Human bromodomain adjacent to zinc finger domain, 2B

Target ID:

BAZ2BA

Aliases:

BAZ2BDKFZP434H071DKFZp762I0516FLJ45644WALp4

Deposition Date:

Wednesday 28th January 2009

Families:

Bromodomain

Related Diseases:

Epigenetics

Structure type:

apo

Download Coordinates:

3G0L

Authors:

Filippakopoulos, P., Keates, T., Salah, E., Burgess-Brown, N., von Delft, F., Arrowsmith, C.H., Edwards, A.M., Weigelt, J., Bountra, C., Knapp, S.

Site:

Oxford

ICM Datapack:

ICM Datapack

3G0L

tabs

Structure Details

BAZ2B belongs to a family of ubiquitously expressed bromodomain containing proteins. Proteins of the BAZ family are characterized by a carboxy-terminal bromodomain adjacent to a PHD finger and a WACZ motif. In addition four other conserved motifs are typically found in the N-terminus of BAZ family members, namely the LH motif (a leucine-rich helical domain), the ZB2 motif and the BAZ 1 and BAZ 2 motifs [1].

The biological function of BAZ2B as not been clarified but it has been suggested that BAZ2B has a similar function as described for the Drosophila Acf1 protein, a protein regulating nucleosome mobilization by the ATP-dependent chromatin remodeling factor ISWI [2]. ISWI complexes mobilize nucleosomes, resulting in alterations in the translational position of the histone and genetic analysis implicates ISWI in transcriptional regulation and maintenance of chromosome structure. The interaction of BAZ2B with ISWI mediated by the BAZ1 motif has recently been described (Jones et al., 2000). Similarly, BAZ2B may also influence transcriptional regulation through its interaction with the CTBP2, (C terminal binding protein 2) [3]. Here we present the structure of the Bromo domain of BAZ2B at 2.0 Å resolution.

Materials and Methods

Entry Clone Source: Synthetic

Entry Clone Accession: n/a

SGC Construct ID: BAZ2BA-c011

GenBank GI number: gi|7304923

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

Entry clone accession/ sequence:
MHHHHHHSSGVDLGTENLYFQ^SMSVKKPK
RDDSKDLALCSMILTEMETHEDAWPFLLPV
NLKLVPGYKKVIKKPMDFSTIREKLSSGQY
PNLETFALDVRLVFDNCETFNEDDSDIGRA
GHNMRKYFEKKWTDTFKVS
^ TEV cleave site

Tags and additions: Cleavable N-terminal His6 tag.

Amplified construct sequence:
TACTTCCAATCCATGAGCGTGAAAAAACCG
AAACGCGATGATAGCAAAGATCTGGCGCTG
TGCAGCATGATTCTGACCGAAATGGAAACC
CATGAAGATGCGTGGCCGTTTCTGCTGCCG
GTGAACCTGAAACTGGTTCCGGGCTATAAA
AAAGTGATTAAAAAACCGATGGATTTTAGC
ACCATTCGTGAAAAACTGAGCAGCGGTCAG
TATCCGAACCTGGAAACCTTTGCGCTGGAT
GTGCGTCTGGTGTTTGATAACTGCGAAACC
TTTAACGAAGATGATAGCGATATTGGCCGT
GCCGGTCATAACATGCGCAAATATTTTGAA
AAAAAATGGACCGATACCTTTAAAGTTAGC
TGACAGTAAAGGTGGATA

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 resuspended 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 21,000 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: 50 mM HEPES, pH 7.5; 150 mM NaCl, 0.5 mM TCEP

Procedure: BAZ2B was concentrated and applied to an S200 16/60 HiLoad gel filtration column equilibrated in 25 mM HEPES, pH 7.5; 150 mM NaCl, 0.5 mM TCEP using an ÄKTA express system.

Mass spec characterization: LC- ESI -MS TOF gave a measured mass of 13598.4 for this construct as predicted from the sequence of this protein.

Protein concentration: Protein was concentrated to 18.43 mg/ml using an Amicon 3 kDa cut-off concentrator.

Crystallization: Crystals grown at 4 °C in 450nl sitting drops from a 1:1 & 1:2 ratio of protein to reservoir solution containing 0.1M MMT pH 6.0 and 30% PEG 1K.

Data Collection: Crystals were cryo-protected using the well solution supplemented with 25% 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 2.1 Å.
Phasing: The structure was solved by molecular replacement.

References

1. Jones, M.H., Hamana, N., Nezu, J., and Shimane, M. (2000). A novel family of bromodomain genes. Genomics 63, 40-45.

2. Eberharter, A., Vetter, I., Ferreira, R., and Becker, P.B. (2004). ACF1 improves the effectiveness of nucleosome mobilization by ISWI through PHD-histone contacts. The EMBO journal 23, 4029-4039.

3. Rual, J.F., Venkatesan, K., Hao, T., Hirozane-Kishikawa, T., Dricot, A., Li, N., Berriz, G.F., Gibbons, F.D., Dreze, M., Ayivi-Guedehoussou, N., et al. (2005). Towards a proteome-scale map of the human protein-protein interaction network. Nature 437, 1173-1178.