Human Histone-lysine N-methyltransferase SETMAR, transposase domain

Target ID:

SETMAR

Aliases:

METNASE

Deposition Date:

Friday 31st October 2008

Families:

Transferases

Related Diseases:

CancerChromatin and epigenetics

Structure type:

apo

Download Coordinates:

3F2K

Authors:

Amaya MF, Dombrovski L, Ni S, Bountra C, Weigelt J, Arrowsmith CH, Edwards AM, Bochkarev A, Min J, Plotnikov AN, Wu H

Site:

Toronto

3F2K

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 histone-lysine N-methyltransferase SETMAR, (also known as SET domain and mariner transposase fusion protein) is a highly expressed fusion between a histone H3 methylase and a mariner family transposase. The mariner transposase region is only present in primates and appeared 40-58 million years ago, after the insertion of a transposon downstream of a preexisting SET gene, followed by the de novo exonization of previously non-coding sequence and the creation of a new intron. Even though SETMAR is unlikely to catalyze transposition in the human genome, the nicking activity may have a role in the DNA repair phenotype. We have solved the crystal structure of the transposase domain of human SETMAR protein at 1.85 Å resolution. The structure of the core domain contains a four-stranded mixed β-sheet, flanked by four helices. Meanwhile, the N- and C-terminal helices form a cap on this core domain.

Materials and Methods

SETMAR

PDB:3F2K

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_006506
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 (Stratagen).

Construct

Prelude:
Sequence:gWVPHELTENQKNRRFEVSSSLILRNHNEPFLDRIVTCDEKWILYDNRRRSAQWLDQEEAPKHFPKPILHPKKVMVTIWWSAAGLIHYSFLNPGETITSEKYAQEIDEMNQKLQRLQLALVNRKGPILLHDNARPHVAQPTLQKLNELGYEVLPHPPYSPDLLPTNYHVFKHLNNFLQGKRFHNQQDAENAFQEFVESQSTDFYATGINQLISRWQKCVDCNGSYFD
Vector:pET28-MHL

Growth

Medium:
Antibiotics:
Procedure:SETMAR transposase domain was expressed in E.coli BL21 (DE3) codon plus RIL in M9 minimal medium in the presence of 50 μg/ml of kanamycin. Cell were grown at 37 degC 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 degC.

Purification

Procedure
The crude extract was cleared by centrifugation. The clarified lysate was loaded onto 5 ml HiTrap Chelating 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 loaded on Superdex200 column (26x60) (Amersham Biosciences), equilibrated with 20 mM Tris-HCl buffer, pH 8.0, and 150 mM NaCl, at flow rate 4 ml/min. TEV protease was added to the pooled fractions containing SETMAR transposase domain and incubated at 4 degC overnight. 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 8.8 mg of the protein per liter of culture.

Extraction

Procedure
Cells were harvested by centrifugation at 12, 227 Xg. The cell pellets were frozen in liquid nitrogen and stored at -80 degC. For purification, the cell paste was thawed and resuspended in lysis buffer with protease inhibitor (1mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:15.8 mg/ml.
Ligand
MassSpec:expected MW (SeMet derivative) = 26797.14 Da
measured MW= 26797.5134 Da.
Crystallization:Purified SETMAR transposase domain was crystallized using the 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% PEG 3,350, 0.2 M di-Na Tartrate.
NMR Spectroscopy:
Data Collection:
Data Processing: