Human bromodomain and PHD finger containing 1, PWWP domain in complex with H3K36me3

Target ID:

BRPF1

Deposition Date:

Thursday 22nd April 2010

Families:

Methyl-lysine readers

Related Diseases:

Chromatin and epigenetics

Structure type:

protein-ligand

Download Coordinates:

3MO8

Authors:

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

Site:

Toronto

3MO8

tabs

Structure Details

Human bromodomain and PHD finger-containing protein 1 (BRPF1) is a multi-domain protein expressed ubiquitously and at the highest level in testes and spermatogonia. It 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. BRPF1 contains a combination of domains usually found in chromatin-associated proteins: two 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 BRPF1 protein in complex with trimethylated H3K36 peptide at 1.69 Å resolution

Materials and Methods

BRPF1

PDB:3MO8

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:51173720
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:gEDSPLDALDLVWAKCRGYPSYPALIIDPKMPREGMFHHGVPIPVPPLEVLKLGEQMTQEAREHLYLVLFFDNKRTWQWLPRTKLVPLGVNQDLDKEKMLEGRKSNIRKSVQIAYHRALQHRSKVQGEQS
Vector:pET28-MHL

Growth

Medium:TB
Antibiotics:
Procedure:BRPF1 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 37oC to an OD600 of 1.5 and induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM and incubated overnight at 15oC.

Purification

Procedure
The lysate was loaded onto 5 ml HiTrap column (Amersham Biosciences), charged with Ni2+. The column was washed with 10 CV of 20 mM HEPES, pH 7.4, containing 500 mM NaCl and 50 mM imidazole, 5% glycerol, and the protein was eluted with elution buffer (20 mM HEPES pH 7.4, 500 mM NaCl, 250 mM imidazole, 5% glycerol). 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 12 mg of the protein per 1L of culture.
Enzymatic treatment: Thrombin

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 (50 mM HEPES, pH 7.4, 500 M NaCl, 2 mM β-mercaptoethanol, 5% glycerol, 0.1% CHAPS) with protease inhibitor (0.1mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:48 mg/ml
Ligand
MassSpec:Expected MW is 15052.51 Da, measured mass is 15052.9917 Da.
Crystallization:Purified BRPF1 was complexed with H3K36me3 peptide
(PATGGVK(me)3KPHRY, protein: peptide molar ratio at 1:5) and 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 3.5 M sodium formate, 0.1 M Tris-HCl, pH 8.5.
NMR Spectroscopy:
Data Collection:
Data Processing: