Human GCN5 general control of amino-acid synthesis 5-like 2

Target ID:

GCN5L2A

Aliases:

GCN5GCN5L2hGCN5KAT2AMGC102791PCAF-b

Deposition Date:

Wednesday 21st May 2008

Families:

Bromodomain

Related Diseases:

Epigenetics

Structure type:

apo

Download Coordinates:

3D7C

Authors:

Filippakopoulos, P., Eswaran, J., Picaud, S., Fedorov, O., Murray, J., von Delft, F., Arrowsmith, C.H., Edwards, A., Bountra, C., Knapp, S.

Site:

Oxford

ICM Datapack:

ICM Datapack

3D7C

tabs

Structure Details

Two related genes code in vertebrates for the histone acetyl transferase (HAT) GCN5: GCN5L2 and PCAF (p300/CBP-associated factor). The two protein share 73% amino acid identity and contain an N-terminal PCAF homology domain, and a C-terminal AT bromodomain which binds to acetyl-lysines (Nagy and Tora, 2007).

GCN5 exerts a global and gene specific HAT activity with histones as main substrates, but this protein has also been found to acetylate a number of transcription factors including Myc (Liu et al., 2008), BRCA1 (Oishi et al., 2006), members of the E2F family (Lang and Hearing, 2003) and p53 (Gamper and Roeder, 2008). GCN5 is also recruited by the viral protein Tat in HIV infection. Association of GCN5 regulates TAT transactivating activity which may trigger chromatin remodeling of proviral genes (Lusic et al., 2003).

Knockout mice of GCN5 die during embryogenesis. Embryos develop normally until day 7.5 dpc but growth is severely retarded by day 8.5 dpc. GCN5-/- embryos fail to form dorsal mesoderm lineages, including chordamesoderm and paraxial mesoderm which is due to a high incidence of apoptosis in these embryos (Xu et al., 2000). Further analysis in mice revealed the importance of GCN5 for development of anteroposterior patterning and anterior neural tube closure (Bu et al., 2007), (Lin et al., 2008). In addition GCN5 is involved in the mechanism of DNA-damage-dependent transcriptional repression as well as in promoting transcriptional activation depending on the transcriptional context.

GCN5 has been considered as a pharmacological target for treatment for metabolic disorders in which hepatic glucose output is deregulated (Lerin et al., 2006). Here we present the structure of the bromo-domain GCN5L2 at 2.1 Å resolution.

Materials and Methods

GCN5L2

PDB:3D7C

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:IMAGE:5575574
Entry Clone Source:MGC
SGC Clone Accession:
Tag:Tag sequence: mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*) N-terminal his6 tag.
Host:BL21(DE3)-R3-pRARE2

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfq*sMELKDPDQLYTTLKNLLAQIKSHPSAWPFMEPVKKSEAPDYYEVIRFPIDLKTMTERLRSRYYVTRKLFVADLQRVIANCREYNPPDSEYCRCASALEKFFYFKLKEGGLIDK
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:Growth medium, induction protocol: 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 over night 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 mls using Centricon concentrators (5 kDa 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
Extraction method: 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:Protein concentration: Centricon with a 5 kDa cut off in SEC-buffer.
Ligand
MassSpec:Mass spec characterization: The mass of the protein was calculated to be 15975 Da and experimentally determined mass was 15975 Da for the His tag containing protein.
Crystallization:Crystallization: Crystals were obtained using the vapor diffusion method. The protein was concentrated in gel filtration buffer to a protein concentration of 8.2 mg/ml. Sitting drops comprising 100 nl of the concentrated protein mixed with 50 nl of a well solution (0.1M TRIS pH 8.5, 25% PEG 3350) were equilibrated against well solution at 4°C. Crystals appeared within 2-3 days.
NMR Spectroscopy:
Data Collection:Data Collection: Crystals were cryo-protected using the well solution supplemented with 25% Ethylene Glycol and flash frozen in liquid nitrogen. Diffraction data were collected from a single crystal on a Rigaku FR-E SuperBright at a single wavelength of 1.5 Å. The structure was solved by molecular replacement and refined to 2.06 Å.
Data Processing:

References

Bu, P., Evrard, Y.A., Lozano, G., and Dent, S.Y. (2007). Loss of Gcn5 acetyltransferase activity leads to neural tube closure defects and exencephaly in mouse embryos. Molecular and cellular biology 27, 3405-3416.
Gamper, A.M., and Roeder, R.G. (2008). Multivalent binding of p53 to the STAGA complex mediates coactivator recruitment after UV damage. Molecular and cellular biology 28, 2517-2527.
Lang, S.E., and Hearing, P. (2003). The adenovirus E1A oncoprotein recruits the cellular TRRAP/GCN5 histone acetyltransferase complex. Oncogene 22, 2836-2841.
Lerin, C., Rodgers, J.T., Kalume, D.E., Kim, S.H., Pandey, A., and Puigserver, P. (2006). GCN5 acetyltransferase complex controls glucose metabolism through transcriptional repression of PGC-1alpha. Cell metabolism 3, 429-438.
Lin, W., Zhang, Z., Chen, C.H., Behringer, R.R., and Dent, S.Y. (2008). Proper Gcn5 histone acetyltransferase expression is required for normal anteroposterior patterning of the mouse skeleton. Development, growth & differentiation 50, 321-330.
Liu, X., Vorontchikhina, M., Wang, Y.L., Faiola, F., and Martinez, E. (2008). STAGA recruits Mediator to the MYC oncoprotein to stimulate transcription and cell proliferation. Molecular and cellular biology 28, 108-121.
Lusic, M., Marcello, A., Cereseto, A., and Giacca, M. (2003). Regulation of HIV-1 gene expression by histone acetylation and factor recruitment at the LTR promoter. The EMBO journal 22, 6550-6561.
Nagy, Z., and Tora, L. (2007). Distinct GCN5/PCAF-containing complexes function as co-activators and are involved in transcription factor and global histone acetylation. Oncogene 26, 5341-5357.
Oishi, H., Kitagawa, H., Wada, O., Takezawa, S., Tora, L., Kouzu-Fujita, M., Takada, I., Yano, T., Yanagisawa, J., and Kato, S. (2006). An hGCN5/TRRAP histone acetyltransferase complex co-activates BRCA1 transactivation function through histone modification. The Journal of biological chemistry 281, 20-26.
Xu, W., Edmondson, D.G., Evrard, Y.A., Wakamiya, M., Behringer, R.R., and Roth, S.Y. (2000). Loss of Gcn5l2 leads to increased apoptosis and mesodermal defects during mouse development. Nature genetics 26, 229-232.