Human PR domain-containing protein 10, methyltransferase domain

Target ID:

PRDM10

Deposition Date:

Friday 31st July 2009

Families:

Methyltransferases

Related Diseases:

CancerChromatin and epigenetics

Structure type:

apo

Download Coordinates:

3IHX

Authors:

Amaya MF, Dombrovski L, Ni S, Weigelt J, Bountra C, Arrowsmith CH, Edwards AM, Botchkarev A, Min J, Plotnikov AN, Wu H

Site:

Toronto

3IHX

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 10 is a member of the PR-domain protein family. All the genes of the 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 some 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.

PRDM10 protein is a transcription factor that contains C2H2-type zinc-fingers. It also contains a positive regulatory domain, which has been found in several other zinc-finger transcription factors including those involved in B cell differentiation and tumor suppression. Studies of the mouse counterpart suggest that this protein may be involved in the development of the central nerve system (CNS), as well as in the pathogenesis of neuronal storage disease. We have solved the crystal structure of the methyltransferase domain of human PRDM10 at 2.5 Å resolution.

Materials and Methods

PRDM10

PDB:3IHX

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:41349458
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal: His-tag with integrated TEV protease site: MGSSHHHHHHSSGLVPRGS
Host:E.coli BL21 (DE3) codon plus RIL (Stratagene).

Construct

Prelude:
Sequence:gKHGPLHPIPNRPVLTRARASLPLVLYIDRFLGGVFSKRRIPKRTQFGPVEGPLVRGSELKDCYIHLKVSLDKGDRKERDLHEDLWFELSDETLCNWMMFVRPAQNHLEQNLVAYQYGHHVYYTTIKNVEPKQELKVWYAASYAEFVNQKIHD
Vector:pET28a-MHL

Growth

Medium:TB
Antibiotics:
Procedure:PRDM10 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 PIPES buffer, pH 6.5, and 250 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 5 mg of the protein per 1L of culture.

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 (1mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:8 mg/ml.
Ligand
MassSpec:Expected MW is 17926.64 Da.
Crystallization:Purified PRDM10 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: