Human BRD4 in complex with a diacetylated histone 4 peptide

Target ID:

BRD4A

Aliases:

CAPHUNKIMCAP

Deposition Date:

Wednesday 30th November 2011

Families:

Bromodomain

Related Diseases:

CancerChromatin and epigeneticsSignalling

Structure type:

protein-protein

Download Coordinates:

3UVY

Authors:

Filippakopoulos, P., Picaud, S., Keates, T., Felletar, I., Chaikuad, A., Krojer, T., Vollmar, M., Pike, A.C.W., von Delft, F., Arrowsmith, C.H., Edwards, A.M., Weigelt, J., Bountra, C., Knapp, S.

Site:

Oxford

3UVY

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.

Using arrays of acetylated peptides (SPOT) we were able to show that BET family members preferentially bind to poly rather than to singly acetylated histone tails. For instance a tetra- acetylated peptide that contained the H4 acetylation sites K5, K8, K12 and K16 bound with single digit µM KD to the first bromodomains of BRD2 and BRD4, increasing affinity at least 20-fold when compared to single marks. In order to determine whether the diverse sequence and spacing of histone Kac residues can be accommodated by a single BRD we systematically determined co-crystal structures of BRD4(1) with the di-acetylated peptides H4K5acK8ac, H4K12acK16ac and H4K16acK20ac. In all cases the two acetylated lysines bound simultaneously and with identical conformations to the BRD4(1) Kac binding site. The N-terminal Kac always formed the anchoring hydrogen bond with the conserved asparagines (N140). In the N-terminal region of H4, flexible glycine residues allow variable peptide conformations with two (H41-11K5acK8ac) or three (H411-21K12acK16ac) linking residues, whereas the large side-chains in H415-25K16acK20ac fit perfectly into surface grooves created by the ZA and BC loops. The generated co-crystal structures explain the similar affinities observed for the various combinations of di- and tri-acetylated H4 peptides. They also suggest that the greater apparent affinity of BRD4(1) and BRD2(1) for tetra acetylated H4 peptides is an avidity effect. However, not all di-acetylated H4 sequences are compatible with this bidentate recognition process. The co-crystal structure of H47-17K8acK12ac with BRD4(1) revealed a canonical mono-acetylated recognition mode suggesting that the H49-11 linker sequence is not suitable for a simultaneous recognition of the two Kac by a single BRD. Consistent with this notion, ITC experiments revealed a binding stoichiometry (N) of 0.5, indicating binding of two BRDs to the H4K8acK12ac peptide whereas only a single binding event with significantly increased affinity was observed for the H4K5acK8ac peptide.

Materials and Methods

BRD4A(1) (3UVY) Materials & Methods

Entry Clone Source: Synthetic

Entry Clone Accession: gi|19718731

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

Amplified construct sequence:
CATATGCACCATCATCATCATCATTC
TTCTGGTGTAGATCTGGGTACCGAGA
ACCTGTACTTCCAATCCATGAACCCC
CCGCCCCCAGAGACCTCCAACCCTAA
CAAGCCCAAGAGGCAGACCAACCAAC
TGCAATACCTGCTCAGAGTGGTGCTC
AAGACACTATGGAAACACCAGTTTGC
ATGGCCTTTCCAGCAGCCTGTGGATG
CCGTCAAGCTGAACCTCCCTGATTAC
TATAAGATCATTAAAACGCCTATGGA
TATGGGAACAATAAAGAAGCGCTTGG
AAAACAACTATTACTGGAATGCTCAG
GAATGTATCCAGGACTTCAACACTAT
GTTTACAAATTGTTACATCTACAACA
AGCCTGGAGATGACATAGTCTTAATG
GCAGAAGCTCTGGAAAAGCTCTTCTT
GCAAAAAATAAATGAGCTACCCACAG
AAGAATGACAGTAAAGGTGGATACGG
ATCCGAA

Final protein sequence (Tag sequence in lowercase):
mhhhhhhssgvdlgtenlyfq^smNP
PPPETSNPNKPKRQTNQLQYLLRVVL
KTLWKHQFAWPFQQPVDAVKLNLPDY
YKIIKTPMDMGTIKKRLENNYYWNAQ
ECIQDFNTMFTNCYIYNKPGDDIVLM
AEALEKLFLQKINELPTEE

^ TEV cleavage site

Tags and additions: Cleavable N-terminal His6 tag

Host: BL21(DE3)-R3-pRARE2

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 cell disruptor. 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 5ml 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:
Gel Filtration buffer: 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 ÅKTAexpress system.

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

Concentration: Protein was concentrated to 8.0 mg/ml using an Amicon 3kDa cut-off concentrator.

Crystallization: Prior to crystallization, protein buffer was exchanged to 50 mM HEPES pH7.5, 150 mM NaCl. Protein was mixed with the peptide at a ratio 50 µM protein for 1.5 mM peptide then concentrated to 8.0 mg/ml using an Amicon 3kDa cut-off concentrator. Crystals were grown at 4°C in 300 nl sitting drops from a 1:2 ratio of protein/peptide to reservoir solution containing 0.20 M NaI, 0.1 M BTProp pH 6.5, 20 % PEG3350 and 10 % EtGly.

Data Collection:
Resolution: 2.02 Å X-ray source: Diamond IO4
Crystals were cryo-protected using the well solution supplemented by 20% ethylene glycol and flash frozen in liquid nitrogen.
X-ray source: Diffraction data were collected from a single crystal at Diamond beamline I03 at a single wavelength of 0.9793 Å and the structure was refined to 2.02 Å
Phasing: The structure was solved by molecular replacement using an ensemble of known bromodomain structures as a starting model.

References

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.
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.
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.
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.
Filippakopoulos, P., Qi, J., Picaud, S., Shen, Y., Smith, W.B., Fedorov, O., Morse, E.M., Keates, T., Hickman, T.T., Felletar, I., Philpott, M., Munro, S., McKeown, M.R., Wang, Y., Christie, A.L., West, N., Cameron, M.J., Schwartz, B., Heightman, T.D., La Thangue, N., French, C.A., Wiest, O., Kung, A.L., Knapp, S*., and Bradner, J.E.* (2010). Selective inhibition of BET bromodomains. Nature 468, 1067-1073.
Panagis Filippakopoulos, Sarah Picaud, Oleg Fedorov, Marco Keller, Matthias Wrobel, Olaf Morgenstern, Franz Bracher, Stefan Knapp. Benzodiazepines and benzotriazepines as protein interaction inhibitors targeting bromodomains of the BET family. Bioorg Med Chem. 2011 Nov 4. In press.