Human bromodomain containing protein 3 - domain 2

Target ID:

BRD3A@2

Deposition Date:

Thursday 25th January 2007

Families:

Bromodomain

Related Diseases:

CancerSignalling and epigenetics

Structure type:

apo

Download Coordinates:

2OO1

Authors:

P.FILIPPAKOPOULOS,A.BULLOCK,E.PAPAGRIGORIOU,T.KEATES,C.COOPER,C.SMEE,E.UGOCHUKWU,J.DEBRECZENI,F.VON DELFT,C.ARROWSMITH,A.EDWARDS,J.WEIGELT,M.SUNDSTROM,S.KNAPP,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2OO1

tabs

Structure Details

BRD3 belongs to the BET subclass of proteins, which are distinguished by two N-terminal bromodomains and one ET (Extra Terminal) domain. BRDs have been found to be associated with chromatin. The poorly characterized ET domain functions as a protein-protein interaction motif and may be part of an atypical serine-kinase activity. The subclass consists of at least four members in mouse and human, Brd2 (also referred to as Fsrg1, RING3), Brd3(Fsrg2, ORFX), Brd4 (Fsrg4, MCAP/HUNK1), and Brdt (Fsrg3, BRD6). BRD proteins are related to the female sterile homeotic protein gene in Drosophila, a gene required maternally for proper expression of other homeotic genes, such as Ubx, which is involved in pattern formation. BRD3 mRNA is ubiquitously expressed in human adult and fetal tissues with highest expression in testis, ovary, placenta, uterus, and brain. BRD3 expression is induced in activated lymphocytes and it is highly expressed in undifferentiated ES cells whereas expression levels are reduced upon endothelial differentiation.

Down regulated expression or loss of BRD3 has been detected in biopsies of nasopharyngeal carcinomas and altered expression levels have been found in bladder cancer. In addition, the BRD3 gene is located to chromosome 9q34, a region susceptible to genomic rearrangement in tumors . BRD3 has also been proposed as a marker for hormone dependent cancer. BRD3 also interacts with LANA-1, the Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen 1, which is required for the replication of episomal viral genomes. Here we report the structure of the second individual bromodomain of BRD3.

Materials and Methods

BRD3: Human bromodomain containing protein 3 (domain 2)

PDB:2OO1

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|11067749
Entry Clone Source:Origene
SGC Clone Accession:
Tag:mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*) N-terminal his6 tag.
Host:Rosetta (DE3)

Construct

Prelude:
Sequence: mhhhhhhssgvdlgtenlyfqsmGKLSE HLRYCDSILREMLSKKHAAYAWPFYKPVD AEALELHDYHDIIKHPMDLSTVKRKMDGR EYPDAQGFAADVRLMFSNCYKYNPPDHEV VAMARKLQDVFEMRFAKMP
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:3ml from a 50 ml overnight culture was used to inoculate each of two flasks containing 1 litre of LB media containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol. Cultures were grown at 37°C to an OD600 of ~0.4 and then cooled to 18°C. Expression was induced for 4 hours using 0.5mM 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; 5 mM imidazole.

Purification

Procedure

Extraction

Procedure
Cell pellets in binding buffer plus 1 mM PMSF, 0.5 mM TCEP were lysed using a high pressure cell disrupter. The lysate was centrifuged at 17,000 rpm for 30 minutes and the supernatant collected for purification. Prior to purification the lysate was passed through a DE52 column (10g/L resin) to remove DNA.
Concentration:
Ligand
MassSpec:
Crystallization:Crystals were obtained in sitting drops using the vapor diffusion method by mixing 150nl of the concentrated protein with 50nl of a well solution 0.2 M NaF; 0.1M BTProp pH 7.5; 20.0% PEG 3350; 10.0% EtGly. Crystals appeared after several days at 4°C.
NMR Spectroscopy:
Data Collection:Crystals were cryo-protected using the well solution and 25% ethylene glycol and flash frozen in liquid nitrogen. Diffraction data (1.6 Å) were collected at the Swiss light source (SLS) at a single wavelength (0.99 nm).
Data Processing:

References

Boyer, A., Lussier, J. G., Sinclair, A. H., McClive, P. J., and Silversides, D. W. (2004). Pre-sertoli specific gene expression profiling reveals differential expression of Ppt1 and Brd3 genes within the mouse genital ridge at the time of sex determination. Biol Reprod 71 , 820-827.
Ishii, H., Mimori, K., Mori, M., and Vecchione, A. (2005). Differentially expressed genes in endothelial differentiation. DNA Cell Biol 24 , 432-437.
Ottinger, M., Christalla, T., Nathan, K., Brinkmann, M. M., Viejo-Borbolla, A., and Schulz, T. F. (2006). Kaposi's sarcoma-associated herpesvirus LANA-1 interacts with the short variant of BRD4 and releases cells from a BRD4- and BRD2/RING3-induced G1 cell cycle arrest. J Virol 80 , 10772-10786.
Thorpe, K. L., Gorman, P., Thomas, C., Sheer, D., Trowsdale, J., and Beck, S. (1997). Chromosomal localization, gene structure and transcription pattern of the ORFX gene, a homologue of the MHC-linked RING3 gene. Gene 200 , 177-183.
Zhou, M., Peng, C., Nie, X. M., Zhang, B. C., Zhu, S. G., Yu, Y., Li, X. L., and Li, G. Y. (2003). Expression of BRD7-interacting proteins,BRD2 and BRD3, in nasopharyngeal carcinoma tissues. Ai Zheng 22 , 123-127.