Human bromodomain containing 4 (bromodomain 1) in complex with JQ1/SGCBD01 probe against BET subfamily

Target ID:

BRD4A

Aliases:

CAPHUNKIMCAP

Deposition Date:

Friday 7th May 2010

Families:

Bromodomain

Related Diseases:

CancerChromatin and epigeneticsSignalling

Structure type:

protein-ligand

Download Coordinates:

3MXF

Authors:

Filippakopoulos, P., Picaud, S., Qi, J., Keates, T., Felletar, I., Fedorov, O., Muniz, J., von Delft, F., Arrowsmith, C., Edwards, A., Weigelt, J., Buntra, C., Bradner, J.E., Knapp, S.

Site:

Oxford

3MXF

tabs

Structure Details

BRD4 belongs to the BET subclass of proteins, which are distinguished by two N-terminal bromodomains and one ET (Extra Terminal) domain. Bromodomains are acetyl-lysine targeting modules commonly found in chromatin-modifying complexes. BET family members bind to mitotic chromosomes and regulate gene activity through cell cycle progression. Bromodomains in BRD4 bind to specific acetylated lysines in histones H3 and H4 and BRD4 has been isolated in complex with the replication factor complex (RFC). Mouse embryos izygous for BRD4 are non-viable due to an early post-implantation defect, suggesting that BRD4 is required for fundamental cellular processes. Disruption of BRD4 function in cell lines has been shown to inhibit progression to S phase.

In many virus-infected cells, the viral genome is similarly packaged into chromatin and chromatin targeting has been shown to be also critical for viral gene expression. BRD4 has been implicated in viral genome segregation and was found associated with E2, a protein encoded by human papillomaviruses (HPVs) and a regulator of the viral E6 oncoprotein. In addition, gene rearrangements of BRD4 have been detected in aggressive carcinoma. Here we determined the structure of the first bromodomain of BRD4 in complex with a BET family specific inhibitor at 1.6 Å resolution.

Materials and Methods

Entry Clone Source: FivePrime

Entry Clone Accession: NM_058243 Variant

SGC Construct ID: BRD4A-c002

GenBank GI number: gi|19718731

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

GI number: gi|19718731

Amplified construct sequence:
CATATGCACCATCATCATCATCATTCTTCT
GGTGTAGATCTGGGTACCGAGAACCTGTAC
TTCCAATCCATGAACCCCCCGCCCCCAGAG
ACCTCCAACCCTAACAAGCCCAAGAGGCAG
ACCAACCAACTGCAATACCTGCTCAGAGTG
GTGCTCAAGACACTATGGAAACACCAGTTT
GCATGGCCTTTCCAGCAGCCTGTGGATGCC
GTCAAGCTGAACCTCCCTGATTACTATAAG
ATCATTAAAACGCCTATGGATATGGGAACA
ATAAAGAAGCGCTTGGAAAACAACTATTAC
TGGAATGCTCAGGAATGTATCCAGGACTTC
AACACTATGTTTACAAATTGTTACATCTAC
AACAAGCCTGGAGATGACATAGTCTTAATG
GCAGAAGCTCTGGAAAAGCTCTTCTTGCAA
AAAATAAATGAGCTACCCACAGAAGAATGA
CAGTAAAGGTGGATACGGATCCGAA

Final protein sequence (tag sequence in lowercase):
mhhhhhhssgvdlgtenlyfq^MNPPPPET
SNPNKPKRQTNQLQYLLRVVLKTLWKHQFA
WPFQQPVDAVKLNLPDYYKIIKTPMDMGTI
KKRLENNYYWNAQECIQDFNTMFTNCYIYN
KPGDDIVLMAEALEKLFLQKINELPTEE

^ TEV cleave site

Tags and additions: Cleavable N-terminal His6 tag.

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

Growth medium, induction protocol: 10 ml from a 50 ml overnight culture containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol were used to inoculate each of two 1 liter cultures of TB containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol. Cultures were grown at 37°C until the OD₆₀₀ reached ~2.5 then the temperature was adjusted to 18°C. Expression was induced overnight using 0.1 mM IPTG at an OD₆₀₀ of 3.0. The cells were collected by centrifugation and the pellet re-suspended in binding buffer and frozen.
Binding buffer:50 mM HEPES pH 7.5; 500 mM NaCl; 10 mM imidazole, 5% glycerol.
Extraction buffer, extraction method: Frozen pellets were thawed and fresh 0.5 mM TCEP, 1 mM PMSF added to the lysate. Cells were lysed using sonication. The lysate was centrifuged at 17,000 rpm for 60 minutes and the supernatant collected for purification.

Column 1: Ni-affinity. Ni-sepharose (Amersham), 5 ml of 50% slurry in 1.5 x 10 cm column, washed with binding buffer.

Column 1 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, 5% glycerol, 50 to 250 mM Imidazole (step elution).

Column 1 Procedure: The supernatant 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 mM, 200 and 250 mM); fractions were collected until essentially all protein was eluted.

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

Column 2: Size Exclusion Chromatography. Superdex S75 16/60 HiLoad.

Column 2 Buffers:10 mM HEPES, pH 7.5; 500 mM NaCl, 5% glycerol.

Column 2 Procedure: The protein was concentrated and applied to an S75 16/60 HiLoad gel filtration column equilibrated in 10 mM HEPES, pH 7.5; 500mM NaCl, 5% glycerol using an ÄKTAexpress system.

Mass spec characterization: LC- ESI -MS TOF gave a measured mass of 15083 for construct as predicted from the sequence of this protein.

Protein concentration: Protein was concentrated to 10.3 mg/ml using an Amicon 3kDa cut-off concentrator.

Crystallization: Crystals were grown at 4°C in 300 nl sitting drops from a 1:1 ratio of protein (10.1 mg/ml + 2 mM ligand JQ1/SGCBD01) to reservoir solution containing 0.2 M NaI, 0.1 M BTProp pH 8.5, 20% PEG3350 and 10% ethylene glycol.

Mass spectrometry characterization: LC- ESI -MS TOF gave a measured mass of 22966 for this construct as predicted from the sequence of this protein.

Protein concentration: Protein was concentrated to 10 mg/ml using an Amicon 10 kDa cut-off concentrator.

Crystallisation: Crystals were grown at 20°C in 300 nl sitting drops from a 1:1 ratio of protein to reservoir solution containing 20% PEG-smear (PEG3350 & PEG MME5K), 0.1 M HEPES pH 7.5

Data Collection: Crystals were cryo-protected using the well solution supplemented by 25% ethylene glycol and flash frozen in liquid nitrogen.
X-ray source: Diffraction data were collected from a single crystal on a Rigaku FRE with an RAXIS IV detector at a single wavelength of 1.542 Å and the structure was refined to 1.6 Å.
Phasing: The structure was solved by molecular replacement using an ensemble of known bromodomain structures as a starting model.

References

1. Abbate, E. A., Voitenleitner, C., and Botchan, M. R. (2006). Structure of the papillomavirus DNA-tethering complex E2:Brd4 and a peptide that ablates HPV chromosomal association. Mol Cell 24 , 877-889.
2. French, C. A., Miyoshi, I., Aster, J. C., Kubonishi, I., Kroll, T. G., Dal Cin, P., Vargas, S. O., Perez-Atayde, A. R., and Fletcher, J. A. (2001). BRD4 bromodomain gene rearrangement in aggressive carcinoma with translocation t(15;19). Am J Pathol 159 , 1987-1992.
3. Houzelstein, D., Bullock, S. L., Lynch, D. E., Grigorieva, E. F., Wilson, V. A., and Beddington, R. S. (2002). Growth and early postimplantation defects in mice deficient for the bromodomain-containing protein Brd4. Mol Cell Biol 22 , 3794-3802.
4. Ilves, I. , Maemets, K., Silla, T., Janikson, K., and Ustav, M. (2006). Brd4 is involved in multiple processes of the bovine papillomavirus type 1 life cycle. J Virol 80 , 3660-3665.