Human cyclin T2 isoform A

Target ID:

CCNT2A

Aliases:

FLJ90560MGC134840

Deposition Date:

Wednesday 21st June 2006

Families:

Miscellaneous

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

2IVX

Authors:

J.E.DEBRECZENI,A.BULLOCK,O.FEDOROV,P.SAVITSKY,G.BERRIDGE,S.DAS,A.C.W.PIKE,A.TURNBULL,E.UGOCHUKWU,E.PAPAGRIGORIOU,F.GORREC,M.SUNDSTROM,A.EDWARDS,C.ARROWSMITH,J.WEIGELT,F.VON DELFT,S.KNAPP

Site:

Oxford

ICM Datapack:

ICM Datapack

2IVX

tabs

Structure Details

Cyclins are regulatory subunits of cyclin-dependent kinases (CDKs). The cyclin T subfamily consists of three isoforms: cyclin T1, cyclin T2a, and T2b which are all structurally similar. Our structure corresponds to the N-terminal cyclin domain of T2 over which cyclin T2a and T2b are identical in sequence (the two proteins just differ in their extreme C-termini). Cyclin T1 has 80% sequence identity with our structure.

The CDK9/cyclin T complex, also known as positive transcription elongation factor b (P-TEFb), is essential for transcriptional elongation in human cells and activates gene expression in a kinase-dependent manner, phosphorylating the carboxy-terminal domain (CTD) of RNA polymerase II. The activity of the P-TEFb complex is regulated by the binding of 7SK RNA and HEXIM1 which sequester the P-TEFb in a large kinase-inactive complex.

CDK9/cyclin T2 complexes are involved in the regulation of terminal differentiation in muscle cells and in different forms of autosomal dominant myopathies. MyoD is reported to recruit CDK9/cyclin T2 to the promoters of muscle-specific genes and PKNalpha, a newly identified binding partner of cyclin T2A cooperates with T2A to enhance MyoD-dependent transcription. Expression of cyclin T2 is regulated during muscle cell differentiation and is undetectable in skeletal muscle cells from patients with centronuclear myopathy but high in normal skeletal muscle. Cyclin T2 is widely expressed in all cell types with high expression levels detected in differentiated tissues like blood, muscle, lymphoid tissue and connective tissue cells.

Materials and Methods

CCNT2: Human Cyclin T2

PDB:2IVX

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|4502629
Entry Clone Source:Origene
SGC Clone Accession:
Tag:mhhhhhhssgvdlgtenlyfq*s TEV-cleavable (*) N-terminal his6 tag.
Host:Rosetta

Construct

Prelude:Note the expressed protein has three changes (shown bold ) compared with gi|4502629.
Sequence: mhhhhhhssgvdlgtenlyfqsLASGRGA SSRWFFTREQLENTPSRRCGVEADKELSC RQQAANLIQEMGQRLNVSQLTINTAIVYM HRFYMHHSFTKFNKNIISSTALFLAAKVE EQARKLEHVIKVAHACLHPLEPLLDTKCD AYLQQTRELVILETIMLQTLGFEITIEHP HTDVVKCTQLVRASKDLAQTSYFMATNSL HLTTFCLQYKPTVIACVCIHLACKWSNWE IPVSTDGKHWWEYVDPTVTLELLDELTHE FLQILEKTPNRLKKIRNWRANQAARKPKV DGQVA
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:1 ml from a 10 ml overnight culture was used to inoculate each of three flasks containing 0.8 L of 2xTY media. Cultures were grown at 37°C until the OD600 reached ~0.7 before the temperature was adjusted to 18°C. Expression was induced for 5 hours using 0.1mM IPTG. The cells were collected by centrifugation and the pellets were frozen. All cultures contained 50 µg/ml kanamycin and 34 µg/ml chloramphenicol.

Purification

Procedure
Column 1: Ni-affinity chromatography.
5 ml of Ni-sepharose slurry was packed in a drip column and equilibrated with binding buffer. The lysate was applied to the column, washed with 100 ml of wash buffer before step elution with increasing imidazole. 10 mM DTT was added to the eluted protein. Fractions were analyzed by SDS-PAGE. The N-terminal his 6 -tag was cleaved by incubating the protein overnight with TEV protease.

Column 2: Size exclusion chromatography (Superdex S75, HiLoad 26/60)
The fractions eluted from the Ni-affinity chromatography were concentrated to ~4 mls using Amicon concentrators (10kDa cut off). The concentrated protein was applied to a Superdex S75 column equilibrated in SEC buffer at a flow rate of 2 ml/min. CCNT2A eluted at a retention time corresponding to the monomeric protein. 10 mM DTT was added to the elution which was >95% pure as judged by SDS-PAGE, and confirmed by mass spectrometry.

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

Extraction

Procedure
Cell pellets were resuspended in 70 ml binding buffer containing 1mM PMSF and 0.5 mM TCEP. Lysis was performed by high pressure homogeniser. The lysate was centrifuged at 16,000 rpm for 40 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:
Ligand
MassSpec:
Crystallization:Native: Crystals were obtained at 4°C using the vapor diffusion method with 150 nl sitting drops. 10 mg/ml protein was mixed at a ratio of 1:2 with the precipitant solution consisting of 0.2M sodium formate, 0.1M BisTrisPropane, 20% PEG 3350, 10% EtGly. Seleno-methionine: SeMet labeled crystals grew from 0.2M sodium fluoride, 20% PEG 3350, 10% EtGly at 4°C.
NMR Spectroscopy:
Data Collection:Diffraction data were collected at the Swiss Light Source X10 beamline to 1.8Å resolution from a crystal that had been cryo-protected with 20% EtGly.
Data Processing:The structure was solved using SeMet-SAD data collected at 0.9794 Å wavelength at the Swiss Light Source X10 beamline.

References

Napolitano, G., Majello, B., and Lania, L. (2002). Role of cyclinT/Cdk9 complex in basal and regulated transcription. Int J Oncol 21 , 171-177.

Napolitano, G., Majello, B., Licciardo, P., Giordano, A., and Lania, L. (2000). Transcriptional activity of positive transcription elongation factor b kinase in vivo requires the C-terminal domain of RNA polymerase II. Gene 254 , 139-145.

De Luca A , De Falco M , Baldi A , Paggi MG (2003) Cyclin T: three forms for different roles in physiological and pathological functions. J Cell Physiol. 194, 101-7.

Nguyen VT, Kiss T, Michels AA, Bensaude O. (2001). 7SK small nuclear RNA binds to and inhibits the activity of CDK9/cyclin T complexes. Nature. 414, 322-5.