Human bromodomain and PHD finger containing 3, PWWP domain

Target ID:

BRPF3

Deposition Date:

Friday 29th October 2010

Families:

Methyl-lysine readers

Related Diseases:

Chromatin and epigenetics

Structure type:

apo

Download Coordinates:

3PFS

Authors:

Bian CB, Lam R, Xu C, Bountra C, Arrowsmith CH, Weigelt J, Edwards AM, Bochkarev A, Min J

Site:

Toronto

3PFS

tabs

Structure Details

Human bromodomain and PHD finger-containing protein 3 (BRPF3) is a component of MOZ/MORF complex. The protein is associated with the histone acetyltransferase MOZ (MYST3) through its N-terminal domain and promotes histone acetylation in vivo. BRPF3 contains a combination of domains usually found in chromatin-associated proteins: one PHD fingers, a bromodomain, and a PWWP domain. The bromodomains 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.

Here we present the crystal structure of the PWWP domain of human BRPF3 protein at 1.9 Å resolution.

Materials and Methods

BRPF3

PDB:3PFS

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:148727368
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:gDYNGSGRSLLLPFEDRGDLEPLELVWAKCRGYPSYPALIIDPKMPREGLLHNGVPIPVPPLDVLKLGEQKQAEAGEKLFLVLFFDNKRTWQWLPRDKVLPLGVEDTVDKLKMLEGRKTSIRKSVQVAYDRAMIHLSRVRG
Vector:pET28-MHL

Growth

Medium:Terrific Broth medium in the presence of 50 μg/ml of kanamycin
Antibiotics:
Procedure:BRPF3 was expressed in E.coli BL21 (DE3) V2RpRARE in growth medium. Cell were grown at 37 °C to an OD₆₀₀ of 1.5 and induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM and incubated overnight at 15 °C.

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 dialysis buffer. TEV protease was added to combined fractions containing BRPF3. 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 20.5 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 degrees Celsius. 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:29.6 mg/ml
Ligand
MassSpec:Expected MW is 16019.6 Da
Measured MW is 16020.4 Da
Crystallization:Purified BRPF3 was crystallized using sitting drop vapor diffusion method at 20 °C by mixing 1 µl of the protein solution (17.3 mg/mL) with 1 µl of the reservoir solution containing 30% PEG 4,000, 0.2 M ammonium sulfate, 0.1 M sodium cacodylate, pH 6.5.
NMR Spectroscopy:
Data Collection:
Data Processing: