Human euchromatic histone methyltransferase 1 in complex with SAH and dimethylated H3K9 peptide

Target ID:

EHMT1

Aliases:

bA188C12.1DKFZp667M072Eu-HMTase1EUHMTASE1FLJ12879FP13812GLPKIAA1876KMT1DRP11-188C12.1

Deposition Date:

Sunday 30th September 2007

Families:

Transferases

Related Diseases:

Cancer and epigenetics

Structure type:

protein-ligand

Download Coordinates:

2RFI

Authors:

Min J, Wu H, Loppnau P, Weigelt J, Sundstrom M, Arrowsmith CH, Edwards AM, Bochkarev A, Plotnikov AN

Site:

Toronto

2RFI

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 euchromatic histone-lysine N-methyltransferase 1 (EHMT1) is a nuclear protein associated with euchromatic regions. It is a histone methyltransferase that methylates Lys-9 of histone H3 (in vitro). H3 Lys-9 methylation represents a specific tag for epigenetic transcriptional repression by recruiting HP1 proteins to methylated histones. EHMT1 contributes to silencing of MYC- and E2F-responsive genes, suggesting a role in G0/G1 transition in cell cycle. Human EHMT1 gene located on chromosome 9 (9q34.3). The sub-telomeric deletion on 9q34 has been found to be associated with recognizable mental retardation syndrome. The critical region responsible for this deletion is ~700 kbp. This region contains at least five genes and EHMT1 is one of them. We have solved the crystal structure of methyltransferase domain of EHMT1 in complex with S-adenosyl homocysteine (SAH) and a dimethylated H3K9 peptide. The structure provides us important information on substrate/co-factor binding.

Materials and Methods

EHMT1

PDB:2RFI

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:MGC
SGC Clone Accession:GI:40217808
Tag:N-terminal: His-tag with integrated thrombin protease site: MGSSHHHHHHSSGLVPRGS
Host:E.coli BL21 (DE3) codon plus RIL (Stratagene).

Construct

Prelude:
Sequence:gsNSQVWSALQMSKALQDSAPDRPSPVERIVSRDIARGYERIPIPCVNAVDSEPCPSNYKYVSQNCVTSPMNIDRNITHLQYCVCIDDCSSSNCMCGQLSMRCWYDKDGRLLPEFNMAEPPLIFECNHACSCWRNCRNRVVQNGLRARLQLYRTRDMGWGVRSLQDIPPGTFVCEYVGELISDSEADVREEDSYLFDLDNKDGEVYCIDARFYGNVSRFINHHCEPNLVPVRVFMAHQDLRFPRIAFFSTRLIEAGEQLGFDYGERFWDIKGKLFSCRCGSPKCRHS
Vector:pET28a-LIC

Growth

Medium:TB
Antibiotics:
Procedure:EHMT1 was expressed in E.coli BL21 (DE3) codon plus RIL in TB medium in the presence of 50 μg/ml of kanamycin. Cell were grown at 37oC to an OD600 of 0.8 and induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM, and incubated overnight at 15oC.

Purification

Procedure
The crude extract was cleared by centrifugation and passing through 20-ml DE52 column equilibrated in 20 mM Tris, pH 8.0, containing 500 mM NaCl and 5% glycerol. The lysate was loaded onto 5 ml HiTrap column (Amersham Biosciences), charged with Ni2+. The column was washed with 10 CV of 20 mM Tris pH 8.0, containing 250 mM NaCl and 50 mM imidazole, 5% glycerol, and the protein was eluted with elution buffer (20 mM Tris pH 8.0, 250 mM NaCl, 250 mM imidazole, 5% glycerol). The protein was dialyzed against 20 mM Tris, pH 8.0, 250 mM NaCl, 5% glycerol, 5mM β-mercaptoethanol in the presence of thrombin (Sigma). The protein was further purified to homogeneity by ion-exchange chromatography on Source 30Q column (10x10) (Amersham Biosciences), equilibrated with buffer 20 mM Tris-HCl, pH 8.0, and eluted with linear gradient of NaCl up to 500 mM concentration (20CV). Purification yield was 5.4 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 (50mM Tris pH 8.0, 0.25 M NaCl, 2 mM β-mercaptoethanol, 5% glycerol, 0.1% Igepal) with protease inhibitor (0.1mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:23 mg/ml
Ligand
MassSpec:Expected MW is 32935.24 Da, measured mass is 32893.84 Da.
Crystallization:EHMT1-AdoHcy-H3K9me complex (protein/AdoHcy/peptide molar ratio of 1:5:10) was crystallized in 16% PEG 4,000, 10 % isopropanol, 0.1 M HEPES (pH 7.5) using hanging drop vapor diffusion method drop at 20 °C by mixing 1.5 µl of the protein solution with 1.5 µl of the reservoir solution.
NMR Spectroscopy:
Data Collection:
Data Processing: