Human polybromo 1 (5th bromodomain)

Target ID:

PB1A

Aliases:

BAF180MGC156155MGC156156PB1PBRM1

Deposition Date:

Wednesday 28th January 2009

Families:

Bromodomain

Related Diseases:

Epigenetics

Structure type:

apo

Download Coordinates:

3G0J

Authors:

Filippakopoulos, P., Keates, T., Picaud, S., von Delft, F., Arrowsmith, C.H., Edwards, A.M., Weigelt, J., Bountra, C., Knapp, S.

Site:

Oxford

3G0J

tabs

Structure Details

PB1, also known as BAF180, is a unique subunit of the Polybromo (PBAF) complex. Localisation of PBAF at the kinetochores of mitotic chromosome is consistent with its requirement for cell cycle progression through mitosis [1]. PB1 resembles closely an analogues complex described in yeast containing the subunits Rsc1, Rsc2 and Rsc3 [2]. Polybromo is composed of 6 Bromodomains, which bind acetylated histones, followed by 2 BAH domains known to act as protein-interaction modules and a C-terminal HMG box. HMGs have been shown to bind nucleosomal DNA. PB1 is widely expressed and thought to target PBAF to chromatin sites, recruit specific effector proteins and alter histone - DNA interactions [1,2].

The phenotype of PB1 knockout models points to an essential role of this protein in cardiac development. Mouse PB1-/- embryos die during development and show hypoplasia of cardiac ventricular free walls and ventricular septal defect and fail to fail to develop the plexus of coronary vessels. Mice also develop trophoblast placental defects [3,4]. Truncating mutations of PB1 have been identified in breast cancer suggesting a tumor suppressor role. Although the precise mechanism for its tumor repressor activity is not known yet it is thought that this occurs at least in part, through its ability to regulate p21 [5].

Here we report the structure of the fifth Bromodomain of PB1 at 1.78 Å resolution.

Materials and Methods

Entry Clone Source: Synthetic

Entry Clone Accession: n/a

SGC Construct ID: PB1A-c044

GenBank GI number: gi|30794372

Vector: pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ]

Entry clone accession/ sequence:
MHHHHHHSSGVDLGTENLYFQ^SMSGISPK
KSKYMTPMQQKLNEVYEAVKNYTDKRGRRL
SAIFLRLPSRSELPDYYLTIKKPMDMEKIR
SHMMANKYQDIDSMVEDFVMMFNNACTYNE
PESLIYKDALVLHKVLLETRRDLEGD
^ TEV cleave site

Tags and additions:Cleavable N-terminal His6 tag.

Amplified construct sequence:
TACTTCCAATCCATGAGCGGCATTAGCCCG
AAAAAAAGCAAATATATGACCCCGATGCAG
CAGAAACTGAACGAAGTGTATGAAGCGGTG
AAAAACTATACCGATAAACGCGGTCGCCGT
CTGAGCGCCATTTTTCTGCGCCTGCCGAGC
CGCAGCGAACTGCCGGATTATTATCTGACC
ATTAAAAAACCGATGGATATGGAAAAAATT
CGCAGCCACATGATGGCGAATAAATATCAG
GATATTGATAGCATGGTTGAAGATTTTGTT
ATGATGTTCAATAACGCGTGCACCTATAAC
GAACCGGAAAGCCTGATCTATAAAGATGCG
CTGGTGCTGCATAAAGTGCTGCTGGAAACC
CGCCGCGATCTGGAAGGCGATTGACAGTAA
AGGTGGATA

Host: BL21 (DE3)R3-pRARE2 (Phage resistant strain)

Growth medium, induction protocol: 5ml from a 50 ml overnight culture containing 50µg/ml kanamycin/34µg/ml chloramphenicol was used to inoculate each of two 1 litre cultures of LB containing 50 µg/ml kanamycin/34µg/ml chloramphenicol. Cultures were grown at 37°C until the OD₆₀₀ reached ~0.5 then the temperature was adjusted to 18°C. Expression was induced overnight using 0.5 mM IPTG at an OD₆₀₀ of 0.9. The cells were collected by centrifugation and the pellet re-suspended in binding buffer and frozen.
Binding buffer: 50mM HEPES pH 7.5; 500 mM NaCl; 5 mM imidazole, 5% glycerol.

Extraction buffer, extraction method: Frozen pellets were thawed and fresh 0.5mM TCEP, 1mM PMSF added to the lysate. Cells were lysed using sonication. The lysate was centrifuged at 16,500 rpm for 60 minutes and the supernatant collected for purification.

