Human hepatoma-derived growth factor-related protein 2, PWWP domain, in complex with an H3K79me3 peptide

Target ID:

HDGF2

Deposition Date:

Friday 28th January 2011

Families:

Methyl-lysine readers

Related Diseases:

CancerChromatin and epigenetics

Structure type:

protein-ligand

Download Coordinates:

3QJ6

Authors:

Zeng H, Amaya MF, Tempel W, Walker JR, Mackenzie F, Bountra C, Weigelt J, Arrowsmith CH, Edwards AM, Min J, Wu H

Site:

Toronto

3QJ6

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.

Among the many PWWP-containing proteins, the largest homologous group is related to the hepatoma-derived growth factor (HDGF), which has growth-stimulating activity in several specific cells. The PWWP domain always occurs at the N-termini of HDGF and the HDGF related proteins. Here we present the crystal structure of the PWWP domain of human HDGF2 protein at 2.24 Å resolution.

Materials and Methods

HDGF2

PDB:3QJ6

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:48255931
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:gMPHAFKPGDLVFAKMKGYPHWPARIDDIADGAVKPPPNKYPIFFFGTHETAFLGPKDLFPYDKCKDKYGKPNKRKGFNEGLWEIQNNPHASYS
Vector:pET28-MHL

Growth

Medium:Terrific Broth
Antibiotics:50 μg/ml of kanamycin
Procedure:HDGF2 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 °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 HDGF2. The cut and uncut protein of HDGF2 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 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:34.4 mg/ml
Ligand
MassSpec:Expected MW is 10696.15 Da
Measured mass is 10696.4774 Da
Crystallization:Purified HDGF2 was complexed with H3K79me3 peptide (EIAQDFK(me)3TDLRY ) and crystallized using hanging drop vapor diffusion method at 20 °C by mixing 1 µl of the protein solution (11.6 mg/mL) with 1 µl of the reservoir solution containing 2.0 M ammonium sulfate, 5% isopropanol.
NMR Spectroscopy:
Data Collection:
Data Processing: