaCl, 5 mM DTT.

Procedure: The fractions eluted of the Ni-affinity chromatography were concentrated to about 4 ml 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.

Mass spec characterization: ESI-MS revealed that the protein had the expected mass of 35585 Da for the uncleaved and unphosphorylated protein.

Protein concentration: 12 mg/ml in SEC buffer using a centricon with a 10kDa cut off

Crystallization: CAMK2AA was crystallized at 4°C using the sitting-drop vapor diffusion method at 10 mg/ml in the presence of 1 mM IDR E804, a cell-permeable indirubin derivative (CalBioChem #402081). Diffraction quality crystals were obtained by mixing 150 nl of protein solution with 50 nl of 12% (w/v) PEG6000 at pH 8.5 in Tris buffer.

Data Collection: Crystals were flash frozen in liquid nitrogen using the crystallization buffer and 30% propylene glycol. Diffraction data were collected to 2.3 Å at the Swiss light source beam-line X10SA.

References

Elgersma, Y., Sweatt, J.D., and Giese, K.P. (2004). Mouse genetic approaches to investigating calcium/calmodulin-dependent protein kinase II function in plasticity and cognition. J Neurosci 24, 8410-8415.

Griffith, L.C. (2004a). Calcium/calmodulin-dependent protein kinase II: an unforgettable kinase. J Neurosci 24, 8391-8393.

Griffith, L.C. (2004b). Regulation of calcium/calmodulin-dependent protein kinase II activation by intramolecular and intermolecular interactions. J Neurosci 24, 8394-8398.

Yuan, K., Chung, L.W., Siegal, G.P., and Zayzafoon, M. (2007). alpha-CaMKII controls the growth of human osteosarcoma by regulating cell cycle progression. Laboratory investigation; a journal of technical methods and pathology 87, 938-950.

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.