Human Casein Kinase I gamma 3 in complex with K00577: a di-substituted diaminothiazole scaffold

Target ID:

CSNK1G3A

Deposition Date:

Wednesday 26th July 2006

Families:

Protein Kinase

Related Diseases:

CancerSignalling

Structure type:

apo

Download Coordinates:

2IZS

Authors:

G.BUNKOCZI,E.SALAH,P.RELLOS,S.DAS,O.FEDOROV,P.SAVITSKY,J.E.DEBRECZENI,O.GILEADI,M.SUNDSTROM,A.EDWARDS,C.ARROWSMITH,J.WEIGELT,F.VON DELFT,S.KNAPP

Site:

Oxford

ICM Datapack:

ICM Datapack

2IZS

tabs

Structure Details

Casein kinase I (CKI) represents a unique group of serine/threonine protein kinases that is ubiquitously expressed in eukaryotic organisms. Seven mammalian CKI isoforms ( α, β, γ1, γ2, γ3, δ and ε ) and various splice variants have been identified to date. CKI family members contain a highly conserved N-terminal catalytic domain coupled to a variable C-terminal region that ranges in size from 40 to 180 amino acids. Casein kinases have been described as monomeric, constitutively active enzymes.

CSNK1G3 is widely expressed with high RNA levels detected in testis and levels found in brain, heart, liver, kidney and muscle . The characterization of the substrate specificity of CKI isoforms initially led to the identification of the canonical consensus sequence S/T(P)-X 1â€“2 -S/T, indicating that modification of serine or threonine residues by CKI requires the preceding phosphorylation of amino acid residues N-terminal of the target site. This requirement of a priming phosphorylation by another kinase restricted CKI to a function in the hierarchical phosphorylation of substrates. However, further studies revealed that a cluster of acidic amino acids N-terminal of the target serine/ threonine and an acidic amino acid in position n-3 could substitute for the phosphoamino acid efficiently. This non-canonical motif consisting of the sequence SLS has been shown to be recognized by CKI in combination with a cluster of acidic amino acid residues C-terminal of the phosphoacceptor site. The CKI substrates NF-AT and b -catenin exhibit such motifs, but phosphorylation of this non-canonical motif is 15â€“25 fold less efficient compared to the motif primed by a phosphoamino acid.

siRNAs targeting of CNK1G3 significantly enhances cell killing by A-443654 , an inhibitor fro the protein kinase Akt. Small molecules targeting CSNK1G3 in addition to Akt may thus exhibit increased efficacy and have the potential for improved therapeutic index. We have crystallized CSNK1G3 in complex with several inhibitors:

A cell-permeable 2,6,9-substituted purine (K00083; CGP74514A; N-(cis-2-Aminocyclohexyl)-N-(3-chlorophenyl)-9-ethyl-9H-purine-2,6-diamine) that acts as a potent, selective inhibitor of Cdk1/cyclin B (IC50 = 25nM). Reported to affect the activities of other kinases only at much higher concentrations (IC50 = 6.1ÂµM, 125ÂµM, and > 10ÂµM for PKCa, PKA, and EGFR, respectively). This purine has been shown to induce mitochondrial damage and apoptosis (= 3ÂµM) in several human leukemia cell lines and at lower concentrations (~ 1ÂµM), an initial G2-M cell cycle arrest was observed in U937 cells, which eventually lead to apoptosis. (SGC structure 2IZT)
Purvalanol A (K00227; 2-(1R-Isopropyl-2-hydroxyethylamino)-6-(3-chloroanilino)-9-isopropyl-purine) A potent, cell-permeable, and selective inhibitor of cyclin-dependent kinases (Cdks; IC50 = 4nM for Cdc2/cyclin B; IC50 = 70nM for Cdk2/cyclin A; IC50 = 35nM for Cdk2/cyclin E; and IC50 = 75nM for Cdk5/p35). (SGC structure 200d, 2izu)
A di-substituted diaminothiazole scaffold related to AG-12286 (K00577) which has been described as CDK4 inhibitors. (This structure)
An related diaminothiazole with different substitution pattern at the two aryl rings (K00576) (SGC structure
2IZR).

