Human SET domain containing 2, methyltransferase domain, in complex with cofactor-site inhibitor

Target ID:

SETD2

Deposition Date:

Monday 18th June 2012

Families:

Transferases

Related Diseases:

Chromatin and epigenetics

Structure type:

protein-ligand

Download Coordinates:

4FMU

Authors:

Dong A, Zeng H, Ibez G, Zheng W, Tempel W, Bountra C, Arrowsmith CH, Edwards AM, Brown PJ, Min J, Luo M, Wu H

Site:

Toronto

4FMU

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 Pr-SNF at 2.1 Å resolution.

Materials and Methods

SETD2

PDB:4FMU

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:197313748
Entry Clone Source:MGC
SGC Clone Accession:SETD2:JMC009-H07:C43809
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:SETD2 was expressed in E.coli BL21(DE3) codon plus RIL in Terrific Broth (TB) medium in the presence of 50 µg/ml ofkanamycin at 37oC 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 15oC.
Antibiotics:
Procedure:

Purification

Procedure
The crude extract was cleared by centrifugation. The clarifiedlysate was loaded onto 5 ml HiTrap Chelating column (Amersham Biosciences), chargedwith Ni2+. The column was washed with 10 CV of 20 mM HEPES buffer, pH 7.4,containing 250 mM NaCl and 50 mM imidazole, and the protein was eluted with elutionbuffer (20 mM HEPES, pH 7.4, 250 mM NaCl, 250 mM imidazole). The protein wasloaded on Superdex200 column (26x60) (Amersham Biosciences), equilibrated with20 mM PIPES buffer, pH 6.5, and 250 mM NaCl, at flow rate 4 ml/min. TEV proteasewas added to the pooled fractions to remove His-tag. The protein was further purifiedto homogeneity by ion-exchange chromatography on Source 30S column (10x10)(Amersham Biosciences), equilibrated with buffer 20 mM PIPES, pH 6.5, and eluted withlinear gradient of NaCl up to 500 mM concentration (20CV). Purification yield was 40mg of the protein per 1L of culture.

TEV cleavage.

Extraction

Procedure
Cells were harvested by centrifugation at 12,227 Xg. The cell pellets were frozen in liquid nitrogen and stored at -80°C. For thepurification, 11 g of the cell paste was thawed and resuspended in 110 ml lysis buffer(50 mM HEPES, pH 7.4, 500 mM NaCl, 5 mM imidazol, 2 mM ß-mercaptoethanol, 5%glycerol) with protease inhibitor (1 mM phenylmethyl sulfonyl fluoride, PMSF). Thecells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:26.1 mg/ml
Ligand
Sinefungin analogMassSpec:expected MW = 31902.3 Da, measured MW= 31901.6 Da.
Crystallization:Purified SETD2 (10.3 mg/ml) was complexed with Pr-SNF at 1:5molar ratio of protein:compound and crystallized using the sitting drop vapor diffusionmethod at 20 °C by mixing 1 µl of the protein solution with 1 µl of the reservoir solutioncontaining 20 % PEG4000, 10 % isoprpanel, 0.1 M HEPES, pH 7.5.
NMR Spectroscopy:
Data Collection:
Data Processing: