Human two AAA domain containing protein

Target ID:

ATAD2A

Aliases:

ANCCAATAD2DKFZp667N1320MGC131938MGC142216MGC29843MGC5254PRO2000

Deposition Date:

Thursday 29th May 2008

Families:

Bromodomain

Related Diseases:

Chromatin and epigeneticsSignalling

Structure type:

apo

Download Coordinates:

3DAI

Authors:

Filippakopoulos, P., Keates, T., Picaud, S., Fedorov, O., Roos, A. K., Von Delft, F., Arrowsmith, C.H., Edwards, A., Bountra, C., Knapp, S.

Site:

Oxford

ICM Datapack:

ICM Datapack

3DAI

tabs

Structure Details

ATAD2 belongs to the family of AAA+ proteins, which are involved in a number of cellular processes ranging from protein degradation and DNA replication to membrane fusion and the movement of microtubule motors. AAA family proteins thereby often perform chaperone-like functions that assist in the assembly, operation, or disassembly of protein complexes. The hallmark of AAA (ATPases Associated with diverse cellular Activities) proteins is a 200-250 amino-acid ATP-binding domain [1]. Human ATAD2 consists of two AAA+ domains and 1 Bromo domain.

ATAD2 directly associates with ER-α (estrogen receptor alpha) in an estrogen dependent manner. It is a co-activator of ER-α and mediates estrogen-stimulated expression of key cell cycle regulators including cyclin D1, c-myc, and E2F1 and it is critical for the recruitment of the co-regulator CBP and histone hyperacetylation at the ER targeted chromatin [2].

ATAD2 is strongly induced by estrogen in human breast cancer cells and is required for estrogen-induced cell proliferation and cell cycle progression [2]. High expression of ATAD2 is correlated with an increased survival rate of high-grade osteosarcoma patients after chemotherapy and it is also differentially expressed in non-small-cell lung cancer [3, 4]. Here we present the structure of the Bromodomain of ATAD2 at 1.89 Å resolution.

Materials and Methods

ATAD2

PDB:3DAI

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|24497618
Entry Clone Source:synthetic
SGC Clone Accession:
Tag:mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*) N-terminal his6 tag.
Host:BL21(DE3)-R3-pRARE2

Construct

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

Growth

Medium:
Antibiotics:
Procedure:The expression plasmid was transformed into the host strain and plated on LB-agar containing 50 µg/ml kanamycin and 35 µg/ml chloramphenicol. Several colonies were combined to inoculate a 1ml culture in TB (+ 50 µg/ml kanamycin, 35 µg/ml chloramphenicol). The culture was grown overnight, glycerol was added to 15% v/v (from a 60% stock), and the resulting glycerol stock was frozen at -80°C in 100 µl aliquots. When required a 50 ml overnight culture was started by inoculating a loopful of cells from the glycerol stock into LB medium containing antibiotic as above. 5ml this overnight culture was used to inoculate similar 1 litre cultures (4L total) which were grown at 37°C until the OD600 reached ~0.5 at which point the temperature was adjusted to 18°C. Expression was induced overnight using 0.5 mM IPTG. 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 mM imidazole, 5% glycerol.)

Purification

Procedure
Column 1: Ion exchange - Nucleic acid removal. DEAE cellulose (DE52, Whatman), 10 g of resin in 2.5 x 20 cm column. The resin was hydrated in 2.5M NaCl, washed with 20 ml binding buffer prior to loading the sample.Buffers: 50mM HEPES pH 7.5; 500 mM NaCl; 5 mM imidazoleProcedure:Supernatant was applied by gravity flow, followed by a wash with 50 ml binding buffer. The column flowColumn 2: Ni-affinity. Ni-NTA (Qiagen), 5 ml of 50% slurry in 1.5 x 10 cm column, washed with binding buffer.Buffers: Binding buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM imidazole, 5% glycerol; 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: The flowthrough from column 1 was loaded by gravity flow on the Ni-NTA column. The column was then washed with 100 ml wash buffer at gravity flow. The protein was eluted by gravity flow by applying 5-ml portions of elution buffer with increasing concentration of imidazole (50 mM, 100 mM, 150 mM and 250 mM); fractions were collected until essentially all protein was eluted. 10 mM DTT was added for overnight storage together with TEV protease for cleavage of the N-terminal hexahistidine tag.Column 3: Size Exclusion Chromatography. Superdex S200 16/60 HiLoadBuffers:25 mM HEPES, pH 7.5; 150 mM NaCl, 0.5 mM TCEPProcedure: Protein was applied to a S200 gel filtration column using an ÄKTAxpress system at 1.0 ml/min. Eluted proteins were collected in 1.8-ml fractions and analyzed on SDS-PAGE. 10 mM DTT was added for storage.Column 4: Ni-affinity. Ni-NTA (Qiagen), 2 ml of 50% slurry in 1.5 x 10 cm column, washed with binding buffer.Procedure:The elution from column 3 (TEV cleaved) was loaded by gravity flow on a Ni-NTA column to remove Ni-binding contaminants. The column was washed with 10 ml wash buffer (as column 2) and proteins eluted as column 2. 10 mM DTT was added for overnight storage.

Extraction

Procedure
Frozen pellets were thawed and fresh 1 mM PMSF and 0.5 mM TCEP were added. Cells were lysed by sonication. The lysate was centrifuged at 21,000 rpm for 60 minutes and the supernatant collected for purification.
Concentration:The cleaved purified protein was concentrated in a VivaSpin500 (5 K MWCO) to 12.2 mg/ml and stored at 4°C. The protein concentration was determined spectrophotometrically.
Ligand
MassSpec:Observed mass without histidine tag, 15430.6 (calculated mass without histidine tag, 15431).
Crystallization:Crystals were obtained using the vapor diffusion method. The protein was concentrated in gel filtration buffer to a protein concentration of 12.2 mg/ml. Sitting drops comprising 75 nl of the concentrated protein mixed with 75 nl of a well solution (2.00M (NH4)2SO4; 0.1M BisTris pH 5.5) were equilibrated against well solution at 4°C. Crystals appeared within 2-3 days.
NMR Spectroscopy:
Data Collection:Crystals were cryo-protected using the well solution supplemented with 2M Li2SO4 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 1.5Å. The structure was solved by molecular replacement and refined to 1.89 Å.
Data Processing:

References

1. Hanson, P.I. and S.W. Whiteheart, AAA+ proteins: have engine, will work. Nat Rev Mol Cell Biol, 2005. 6(7): p. 519-29.

2. Zou, J.X., et al., ANCCA, an estrogen-regulated AAA+ ATPase coactivator for ERalpha, is required for coregulator occupancy and chromatin modification. Proc Natl Acad Sci U S A, 2007. 104(46): p. 18067-72.

3. Fellenberg, J., et al., Identification of drug-regulated genes in osteosarcoma cells. Int J Cancer, 2003. 105(5): p. 636-43.

4. Petroziello, J., et al., Suppression subtractive hybridization and expression profiling identifies a unique set of genes overexpressed in non-small-cell lung cancer. Oncogene, 2004. 23(46): p. 7734-45.