Human bromodomain, testis-specific [isoform a; tv1]

Target ID:

BRDTA

Aliases:

BRD6BRDT

Deposition Date:

Sunday 30th September 2007

Families:

Bromodomain

Related Diseases:

Epigenetics

Structure type:

apo

Download Coordinates:

2RFJ

Authors:

Filippakopoulos, P., Salah, E., Savitsky, P., Keates, T., Parizotto, E., Elkins, J., Pike, A.C.W., Ugochukwu, E., von Delft, F., Arrowsmith, C.H., Edwards, A., Weigelt, J., Sundstrom, M., Knapp, S.

Site:

Oxford

ICM Datapack:

ICM Datapack

2RFJ

tabs

Structure Details

The BET proteins constitute a family of bromodomain-containing proteins characterized by the presence of two bromodomains and a C-terminal domain designated the extra terminal (ET) motif. There are four BET sub-family members in the human genome: Brd2, Brd3, Brd4 and Brdt. Brdt is a testis-specific member of the BET family with a narrow expression pattern restricted to the germ line, specifically to pachytene and diplotene spermatocytes and early spermatids. BRDT specifically binds hyperacetylated histone H4 tails and the protein is essential for normal spermatogenesis. Specific deletion of the first bromodomain in mice results in abnormal spermatids and complete sterility. A recently published genome-wide association study of idiopathic male infertility identified single-nucleotide polymorphisms of BRDT as significantly associated with oligozoospermia or azoospermia in European men.

Aberrant expression of BRDT has been described in several cases of non-small cell lung cancer, as well as in a case of squamous cell carcinoma of the head and neck and esophagus.

The essential role of BRDT in spermatogenesis establishes a compelling rationale to target BRDT for a contraceptive effect. To enable development of BRDT specific inhibitors we determined the structure of the first Bromodomain of BRDT in complex with the pan-BET inhibitor (+)-JQ1.

Here we describe the structure of the first Bromodomain of BRDT at 2 Å resolution.

Materials and Methods

BRDT

PDB:2RFJ

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|46399198
Entry Clone Source:IMAGE
SGC Clone Accession:IMAGE:5742670
Tag:mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*) N-terminal his6 tag.
Host:BL21(DE3)-R3-pRARE2

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfq*smNTKKNGRLTNQLQYLQKVVLKDLWKHSFSWPFQRPVDAVKLQLPDYYTIIKNPMDLNTIKKRLENKYYAKASECIEDFNTMFSNCYLYNKPGDDIVLMAQALEKLFMQKLSQMPQEE
Vector: pNIC28-Bsa4.

Growth

Medium:
Antibiotics:
Procedure:1ml from a 10 ml overnight culture containing 50 µg/ml kanamycin was used to inoculate 1 liter of TB media containing 50 µg/ml kanamycin. Cultures were grown at 37°C until the OD600 reached ~2.0. After that the temperature was adjusted to 18°C. Expression was induced overnight using 1mM IPTG. The cells were collected by centrifugation and the pellets were frozen.

Purification

Procedure
Column 1: Ni-affinity chromatography ( HisTrap FF crude, 5 ml )All purification steps were carried out using an AKTAexpress system (GE Healthcare) at 7ºC. The lysate was loaded on a pre-equilibrated His-trap column at 0.8 ml/min, using a standard purification method. After loading, the column was washed at 0.8 ml/min with 10 ml binding buffer, then 20 ml wash buffer, and protein was eluted with 5 ml of elution buffer. The peak fraction was collected automatically according to A280.Column 2: Size exclusion chromatography (HiLoad 16/60 Superdex S75)The BRDTA containing fraction eluted of the Ni-affinity chromatography was automatically loaded on the SEC column at 1.2 ml/min. BRDTA eluted at a retention time corresponding to the monomeric protein. Eluted fractions were 95% pure as judged by SDS-PAGE , and confirmed by mass spectrometer (expected mass: 16614 Da).Tag cleavage and column 3: The protein was incubated with TEV protease overnight at 4ºC. After cleavage, the protein was passed through a Ni-sepharose column (0.5 ml bed volume) to capture the cleaved tag and other contaminants. The molecular mass after tag cleavage was confirmed by mass spectrometry (14149 Da).

Extraction

Procedure
The cell pellet (35 g) from 2 L cultue was re-suspended in one volume (35 ml) of 2x extraction buffer. The re-suspended cells were lysed by one passage through a high-pressure cell breaker (Constant Systems) and subsequent sonication; the cell breaker was washed with 1x extraction buffer, bringing the total volume to 120 ml. DNA was precipitation by addition of PEI (polyethyleneimine, pH 7.5) to a final concentration of 0.15 % during an incubation time of 30 min on ice, followed by a centrifugation at 17,000 rpm (4°C); The supernatant was further cleared by filtration through a 0.2 µm filter (serum Acrodisc).
Concentration:The protein was concentrated to 35 mg/ml using a Centricon device (5 kDa cut off).
Ligand
MassSpec:
Crystallization:Crystals were obtained using the vapor diffusion method. Drops were setup in 96 well sitting drop plates by mixing 100nl of the concentrated protein (35 mg/ml) with 100nl of a well solution containing 0.20M Na/K(tart); 20.0% PEG 3350; 10.0% EtGly.
NMR Spectroscopy:
Data Collection:Diffraction data were collected at 2.0 Å resolution from a crystal that was cryo-protected using 20% ethylene glycol (end concentration) at the SLS beam-line SAX10.
Data Processing:

References

Pivot-Pajot, C., Caron, C., Govin, J., Vion, A., Rousseaux, S., and Khochbin, S. (2003). Acetylation-dependent chromatin reorganization by BRDT, a testis-specific bromodomain-containing protein. Molecular and cellular biology 23 , 5354-5365.
Scanlan, M.J., Altorki, N.K., Gure, A.O., Williamson, B., Jungbluth, A., Chen, Y.T., and Old, L.J. (2000). Expression of cancer-testis antigens in lung cancer: definition of bromodomain testis-specific gene (BRDT) as a new CT gene, CT9. Cancer letters 150, 155-164.
Shang, E., Nickerson, H.D., Wen, D., Wang, X., and Wolgemuth, D.J. (2007). The first bromodomain of Brdt, a testis-specific member of the BET sub-family of double-bromodomain-containing proteins, is essential for male germ cell differentiation. Development (Cambridge , England ) 134 , 3507-3515.
Shang, E., Salazar, G., Crowley, T.E., Wang, X., Lopez, R.A., Wang, X., and Wolgemuth, D.J. (2004). Identification of unique, differentiation stage-specific patterns of expression of the bromodomain-containing genes Brd2, Brd3, Brd4, and Brdt in the mouse testis. Gene Expr Patterns 4, 513-519.
Zheng, Y., Yuan, W., Zhou, Z., Xu, M., and Sha, J.H. (2005). Molecular cloning and expression of a novel alternative splice variant of BRDT gene. International journal of molecular medicine 15 , 315-321.
Aston, K.I., Krausz, C., Laface, I., Ruiz-Castane, E., and Carrell, D.T. (2010). Evaluation of 172 candidate polymorphisms for association with oligozoospermia or azoospermia in a large cohort of men of European descent. Hum. Reprod. 25, 1383-1397.
Filippakopoulos, P., Qi, J., Picaud, S., Shen, Y., Smith, W.B., Fedorov, O., Morse, E.M., Keates, T., Hickman, T.T., Felletar, I., et al. (2010). Selective inhibition of BET bromodomains. Nature 468, 1067-1073.
Matzuk,MM., McKeown, MR.,Filippakopoulos, P. Li, Q., Ma,L., Agno,JE., Lemieux, ME., Picaud,S., Yu, RN., Qi, J., Knapp, S., Bradner, JE. (2012). Small-molecule inhibition of BRDT for male contraception. Cell, 150, 673-684.