Structural comparison showed that both diaminothiazole inhibitors rearranged the glycine rich loop when compared to the purine type inhibitors. In addition to this structural rearrangement the extended region between the activation loop and the P1 loop rotated towards the solvent in structures in complex with the diaminothiazole type inhibitors.

Materials and Methods

Entry clone source: Origene

Entry clone accession : NM_001031812

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

Sequence, tags and additions:
mhhhhhhssgvdlgtenlyfq*sMGVLMVGPNFRVGKKIG
CGNFGELRLGKNLYTNEYVAIKLEPMKSRAPQLHLEYRFY
KQLGSGDGIPQVYYFGPCGKYNAMVLELLGPSLEDLFDLC
DRTFSLKTVLMIAIQLISRMEYVHSKNLIYRDVKPENFLI
GRPGNKTQQVIHIIDFALAKEYIDPETKKHIPYREHKSLT
GTARYMSINTHLGKEQSRRDDLEALGHMFMYFLRGSLPWQ
GLKADTLKERYQKIGDTKRATPIEVLCENFPEMATYLRYV
RRLDFFEKPDYDYLRKLFTDLFDRKGYMFDYEYDWIGKQL
PTPVGAVQQDPALSSNREAHQHRDKMQQSKNQ

TEV-cleavable (*) N-terminal his6 tag.

Host : BL-21(DE3)R3 phage resistant

Growth medium, induction protocol: 1ml from a 10 ml overnight culture containing 50 µg/ml kanamycin was used to inoculate 1 liter of TB media containing 50 µg/ml kanamycin. Cultures were grown at 37°C until the OD 600 reached ~2.0. After that the temperature was adjusted to 25°C. Expression was induced for 4 hours using 1mM IPTG. The cells were collected by centrifugation and the pellet were frozen.

Extraction buffer, extraction method: Cell pellets were resuspended in 50 ml binding buffer lysed using sonication . The lysate was centrifuged at 19,000 rpm for 60 minutes and the supernatant collected for purification.

Column 1 : Ni-affinity, HisTrap, 1 ml (GE/Amersham)

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. Elution buffer: 50mM HEPES pH 7.5, 300mM NaCl, 150 mM Imidazole.

Procedure: The column was equilibrated with binding buffer. The lysate was applied to the column which was subsequently washed with wash buffer 1. CSNK1G3 was eluted with elution buffer. The eluted protein was collected and analyzed by SDS-PAGE. DTT was added to the protein sample to a final concentration of 5mM. The N-terminal his 6 -tag was cleaved by incubating the protein overnight with TEV protease, shrimp alkaline phosphatase and lambda phosphatase.

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

Buffers : Gel Filtration Buffer: 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 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. CSNK1G3 eluted at a retention time corresponding to the monomeric protein. Eluted fractions were 95% pure as judged by SDS-PAGE, and confirmed by mass spectrometry as the unphosphorylated protein.

Protein concentration: Centricon with a 10kDa cut off in SEC-buffer

Crystallisation & Data Collection: Crystals were obtained using the vapor diffusion method. di-substituted Diaminothiazole complexes were crystallized using a protein concentration of 16 mg/ml containing 1 mM of inhibitor. Drops were setup in 96 well sitting drop plates by mixing either 100nl of the concentrated protein with 50nl of a well solution containing 10% PEG10K, 0.2 M MgCl₂, 0.1M Tris pH 7.0 or 75 nl of protein solution with 75 nl of well solution. Diffraction data were collected at 1.30 Å and 1.95 Å resolution from a crystal that was cryo-protected with 20% propylene glycole using the SLS beamline X10SA or an FRE X-ray generator (Rigaku) and a HTC detector, respectively.

References

Knippschild U , Gocht A , Wolff S , Huber N , Lohler J , Stoter M . (2005) The casein kinase 1 family: participation in multiple cellular processes in eukaryotes. Cell Signal 17(6), 675-689.
Morgan-Lappe S , Woods KW , Li Q , Anderson MG , Schurdak ME , Luo Y , Giranda VL , Fesik SW , Leverson JD (2006). RNAi-based screening of the human kinome identifies Akt-cooperating kinases: a new approach to designing efficacious multitargeted kinase inhibitors. Oncogene 25,1340-1348.