Human enhancer of zeste homolog 2, CXC and methyltransferase domains

Target ID:

EZH2

Deposition Date:

Friday 30th August 2013

Families:

Methyltransferases

Related Diseases:

CancerChromatin and epigenetics

Structure type:

apo

Download Coordinates:

4MI0

Authors:

Dong A, Zeng A, Wernimont A, He H, Bountra C, Arrowsmith CH, Edwards AM, Brown PJ, Wu H

Site:

Toronto

4MI0

tabs

Structure Details

Enhancer of zeste homolog 2 (EZH2) is the catalytic component of polycomb repressive complex 2 (PRC2), an epigenetic regulator of stem cell pluripotency, and expression of tissue-specific genes involved in cellular differentiation and developmental programs. EZH2 carries out a key function of the PRC2 complex, namely the sequential mono-, di- and trimethylation of Lysine 27 of histone H3 (H3K27) within chromatin. H3K27me3 is an epigenetic mark associated with transcriptional repression and contributes to repression of developmental genes, thereby participating in the maintenance of stem cell pluripotency. Overexpression or activating mutations of EZH2, the catalytic component of the PRC2 complex, are linked to hyper-trimethylation of lysine 27 of histone H3 (H3K27me3) in many cancers. Unlike most SET domain methyltranserases, EZH2 requires PRC2 components, SUZ12 and EED, for activity, but the mechanism by which catalysis is promoted in the PRC2 complex is unknown. We solved the 2.0 Å crystal structure of the EZH2 methyltransferase domain, revealing that most of the canonical structural features of SET domain methyltransferase structures are conserved. The site of methyl transfer is in a catalytically competent state, and the structure clarifies the structural mechanism underlying oncogenic hyper-trimethylation of H3K27 in tumors harboring mutations at Y641 or A677. On the other hand, the I-SET and post-SET domains occupy atypical positions relative to the core SET domain resulting in incomplete formation of the cofactor binding site and occlusion of the substrate binding groove. A novel CXC domain N-terminal to the SET domain may contribute to the apparent inactive conformation.

Materials and Methods

EZH2

PDB:4MI0

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:21361095
Entry Clone Source:MGC
SGC Clone Accession:
Tag:C-terminal non-cleavable His-tag.
Host:SF9

Construct

Prelude:
Sequence:mYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEYCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIPhhhhhh
Vector:pFBOHMHL

Growth

Medium:
Antibiotics:
Procedure:EZH2 was expressed in SF9 cells. The cells at density 4mln/ml were infected with 20ml of P3 viral stocks and shaken at 100 RPM at 27˚C for 48-72 hours until the viability drops to 70-80%.

Purification

Procedure
For purification the cell paste was thawed and resuspended in lysis buffer containing 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM imidazol, 2 mM ß-mercaptoethanol, 5% glycerol, 0.6% NP-40, protease inhibitor cocktail (Roche), 3000 U of benzonase (Novagen). Cells were lyzed by brief sonication. The clarified lysate was loaded onto a 2 mL TALON column (Clonetech). The column was washed with 50 column volumes of 20 mM Tris-HCl buffer, pH 8.0, containing 500 mM NaCl, 5% glycerol and 5 mM imidazole, and the protein was eluted with elution buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5% glycerol, 250 mM imidazole). The eluted protein was further purified to homogeneity on a Superdex200 column (GE Healthcare), equilibrated with 20 mM Tris-HCl buffer, pH 8.0, and 500 mM NaCl.

Extraction

Procedure
Cells were harvested by centrifugation at 5,000 rpm. The cell pellets were frozen in liquid nitrogen and stored at -80˚C.
Concentration:12 mg/ml
Ligand
MassSpec:expected mass is 26797.4 Da, measured mass is 26839.98 Da.
Crystallization:Purified EZH2 (10 mg/ml) was mixed with SAM at 1:10 molar ratio of protein:compound and crystallized using sitting drop vapor diffusion method at 20 °C by mixing 1 µl of the protein solution with 1 µl of the reservoir solution containing 20 % PEG3350, 0.1 M HEPES, pH 7.5, 0.2 M Li SO4.
NMR Spectroscopy:
Data Collection:
Data Processing: