Human SET and MYND domain containing 3, methyltransferase domain, in complex with S-adenosyl-methionine and glycerol

Target ID:

SMYD3

Deposition Date:

Monday 28th February 2011

Families:

Transferases

Related Diseases:

CancerChromatin and epigenetics

Structure type:

protein-ligand

Download Coordinates:

3QWP

Authors:

Dong A, Dombrovski L, Li Y, Wernimont A, Weigelt J, Bountra C, Arrowsmith CH, Edwards AM, Min J, Wu H

Site:

Toronto

3QWP

tabs

Structure Details

Histone methylation plays an important role in transcriptional regulation of gene expression. It can either activate or repress transcription, depending on the residue modified and the degree of methylation of its ε-amino group. Methylation of histone H3K4, H3K36 and H3K79 has been shown to be associated with transcriptional activation, while methylation of H3K9, H3K27 and H4K20 associate with transcriptional repression.

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. SET-domain containing proteins can be classified into multiple sub-families based on their domain structure and substrate specificity. Among all the subfamilies, they all share a canonical core domain flanked by variable structural content. SMYD (SET and MYND) proteins belong to one of the sub-families with a unique domain architecture. Five members have been identified in SMYD family with different specificity towards different histone marks: SMYD1 and SMYD3 methylate histone H3 at Lys4; SMYD2 modifies histone H3 at residue Lys36; the sites of modification by SMYD4 and SMYD5 remain unclear. Many of the SMYD proteins are direct regulators of certain cancers and essential developmental process through its chromatin modification activity.

SMYD3 is a H3K4-specific methyltransferase which is frequently over-expressed in human colorectal, liver and breast cancer. This high level of expression of SMYD3 is essential for the growth of cancer cells. But how SMYD3 regulates the development and progression of these malignancies remains elusive. Meanwhile, SMYD3 is also found to be involved in estrogen receptor (ER)-mediated transcription via its histone methyltransferase activity in estrogen signaling pathway. Previous studies have also shown that the N-terminus of SMYD3 plays a critical role in modulation of SMYD3's HMT activity.

In order to investigate the mechanism of the modulation of SMYD3 HMTase activity, we have solved the crystal structure of human SMYD3 in complex with S-adenosyl-L-methionine (SAM) at 2.09 Å resolution. The structure of SMYD3 contains a conserved but split SET domain, a central MYND Zn-binding motif and a C-terminal TPR-like domain. The latter two domains are characteristic SMYD family proteins, and wrap around the core SET domain. These two domains provide the potential binding surfaces for the cellular binding partners of SMYD3 such as ERa and Hsp90.

Materials and Methods

SMYD3

PDB:3QWP

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:19570495
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:gMEPLKVEKFATANRGNGLRAVTPLRPGELLFRSDPLAYTVCKGSRGVVCDRCLLGKEKLMRCSQCRVAKYCSAKCQKKAWPDHKRECKCLKSCKPRYPPDSVRLLGRVVFKLMDGAPSESEKLYSFYDLESNINKLTEDrKEGLRQLVMTFQHFMREEIQDASQLPPAFDLFEAFAKVICNSFTICNAEMQEVGVGLYPSISLLNHSCDPNCSIVFNGPHLLLRAVRDIEVGEELTICYLDMLMTSEERRKQLRDQYCFECDCFRCQTQDKDADMLTGDEQVWKEVQESLKKIEELKAHWKWEQVLAMCQAIISSNSERLPDINIYQLKVLDCAMDACINLGLLEEALFYGTRTMEPYRIFFPGSHPVRGVQVMKVGKLQLHQGMFPQAMKNLRLAFDIMRVTHGREHSLIEDLILLLEECDANIRAS
Vector:pET28-MHL

Growth

Medium:M9 minimal medium
Antibiotics:50 μg/ml of kanamycin
Procedure:SMYD3 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 °C 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 °C.

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 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 6 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 degrees Celsius. For the purification, 11 g of the cell paste was thawed and resuspended in 110 ml 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 mg/ml
Ligand
MassSpec:Expected MW = 49920.25 Da
Measured MW = 49920.7725 Da
Crystallization:Purified SMYD3 (10 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 20% PEG 3,350, 0.2 M calcium chloride.
NMR Spectroscopy:
Data Collection:
Data Processing: