Human PR domain containing 12

Target ID:

PRDM12

Deposition Date:

Tuesday 30th September 2008

Families:

Transferases

Related Diseases:

CancerChromatin and epigenetics

Structure type:

apo

Download Coordinates:

3EP0

Authors:

Amaya MF, Zeng H, Loppnau P, Bountra C, Weigelt J, Arrowsmith CH, Edwards AM, Bochkarev A, Min J, Plotnikov AN, Wu H

Site:

Toronto

ICM Datapack:

ICM Datapack

3EP0

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 (HKMTs). Majority of the HKMTs contain a ~150 amino-acid-long SET domain. The SET-domain containing HKMTs can be classified into 7 major families according to the sequences surrounding the SET domain: the SUV39, SET1, SET2, EZ, RIZ(PR domain-containing proteins), SMYD and SUV420 families.

Human PR domain containing protein 12 is a member of the PR-domain protein family. Although little is known about the function of PRDM12 protein, all the genes of the other members of this family are located to chromosomal regions that commonly undergo deletion in human cancer. PRDM family members share the feature of expressing two proteins that differ in the presence and absence of the PR domain. The studies of the other members of this family have shown that a variety of tumor types often overexpress the PR-domain negative protein instead of the full-length protein. We have solved the crystal structure of the methyltransferase domain of human PRDM12 at 2.1 Å resolution.

Materials and Methods

PRDM12

PDB:3EP0

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_067632
Entry Clone Source:MGC
SGC Clone Accession:GI:15042949
Tag:N-terminal: His-tag with integrated thrombin protease site: MGSSHHHHHHSSGLVPRGS
Host:E.coli BL21 (DE3) codon plus RIL (Stratagene).

Construct

Prelude:
Sequence:gsKTAFTAEVLAQSFSGEVQKLSSLVLPAEVIIAQSSIPGEGLGIFSKTWIKAGTEMGPFTGRVIAPEHVDICKNNNLMWEVFNEDGTVRYFIDASQEDHRSWMTYIKCARNEQEQNLEVVQIGTSIFYKAIEMIPPDQELLVWYGNSHNTFLGIPGVPGLEEDQKKNKHED
Vector:pET28a-LIC

Growth

Medium:
Antibiotics:
Procedure:PRDM12 was expressed in E.coli BL21 (DE3) codon plus in Terrific Broth (TB) medium in the presence of 50 μg/ml of kanamycin. Cell were grown at 37 degC to an OD600 of 1.5 and induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM, and incubated overnight at 15 degC.

Purification

Procedure
The lysate was loaded onto 5 ml HiTrap 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 14.5 mg of the protein per 1L of culture.

Enzymatic treatment: Thrombin.

Extraction

Procedure
Cells were harvested by centrifugation at 7,000 rpm. The cell pellets were frozen in liquid nitrogen and stored at -80 degC. For the purification the cell paste was thawed and resuspended in lysis buffer with protease inhibitor (0.1mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:39.8 mg/ml
Ligand
MassSpec:Expected MW is 19219.67 Da, measured mass is 19219.9691 Da.
Crystallization:Purified PRDM12 was crystallized using hanging drop vapor diffusion method at 20 °C by mixing 1 µl of the protein solution (5.0 mg/mL) with 1 µl of the reservoir solution containing 19% PEG 2,000 MME, 0.1 M KSCN. Crystal was frozen in liquid nitrogen using glycerol as cryoprotectant.
NMR Spectroscopy:
Data Collection:
Data Processing: