Human suppressor of variegation 4-20 homolog 2, methyltransferase domain, in complex with S-adenosyl-L-methionine

Target ID:

SUV420H2

Deposition Date:

Wednesday 27th April 2011

Families:

Transferases

Related Diseases:

Chromatin and epigenetics

Structure type:

protein-ligand

Download Coordinates:

3RQ4

Authors:

Dong A, Zeng H, Tempel W, Loppnau P, Bountra C, Weigelt J, Arrowsmith CH, Edwards AM, Min J, Wu H

Site:

Toronto

3RQ4

tabs

Structure Details

Histone methyltransferases play an important role in controlling gene expression through site-specific methylation of lysines in core and linker histones within chromatin. Histone lysine methylation can either activate or repress transcription, depending on the residue modified and the degree of methylation of its ε-amino group. To date, there are five lysines within histone H3 (K4, K9, K27, K36 and K79) and one lysine within histone H4 (K20) have been shown to be methylated by specific histone lysine methyltransferases.

Human suppressor of variegation 4-20 homolog 2 protein (SUV420H2) contains a methyltransferase domain (SET domain). This protein methylates Lys20 of histone H4. H4 Lys20 methylation represents a specific tag for epigenetic transcriptional repression. SUV420H2 is associated with pericentric heterochromatin through interactions with HP1 proteins CBX1, CBX3 and CBX5, thereby it mainly functions in these regions and plays a central role in the establishment of constitutive heterochromatin in these regions. SUV420H2-generated dimethylation can facilitate DNA damage response and the selective trimethylation is involved in heterochromatin formation. It has been found that in breast cancer cells, the expression of SUV420H2 was significantly down regulated, accompanied by decreasing of trimethylation of H4K20.

We have solved the crystal structure of methyltransferase domain of human SUV420H2 in complex with S-adenosyl-L-methionine (SAM) at 1.8 Å resolution. The SUV420H2 structure has a typical SET domain fold, which comprised largely of β strands.

Materials and Methods

SUV420H2

PDB:3RQ4

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:31543169
Entry Clone Source:MGC
SGC Clone Accession:APC065C12
Tag:N-terminal: His-tag with integrated TEV protease site: MHHHHHHSSGRENLYFQG
Host:E.coli BL21 (DE3) V2R-pRARE

Construct

Prelude:
Sequence:gGPDRVTARELCENDDLATSLVLDPYLGFRTHKMNVSPVPPLRRQQHLRSALETFLRQRDLEAAYRALTLGGWTARYFQSRGPRQEAALKTHVYRYLRAFLPESGFTILPCTRYSMETNGAKIVSTRAWKKNEKLELLVGCIAELREADEGLLRAGENDFSIMYSTRKRSAQLWLGPAAFINHDCKPNCKFVPADGNAACVKVLRDIEPGDEVTCFYGEGFFGEKNEHCECHTCERKGEGAFRTRPRE
Vector:pET28-MHL

Growth

Medium:M9 minimal medium
Antibiotics:50 µg/ml of kanamycin
Procedure:SUV420H2 was expressed in E.coli BL21 (DE3) V2RpRARE in M9 minimal medium in the presence of 50 µg/ml of kanamycin. Cell were grown at 37°C to an OD600 of 0.8 and induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM, in the presence of 50 mg/L of SeMet 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 20 mM HEPES, pH 7.4, containing 250 mM NaCl and 50 mM imidazole, 5% glycerol, and the protein was eluted with elution buffer (20 mM HEPES pH 7.4, 250 mM NaCl, 250 mM imidazole, 5% glycerol). The protein was then loaded on to a Superdex200 (26X60, Amersham Biosciences) column equilibrated in 20 mM PIPES, pH 6.5 buffer containing 250 mM NaCl. TEV protease was added to combined fractions containing SUV420H1. 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 24 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°C. For the purification the cell paste was thawed and resuspended in lysis buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 2 mM β-mercaptoethanol, 5% glycerol, 0.1% CHAPS) with protease inhibitor (1mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:15 mg/ml
Ligand
MassSpec:Enzymatic treatment: TEV - Expected MW is 28137.0 Da, measured mass is 28137.3 Da.
Crystallization:Purified SUV420H2 (10 mg/mL) was complexed with S-adenosyl-L-methionine (SAM) (Sigma) at 1:10 molar ratio of protein:SAM and crystallized using hanging drop vapor diffusion method at 20 °C by mixing 1 µl of the protein solution with 1 µl of the reservoir solution containing 12% PEG8K, 0.1 M HEPES pH 7.2, 12% Ethylene glycol.
NMR Spectroscopy:
Data Collection:
Data Processing: