Human Wolf-Hirschhorn syndrome candidate 1-like 1 (WHSC1L1/NSD3), methyltransferase domain, in complex with the cofactor S-adenosylmethionine

Target ID:

WHSC1L1

Deposition Date:

Wednesday 25th March 2015

Families:

Methyltransferases

Related Diseases:

CancerChromatin and epigenetics

Structure type:

apo

Download Coordinates:

Superceded by 5UPD

Authors:

Tempel W, Yu W, Dong A, Cerovina T, Bountra C, Arrowsmith CH, Edwards AM, Brown PJ, Wu H

Site:

Toronto

4YZ8

tabs

Structure Details

Methylation of histone lysine residues is an important feature of epigenetic regulation of chromatin structure and function. The enzymes that catalyze the methylation reactions (HKMTs) have been implicated in numerous diseases. The nuclear receptor-binding SET domain (NSD) family of histone lysine methyltransferases consists of three members: NSD1, NSD2 (MMSET, WHSC1), and NSD3 (WHSC1L1). Dysfunctions of NSD proteins can trigger developmental defects such as Sotos syndrome and Wolf-Hirschhorn syndrome. A growing number of studies have shown that NSDs are oncogenes. Alteration and/or amplification of NSDs are associated with various cancers. The biological importance of NSD proteins in turmorigenesis makes them attractive therapeutic targets. Much effort has been put into discovery of inhibitors for NSD catalytic activity. However, the molecular mechanism of NSD protein functions is not fully understood. We have solved the crystal structure of NSD3 catalytic domain, bound with co-factor SAM at 1.8 Å resolution. The structure of NSD3 revealed a canonical SET-domain methyltransferase fold, containing pre-SET, SET and post-SET domains. One co-factor, SAM, was bound to the well-formed pocket between the SET and post-SET domain. Unlike the previously reported H3K36 methyltransferase structures, the auto-regulatory loop of NSD3 is rather closer to an active state than in an auto-inhibitory state. Our structure helps to understand the substrate recognition of H3K36 methyltransferases. It also sheds a light on designing of specific inhibitors for this family of HKMTs.

Materials and Methods

WHSC1L1

PDB:4YZ8

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC101717
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal: His-tag with integrated TEV protease site: MHHHHHHSSGRENLYFQG
Host:E.coli BL21 (DE3) V2R-pRARE

Construct

Prelude:
Sequence:gQRESKEALEIEKNSRKPPPYKHIKANKVIGKVQIQVADLSEIPRCNCKPADENPCGLESECLNRMLQYECHPQVCPAGDRCQNQCFTKRLYPDAEIIKTERRGWGLRTKRSIKKGEFVNEYVGELIDEEECRLRIKRAHENSVTNFYMLTVTKDRIIDAGPKGNYSRFMNHSCNPNCETQKWTVNGDVRVGLFALCDIPAGMELTFNYNLDCLGNGRTECHCGADNCSGFLG
Vector:pET28-MHL

Growth

Medium:
Antibiotics:
Procedure:WHSC1L1 was expressed in E.coli BL21 (DE3) V2R-pRARE cells. The transformed cells were grown in TB (terrific broth) medium in the presence of 50 μg/ml of kanamycin and chloramphenicol at 37oC to an OD600 of 3.0. The expression of WHSC1L1 was induced by addition of 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 the protein was purified by Ni-NTA column (Qiagen). The eluted protein fraction in 20 mM Tris-HCl, 250 mM NaCl, 250 mM imidazole) was loaded into a gel filtration column (Superdex 200, 16/60, GE Healthcare) which was pre-equilibrated with a buffer containing 200 mM PIPES, 250 mM NaCl, pH 6.5. The protein was then processed by in-house produced TEV protease overnight to remove His tag. The protein was further purified to homogeneity with a HiTrap SP FF (GE Healthcare) column, equilibrated with 50 mM PIPES, 10 mM NaCl, 2 mM DTT, pH 6.5 and eluted with linear gradient of NaCl up to 500 mM concentration. The purified protein was stored in 50 mM Tris-HCl, 20 mM NaCl, 1 mM TCEP, pH 8.5.

Enzymatic treatment: TEV cleavage.

Extraction

Procedure
Cells were harvested by centrifugation at 12, 227 Xg for 10 minutes at 4 oC. The cell pellets were frozen in liquid nitrogen and stored at -80˚C. For purification, the cell paste was thawed and resuspended in lysis buffer (20 mM Tris-HCl, pH 8.0, 250 mM NaCl, 2 mM mercaptoethanol, 5% glycerol, 20 mM imidazole) with protease inhibitor (1 mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:5.9 mg/ml
Ligand
MassSpec:expected MW = 26455.1 Da, measured MW= 26455.7275 Da.
Crystallization:Purified WHSC1L1 (5.6 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 0.5 µl of WHSC1L1/SAM solution with 0.5 μl reservoir solution (20% PEG 5K MME, 0.1 M Bis-Tris, pH 6.5) and 0.2 μl sarcosine (0.1 M; Hampton research). The crystals were flash frozen in liquid nitrogen using 15% glycerol as cryoprotectant.
NMR Spectroscopy:
Data Collection:
Data Processing: