Human myotonin-protein kinase, Form I

Target ID:

DMPK1A

Aliases:

DMDM1DM1PKDMKDMPKMDPKMT-PK

Deposition Date:

Sunday 30th September 2007

Families:

Protein Kinase

Related Diseases:

Signalling

Structure type:

protein-ligand

Download Coordinates:

2VD5

Authors:

A.C.W.PIKE,A.AMOS,J.ELKINS,A.BULLOCK,K.GUO,O.FEDOROV,G.BUNKOCZI,P.FILIPPAKOPOULOS,E.S.PILKA,E.UGOCHUKWU,C.UMEANO,F.NIESEN,M.SUNDSTROM,J.WEIGELT,A.EDWARDS,C.H.ARROWSMITH,F.VON DELFT,S.KNAPP

Site:

Oxford

ICM Datapack:

ICM Datapack

2VD5

tabs

Structure Details

DMPK is a Ser/Thr kinase of the AGC group of protein kinases. This kinase is expressed in skeletal muscle at neuromuscular junctions, myotendinous junctions, terminal cisternae of the sarcoplasmic reticulum (SR), intercalated discs in the cardiac muscle as well as in liver.

DMPK is activated in response to G protein second messengers but in contrast to other members of this family no GTPase binding domain has been described. When expressed it is maintained in an inactive conformation by an auto-regulatory C-terminal coiled-coil region.

DMPK is critical to the modulation of cardiac contractility and to the maintenance of proper cardiac conduction. It also plays a role in muscle differentiation by controlling the rearrangement of the cytoskeleton that takes place during elongation and fusion of myblasts into myotubes.

Low DMPK activity has been associated to the development of myotonic dystrophy (DM, Steinert disease), a neurodegenerative disorder characterized by myotonia, muscle wasting in the distal extremities, cataract, hypogonadism, defective endocrine functions and cardiac arrhythmias. Low DMPK expression levels in diseased tissue are the result of an amplified trinucleotide CTG repeat in the 3-prime untranslated region. Unaffected individuals have between 5 and 37 copies whereas DM patients who are minimally affected have at least 50 repeats, while more severely affected patients have expansion of the repeat-containing segment up to several kilobases. The CTG repeats are thought to alter local chromatin structure resulting in hyperstable nucleosomes that inhibit the passage of transcription complexes.

Here we present the structure of the kinase domain of DMPK in complex with the bisindoyl maleide inhibitor BIM VIII.

Materials and Methods

DMPK1

PDB:2VD5

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|976144
Entry Clone Source:MGC
SGC Clone Accession:IMAGE:6480902
Tag:N-terminal, TEV cleavable hexahistidine tag
Host:BL21(DE3)-R3-pRARE2

Construct

Prelude:
Sequence:smQQLVLDPGFLGLEPLLDLLLGVHQELGASELAQDKYVADFLQWAEPIVVRLKEVRLQRDDFEILKVIGRGAFSEVAVVKMKQTGQVYAMKIMNKWDMLKRGEVSCFREERDVLVNGDRRWITQLHFAFQDENYLYLVMEYYVGGDLLTLLSKFGERIPAEMARFYLAEIVMAIDSVHRLGYVHRDIKPDNILLDRCGHIRLADFGSCLKLRADGTVRSLVAVGTPDYLSPEILQAVGGGPGTGSYGPECDWWALGVFAYEMFYGQTPFYADSTAETYGKIVHYKEHLSLPLVDEGVPEEARDFIQRLLCPPETRLGRGGAGDFRTHPFFFGLDWDGLRDSVPPFTPDFEGATDTCNFDLVEDGLTAMVSGGGETLSDIREGAPLGVHLPFVGYSYSCMALRDSEVPGPTP
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:Transformation: The construct DNA was transformed into homemade chemically competent cells of the expression strain by a standard heat shock procedure.Glycerol stock prepataion: A number of colonies from the transformation were used to innoculate 1 ml of LB media containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol, which was placed in a 37°C shaker overnight. The next day glycerol stocks were prepared from this overnight culture.Expression: A glycerol stock was used to innoculate 10 ml of TB media containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol, which was placed in a 37°C shaker overnight. The next day this starter culture was used to innoculate 1L of LB media containing 50 µg/ml kanamycin. After growth at 37°C until the OD600 was around 0.5, the temperature was reduced to 18°C. The cells were induced by the addition of 1.0 mM IPTG when the OD600 was around 0.8. The expression was continued overnight. Two of these 1L growths were combined for the subsequent protein purification.