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, then washed with 20 ml binding buffer prior to loading the sample.

Buffers: 50mM HEPES pH 7.5; 500 mM NaCl; 5 mM imidazole, 5% glycerol

Procedure: Supernatant was applied by gravity flow, followed by a wash with 50 ml binding buffer. The column flow-through was collected.

Column 2: Ni-affinity. Ni-sepharose (Amersham), 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 (step elution).

Procedure: The flowthrough from column 1 was loaded by gravity flow on the Ni-sepharose column. The column was then washed with 30 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 and 250 mM); fractions were collected until essentially all protein was eluted. 10 mM DTT was added for overnight storage.

Enzymatic treatment: The N-terminal His tag was cleaved by treatment with TEV protease

Column 3: Size Exclusion Chromatography. Superdex S200 16/60 HiLoad

Buffers: 25 mM HEPES, pH 7.5; 300 mM NaCl, 0.5 mM TCEP

Procedure: PB1 was concentrated and applied to an S200 16/60 HiLoad gel filtration column equilibrated in 25mM HEPES, pH 7.5; 300mM NaCl, 0.5 mM TCEP using an ÄKTAexpress system.

Column 4: Ni-affinity. Ni-sepharose (Amersham), 2 ml of 50% slurry in a Bio-rad poly-prep column, washed with binding buffer.

Buffers:
Binding buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM imidazole, 5% Glycerol
Wash buffer: 25 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM TCEP
Elution buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 250 mM Imidazole , 5% Glycerol

Procedure: Gel filtration fractions containing PB1 were pooled and loaded by gravity flow on the Ni-sepharose column. After loading a further 4 ml of binding buffer was added and the full flow through was collected. The column was then washed with 5 ml wash buffer at gravity flow. Finally, 6 ml of elution buffer was added. Flow through, wash and elution fractions were analysed by SDS PAGE. The TEV-cleaved PB1protein was mainly found in the wash fraction. 10 mM DTT was added for overnight storage.

Mass spec characterization: LC- ESI -MS TOF gave a measured mass of 14626 for this construct as predicted from the sequence of this protein.

Protein concentration: Protein was concentrated to 11.49 mg/ml using an Amicon 3 kDa cut-off concentrator.

Crystallization: Crystals were grown at 4 °C in 600nl sitting drops from a 1:1 & 1:2 ratio of protein to reservoir solution 0.2M Na Nitrate; 25w/v PEG3350; 5v/v ethylene glycol.

Data Collection: Crystals were cryo-protected using the well solution supplemented with 20% ethylene glycol and flash frozen in liquid nitrogen.
X-ray source: Diffraction data were collected from a single crystal on a Rigaku FR-E SuperBright at a single wavelength of 1.5 Å and the structure was refined to 1.75 Å
Phasing: The structure was solved by molecular replacement using an ensemble of known bromodomain structures as a starting model.

References

1. Thompson, M. (2008). Polybromo-1: The chromatin targeting subunit of the PBAF complex. Biochimie.

2. Xue, Y., Canman, J.C., Lee, C.S., Nie, Z., Yang, D., Moreno, G.T., Young, M.K., Salmon, E.D., and Wang, W. (2000). The human SWI/SNF-B chromatin-remodeling complex is related to yeast rsc and localizes at kinetochores of mitotic chromosomes. Proceedings of the National Academy of Sciences of the United States of America 97, 13015-13020.

3. Wang, Z., Zhai, W., Richardson, J.A., Olson, E.N., Meneses, J.J., Firpo, M.T., Kang, C., Skarnes, W.C., and Tjian, R. (2004). Polybromo protein BAF180 functions in mammalian cardiac chamber maturation. Genes & development 18, 3106-3116.

4. Huang, X., Gao, X., Diaz-Trelles, R., Ruiz-Lozano, P., and Wang, Z. (2008). Coronary development is regulated by ATP-dependent SWI/SNF chromatin remodeling component BAF180. Developmental biology 319, 258-266.

5. Xia, W., Nagase, S., Montia, A.G., Kalachikov, S.M., Keniry, M., Su, T., Memeo, L., Hibshoosh, H., and Parsons, R. (2008). BAF180 is a critical regulator of p21 induction and a tumor suppressor mutated in breast cancer. Cancer research 68, 1667-1674.