Human TTK protein kinase

Target ID:

TTKA

Aliases:

ESKFLJ38280MPS1MPS1L1PYTTTK

Deposition Date:

Friday 29th February 2008

Families:

Protein Kinase

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

3CEK

Authors:

Filippakopoulos, P., Soundararajan, M., Keates, T., Elkins, J. M., King, O., Fedorov, O., Picaud, S. S., Pike, A.C.W., Roos, A., Pilka, E., von Delft, F., Arrowsmith, C.H., Edwards, A., Weigelt, J., Bountra, C., Knapp, S.

Site:

Oxford

ICM Datapack:

ICM Datapack

3CEK

tabs

Structure Details

TTK is the human orthologue of the yeast MPS1 (monopolar spindle) kinase, which is required for spindle pole duplication and the spindle checkpoint in yeast.

TTK is highly expressed in rapidly proliferating cells like testis, bone marrow and thymus and expression is enhanced in IL-2 responsive PBL blasts upon IL-2 treatment. TTK expression is cell cycle dependent and is negatively regulated by p53. The catalytic domain of TTK displays affinity for microtubules and microtubule binding is discussed to contribute to the regulation of kinase activity. Association to kinetochores of TTK is mediated by the amino-terminal, non-catalytic domain and specifically requires the presence of the Hec1/Ndc80-Nuf2 complex located at the kinetochore. TTK coordinates spindle attachment error corrections and checkpoint signaling. It is required for centrosome duplication, for normal progression of mitosis and the spindle checkpoint.

TTK has been suggested as potential target for cancer therapy. SNPs in TTK have been associated with breast cancer risk and elevated expression levels been reported in thyroid papillary carcinoma as well as in gastric cancer tissue and bronchogenic carcinoma. Here we report the apo-structure of the TTK kinase domain at 2.3Å resolution.

Materials and Methods

TTK

PDB:3CEK

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:IMAGE:5270012
Entry Clone Source:MGC
SGC Clone Accession:
Tag:Tag sequence: mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*) N-terminal his6 tag.
Host:BL21 (DE3) phage resistant Rosetta strain

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfq*smSVKGRIYSILKQIGSGGSSKVFQVLNEKKQIYAIKYVNLEEADNQTLDSYRNEIAYLNKLQQHSDKIIRLYDYEITDQYIYMVMECGNIDLNSWLKKKKSIDPWERKSYWKNMLEAVHTIHQHGIVHSDLKPANFLIVDGMLKLIDFGIANQMQPDTTSVVKDSQVGTVNYMPPEAIKDMSSSRENGKSKSKISPKSDVWSLGCILYYMTYGKTPFQQIINQISKLHAIIDPNHEIEFPDIPEKDLQDVLKCCLKRDPKQRISIPELLAHPYVQIQTH[L]VNQMAKGTTEEThe construct contains a mutation P797L highlighted in square brackets.
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:1ml from a 10 ml overnight culture containing 50 µg/ml kanamycin and 35 µg/ml chloramphenicol was used to inoculate 1 liter of LB media containing the same concentration of antibiotics. Cultures were grown at 37°C until the OD600 reached ~0.3. After that the temperature was adjusted to 20°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; 500 mM NaCl; 5% glycerol, 10 mM imidazole.

Purification

Procedure
Column 1: Ni-affinity chromatography.Buffers: Binding buffer: Binding buffer: 50 mM HEPES pH 7.5, 500 mM NaCl , 5% Glycero, 10mM Imidazole. Wash buffer: 50 mM HEPES pH 7.5, 500 mM NaCl , 30 mM Imidazole , 5% glycerol. Elution buffer: 50 mM HEPES pH 7.5, 500 mM NaCl , 50 to 250 mM imidazole, 5% Glycerol.Procedure: 5 ml of 50% Ni-NTA slurry (Qiagen) 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 1 and 2. The protein was eluted by gravity flow by applying 5 ml portions of elution buffer with increasing concentration of imidazole (50 mM, 100 mM, 150mM, 250 mM); fractions were collected until essentially all protein was eluted. The eluted protein was analyzed by SDS-PAGE. DTT was added to the protein sample to a final concentration of 5mM.Column 2: Size exclusion chromatography (Superdex S75, 60 x 1cm)SEC-Buffers: 50 mM Hepes, pH 7.5, 150 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.

Extraction

Procedure
Cell pellets were lysed by C5 high pressure homogenizer (Avestin). The lysate was centrifuged at 21,000 rpm for 60 minutes and the supernatant collected for purification.
Concentration:Centricon with a 30 kDa cut off in SEC-buffer.
Ligand
MassSpec:The mass of the protein was calculated to be 36064 Da and experimentally determined mass was 36785 Da for the His tag containing protein. The identity of the protein was reconfirmed to be correct by DNA sequencing both DNA strands of this expression construct. It is therefore likely that the difference in Mass is due to posttranslational modifications of the protein in regions not visible in the electron density.
Crystallization:Crystals were obtained using the vapor diffusion method and a protein concentration of 11 mg/ml by mixing 150nl of the concentrated protein with 150 nl of a well solution containing: 55w/v PEG 300; 0.25M NaCl; 6.4 pH Na/K phosphate.
NMR Spectroscopy:
Data Collection:Crystals were cryo-protected using the well solution supplemented with an additional 25% ethylene glycol and flash frozen in liquid nitrogen. Diffraction data were collected at the SLS beam line SAX10. Resolution: 2.4 Å.
Data Processing:

References

Abrieu, A., Magnaghi-Jaulin, L., Kahana, J.A., Peter, M., Castro, A., Vigneron, S., Lorca, T., Cleveland, D.W., and Labbe, J.C. (2001). Mps1 is a kinetochore-associated kinase essential for the vertebrate mitotic checkpoint. Cell 106, 83-93.

Hardwick, K.G., Weiss, E., Luca, F.C., Winey, M., and Murray, A.W. (1996). Activation of the budding yeast spindle assembly checkpoint without mitotic spindle disruption. Science (New York, NY 273, 953-956.

Kang, J., Chen, Y., Zhao, Y., and Yu, H. (2007). Autophosphorylation-dependent activation of human Mps1 is required for the spindle checkpoint. Proceedings of the National Academy of Sciences of the United States of America 104, 20232-20237.

Mattison, C.P., Old, W.M., Steiner, E., Huneycutt, B.J., Resing, K.A., Ahn, N.G., and Winey, M. (2007). Mps1 activation loop autophosphorylation enhances kinase activity. The Journal of biological chemistry 282, 30553-30561.

Salvatore, G., Nappi, T.C., Salerno, P., Jiang, Y., Garbi, C., Ugolini, C., Miccoli, P., Basolo, F., Castellone, M.D., Cirafici, A.M., et al. (2007). A cell proliferation and chromosomal instability signature in anaplastic thyroid carcinoma. Cancer research 67, 10148-10158.

Schmidt, M., Budirahardja, Y., Klompmaker, R., and Medema, R.H. (2005). Ablation of the spindle assembly checkpoint by a compound targeting Mps1. EMBO reports 6, 866-872.