Purification

Procedure
Purification: The protein was purified by Ni-affinity chromatography, TEV protease digestion to remove the hexahistidine tag, and gel filtration.Column 1: Ni-NTA (2 ml)The clarified supernatent was passed through a column of DE52 resin to bind the DNA and then through a 2 ml volume Ni-NTA column. The Ni-NTA column was washed with Lysis Buffer, and then eluted with Binding Buffer containing 30 mM, then 50 mM, then 250 mM imidazole. The DMPK1A appeared in both the 50 mM and 250 mM imidazole elution fractions.Column 2: S200 16/60 Gel filtration.The GF column was pre-equilibrated with GF Buffer. The TEV protease digested Ni-NTA eluant was concentrated to a volume of about 8 ml. Two gel filtration runs were made, each loading half of the concentrated Ni-NTA eluant. The flow rate was 1.0 ml/min. Eluted proteins were collected in 1.8 ml fractions. The fractions containing protein were identified on a coomasie blue stained gel.

Extraction

Procedure
Cell harvest: Cells were spun down, resuspended in Lysis Buffer, and frozen at -20°C.Cell Lysis: The resuspended cells were thawed and lysed by sonication. The cell debris was spun down.
Concentration:The DMPK1A was exchanged into a buffer of 50 mM BisTrisPropane pH 6.5, 100 mM KCl, 0.5 mM TCEP and concentrated to 11 mg/ml (measured using a nanodrop machine) . The inhibitor BIM 8 (bisindolylmaleimide VIII, acetate salt) was added to a concentration of 0.5 mM.
Ligand
BIM 8 (bisindolylmaleimide VIII, acetate salt)MassSpec:Measured: 46061; Expected: 46059
Crystallization:Crystals grew from a 3:1 ratio of protein to precipitant solution (15% PEG3350, 0.2M MgCl 2 , 0.1M BisTris pH 6.3), using the vapour diffusion method.
NMR Spectroscopy:
Data Collection:Crystals were cryo-protected by equilibration into precipitant solution containing 25% ethylene glycol, and then flash frozen in liquid nitrogen. Data was collected at the Swiss Light Source beamline X10. Resolution: 2.8 Å.
Data Processing:

References

Bush, E.W., Helmke, S.M., Birnbaum, R.A., and Perryman, M.B. (2000). Myotonic dystrophy protein kinase domains mediate localization, oligomerization, novel catalytic activity, and autoinhibition. Biochemistry 39 , 8480-8490.

Dunne, P.W., Walch, E.T., and Epstein, H.F. (1994). Phosphorylation reactions of recombinant human myotonic dystrophy protein kinase and their inhibition. Biochemistry 33 , 10809-10814.

Furling, D., Lemieux, D., Taneja, K., and Puymirat, J. (2001). Decreased levels of myotonic dystrophy protein kinase (DMPK) and delayed differentiation in human myotonic dystrophy myoblasts. Neuromuscul Disord 11 , 728-735.

Garcia, P., Ucurum, Z., Bucher, R., Svergun, D.I., Huber, T., Lustig, A., Konarev, P.V., Marino, M., and Mayans, O. (2006). Molecular insights into the self-assembly mechanism of dystrophia myotonica kinase. Faseb J 20 , 1142-1151.

Dunne, P.W., Walch, E.T., and Epstein, H.F. (1994). Phosphorylation reactions of recombinant human myotonic dystrophy protein kinase and their inhibition. Biochemistry 33 , 10809-10814.

Mooers, B.H., Logue, J.S., and Berglund, J.A. (2005). The structural basis of myotonic dystrophy from the crystal structure of CUG repeats. Proceedings of the National Academy of Sciences of the United States of America 102 , 16626-16631.

Reddy, S., Smith, D.B., Rich, M.M., Leferovich, J.M., Reilly, P., Davis, B.M., Tran, K., Rayburn, H., Bronson, R., Cros, D., et al. (1996). Mice lacking the myotonic dystrophy protein kinase develop a late onset progressive myopathy. Nature genetics 13 , 325-335.

Salvatori, S., Fanin, M., Trevisan, C.P., Furlan, S., Reddy, S., Nagy, J.I., and Angelini, C. (2005). Decreased expression of DMPK: correlation with CTG repeat expansion and fibre type composition in myotonic dystrophy type 1. Neurol Sci 26 , 235-242.

Thornton, C.A., Wymer, J.P., Simmons, Z., McClain, C., and Moxley, R.T., 3rd (1997). Expansion of the myotonic dystrophy CTG repeat reduces expression of the flanking DMAHP gene. Nature genetics 16 , 407-409.

Tiscornia, G., and Mahadevan, M.S. (2000). Myotonic dystrophy: the role of the CUG triplet repeats in splicing of a novel DMPK exon and altered cytoplasmic DMPK mRNA isoform ratios. Molecular cell 5 , 959-967.