Human bromodomain containing 1, PWWP domain

Target ID:

BRD1

Deposition Date:

Friday 26th February 2010

Families:

Methyl-lysine readers

Related Diseases:

Chromatin and epigenetics

Structure type:

apo

Download Coordinates:

3LYI

Authors:

Lam R, Zeng H, Ni S, Bountra C, Weigelt J, Arrowsmith CH, Edwards AM, Bochkarev A, Min J, Wu H

Site:

Toronto

3LYI

tabs

Structure Details

Human bromodomain containing protein 1 (BRD1) is a component of MOZ/MORF complex, which composed at least of ING5, MYST3/MOZ, MYST4/MORF, MEAF6 and one of BRPF1, BRD1/BRPF2 and BRPF3. BRD1 contains a combination of domains usually found in chromatin-associated proteins: a PHD finger, a bromo domain, and a PWWP domain. The bromo domains have been shown to interact with acetylated lysine of histone tails and other proteins, while the PHD fingers bind to methylated lysine 4 of histone H3. PWWP domain (named after the conserved Pro-Trp-Trp-Pro motif), is composed of ~90 residues and is present within all eukaryotes. PWWP domains have been found in more than 60 proteins which are involved in the processes of DNA methylation, DNA repair and transcriptional regulation. Even though PWWP domain is believed to be a chromatin targeting module and the PWWP-mediated chromatin targeting is essential for the enzyme function during development, the exact function of the PWWP has remained elusive.

We have solved the crystal structure of the PWWP domain of human BRD1 protein at 2.1 Å resolution.

Materials and Methods

BRD1

PDB:3LYI

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:11321642
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal: His-tag with integrated TEV protease site: MHHHHHHSSGRENLYFQG
Host:E. coli BL21 (DE3) codon plus RIL (Stratagene).

Construct

Prelude:
Sequence:gSVLEPLKVVWAKCSGYPSYPALIIDPKMPRVPGHHNGVTIPAPPLDVLKIGEHMQTKSDEKLFLVLFFDNKRSWQWLPKSKMVPLGIDETIDKLKMMEGRNSSIRKAVRIAFDRAMNHLSRVHGE
Vector:pET28-MHL

Growth

Medium:
Antibiotics:
Procedure:BRD1 was expressed in E.coli BL21 (DE3) codon plus in Terrific Broth medium in the presence of 50 μg/ml of kanamycin. Cell were grown at 37 degC to an OD600 of 1.5 and induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM and incubated overnight at 15 degC.

Purification

Procedure
The lysate was loaded onto 5 ml HiTrap column (Amersham Biosciences), charged with Ni2+. The column was washed with 10 CV of Wash buffer, and the protein was eluted with elution buffer. The protein was dialyzed against buffer containing 20 mM HEPES, pH 7.4, 500 mM NaCl and 5% glyceral. TEV protease was added to combined fractions containing BRPF1. The protein was further purified to homogeneity by ion-exchange chromatography on Source 30S column (10x10) (Amersham Biosciences), equilibrated with buffer 20 mM PIPES, pH 6.5, and eluted with linear gradient of NaCl up to 500 mM concentration (20CV). Purification yield was 23 mg of the protein per 1L of culture.

Extraction

Procedure
Cells were harvested by centrifugation at 7,000 rpm. The cell pellets were frozen in liquid nitrogen and stored at -80˚C. For the purification the cell paste was thawed and resuspended in lysis buffer with protease inhibitor (0.1mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:53.3 mg/ml.
Ligand
MassSpec:Expected MW is 14284.85 Da, measured mass is 14286.2899 Da.
Crystallization:Purified BRD1 was crystallized using sitting drop vapor diffusion method at 20 °C by mixing 1 µl of the protein solution (10 mg/mL) with 1 µl of the reservoir solution containing 30% PEG2K-MME, 0.20 M KBr.
NMR Spectroscopy:
Data Collection:
Data Processing: