Human euchromatic histone methyltransferase 1 in complex with SAH and mono-Methylated H3K9 peptide

Target ID:

EHMT1

Aliases:

bA188C12.1DKFZp667M072Eu-HMTase1EUHMTASE1FLJ12879FP13812GLPKIAA1876KMT1DRP11-188C12.1

Deposition Date:

Saturday 30th May 2009

Families:

Transferases

Related Diseases:

Chromatin and epigenetics

Structure type:

protein-ligand

Download Coordinates:

3HNA

Authors:

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

Site:

Toronto

3HNA

tabs

Structure Details

The methylation of a variety of biologically active molecules is a common theme in biology and RNA methylation is one of them. RNA methylation has been observed in different species of RNA, including mRNAs, tRNAs, and non-coding RNAs. The functional roles of RNA methylation are not completely understood. Recent studies have indicated that RNA methylation could be an epigenetic mechanism for regulation of gene expression and other cellular process. The methylation of various RNA molecules is catalyzed by a variety of RNA mathyltransferases and the modification can occur at different positions in RNA molecules. N6-methyladenosine (m6A) and 5-methycytosine (5-mC) are the most common and abundant methylation modification in RNA molecules in eukaryotes. Studies have shown that m6A and 5-mC RNA methylation can affect RNA stability and mRNA translation.

Human chromosome 9 open reading frame 114 (C9orf114) encodes a 376-aa protein of unknown function. We have solved the crystal structure of C9orf114 in complex with SAH at 1.86 Å resolution. The structure of C9orf114 contains a TIM-barrel-like fold, with a deep trefoil knot near the C-terminus. This structural arrangement is the characteristic of a family of RNA methyltransferase. Structural analysis by using the program DALI also showed that C9orf114 shares remarkable structural homology with a number of RNA methyltransferases.

Materials and Methods

EHMT1

PDB:3HNA

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:40217808
Entry Clone Source:MGC
SGC Clone Accession:
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: