Human DNA (cytosine-5-)-methyltransferase 3 beta (DNMT3B), PWWP domain

Target ID:

DNMT3B

Deposition Date:

Monday 29th December 2008

Families:

Transferases

Related Diseases:

Epigenetics

Structure type:

apo

Download Coordinates:

3FLG

Authors:

Amaya MF, Zeng H, MacKenzie F, Weigelt J, Bountra C, Arrowsmith CH, Edwards AM, Botchkarev A, Min J, Wu H

Site:

Toronto

3FLG

tabs

Structure Details

PWWP domain, which was 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. However, the exact function of the PWWP has remained elusive. Recently, the function of the PWWP domain as a chromatin targeting module has been indicated in the mammalian DNA methyltransferases. The PWWP-mediated chromatin targeting is essential for the enzyme function during development. The functional importance of the PWWP-mediated chromatin targeting was also suggested by the fact that a missense mutation in the PWWP domain causes ICF (immunodeficiency, centromeric heterochromatin instability and facial anomalies) syndrome, which is characterized by hypomethylation in classical satellite DNA. Experiments revealed that the mutant protein had completely lost its chromatin targeting capacity. However, the ligand and its functional interaction mechanism are still unknown.

DNMT3B is a member of the DNMT3 family. This protein is essential: inactivation of DNMT3B abolishes de novo methylation in mouse embryo. DNMT3B is required for methylation of centromeric minor satellite repeats. Some human diseases such as ICF syndrome and cancer have been linked to DNMT3B. DNMT3B is a multi-domain protein. It contains a catalytic domain at the C-terminus and a PWWP domain near the N-terminus. We present the crystal structure of the PWWP domain of human DNMT3B protein at 1.8 Å resolution. The structure can be divided into two parts: the N-terminal stranded structure and the C-terminal helix bundle. The two parts are packed against each other to form a structure module.

Materials and Methods

DNMT3B

PDB:3FLG

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_008823
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:gEADSGDGDSSEYQDGKEFGIGDLVWGKIKGFSWWPAMVVSWKATSKRQAMSGMRWVQWFGDGKFSEVSADKLVALGLFSQHFNLATFNKLVSYRKAMYHALEKARVRAGKTFPSSPGDSLEDQLKPMLEWAHGGFKPTGIEGLKPNNTQP
Vector:pET28-MHL

Growth

Medium:
Antibiotics:
Procedure:The PWWP domain of DNMT3B 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 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 then loaded on to a Superdex200 (60X60, Amersham Biosciences) column equilibrated in 20 mM HEPES, pH 7.4 buffer containing 250 mM NaCl. TEV protease was added to combined fractions containing DNMT3B. The cut and uncut protein of DNMT3B were separated on a Ni column. 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 8 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 degC. For the purification the cell paste was thawed and resuspended in lysis buffer with protease inhibitor (1mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:45.5 mg/ml.
Ligand
MassSpec:Expected MW is 16733.83 Da, measured mass is 16734.24 Da.
Crystallization:Purified PWWP domain of DNMT3B was crystallized using sitting drop vapor diffusion method at 20 °C by mixing 1 µl of the protein solution (22 mg/ml) with 1 µl of the reservoir solution containing 30% PEG 2,000 MME, 0.2 M KBr. Crystals were frozen in liquid nitrogen using glycerol as cryoprotectant.
NMR Spectroscopy:
Data Collection:
Data Processing: