Human SET domain-containing protein 2, methyltransferase domain, in complex with SAM

Target ID:

SETD2

Deposition Date:

Saturday 31st January 2009

Families:

Transferases

Related Diseases:

CancerChromatin and epigenetics

Structure type:

protein-ligand

Download Coordinates:

4H12

Authors:

Amaya MF, Zeng H, Mackenzie F, Bountra C, Weigelt J, Arrowsmith CH, Edwards AM, Bochkarev A, Min J, Wu H

Site:

Toronto

4H12

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. The methylation of histone lysine residues are mostly catalyzed by a family of SET-domain containing proteins. The SET domain is an evolutionarily conserved ~130 residues motif. In addition to the SET domain, most of the histone methyltransferases also carry other functional domains.

Human SET domain containing protein 2 (SETD2, also known as Huntingtin-interacting protein B (HYPB)) belongs to a class of proteins that interact with the Huntington's disease (HD) protein huntingtin. HD is a neurodegenerative disorder characterized by loss of striatal neurons, caused by an expansion of a polyglutamine tract in the HD protein huntingtin. SETD2 has been identified as a DNA binding protein. The SET domain of SETD2 methylates Lys-36 of histone H3. H3 Lys-36 methylation represents a specific tag for epigenetic transcriptional activation. SETD2 may act as a transcription activator that binds to promoters. It also plays a role in chromatin structure modulation during elongation via its interaction with hyperphosphorylated POLR2A. In addition, SETD2 binds to the promoters of adenovirus 12 E1A gene in case of infection, possibly lead to regulation of its expression.

We have solved the crystal structure of the SET domain of human SETD2 in complex with AdoMet at 2.0 Å resolution.

Materials and Methods

SETD2

PDB:4H12

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_054878
Entry Clone Source:MGC
SGC Clone Accession:GI:197313748
Tag:N-terminal: His-tag with integrated TEV protease site: MHHHHHHSSGRENLYFQG
Host:E.coli BL21 (DE3) codon plus RIL (Stratagen).

Construct

Prelude:
Sequence:gETSVPPGSALVGPSCVMDDFRDPQRWKECAKQGKMPCYFDLIEENVYLTERKKNKSHRDIKRMQCECTPLSKDERAQGEIACGEDCLNRLLMIECSSRCPNGDYCSNRRFQRKQHADVEVILTEKKGWGLRAAKDLPSNTFVLEYCGEVLDHKEFKARVKEYARNKNIHYYFMALKNDEIIDATQKGNCSRFMNHSCEPNCETQKWTVNGQLRVGFFTTKLVPSGSELTFDYQFQRYGKEAQKCFCGSANCRGYLGGENRVSIRAAGGKMKKERSRK
Vector:pET28-MHL

Growth

Medium:
Antibiotics:
Procedure:SETD2 was expressed in E.coli BL21 (DE3) codon plus RIL in Terrific Broth (TB) medium in the presence of 50 μg/mL of kanamycin at 37 ºC to an OD600 of 1.5. Cells were then induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM, and incubated overnight at 15 ºC.

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. 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 40 mg of the protein per 1L 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 ºC. For the purification, 11 g of the cell paste was thawed and resuspended in 110 mL lysis buffer with protease inhibitor (1 mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:26.1 mg/ml
Ligand
MassSpec:Expected MW = 31902.3 Da.
Measured MW= 31901.6 Da.
Crystallization:Purified SETD2 (10.3 mg/mL) was complexed with S-adenosyl-L-methionine (SAM) (Sigma) at 1:10 molar ratio of protein:SAM and 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 30% PEG 2K MME, 0.1 M KSCN.
NMR Spectroscopy:
Data Collection:
Data Processing: