Human PR domain containing 11, methyltransferase domain

Target ID:

PRDM11

Deposition Date:

Thursday 31st March 2011

Families:

Transferases

Related Diseases:

Chromatin and epigenetics

Structure type:

apo

Download Coordinates:

3RAY

Authors:

Dong A, Zeng H, Walker JR, Loppnau P, Bountra C, Weigelt J, Arrowsmith CH, Edwards AM, Min J, Wu H

Site:

Toronto

3RAY

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 11 (PRDM11) is a member of the PR-domain protein family. This sub-family of HKMTs consists of 17 family members. Several of the PRDMs have been characterized as tumor suppressors, including PRDM2, PRDM3, PRDM5 and PRDM16, but the mechanisms are largely unknown. Although little is known about the function of PRDM11 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 SET domain of human PRDM11 at 1.73 Å resolution.

Materials and Methods

PRMD11

PDB:3RAY

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:41349466
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal: His-tag with integrated TEV protease site: MHHHHHHSSGRENLYFQ*G
Host:E.coli BL21 (DE3) V2RpRARE

Construct

Prelude:
Sequence:gDSSAMEVEPKKLKGKRDLIVPKSFQQVDFWFCESCQEYFVDECPNHGPPVFVSDTPVPVGIPDRAALTIPQGMEVVKDTSGESDVRCVNEVIPKGHIFGPYEGQISTQDKSAGFFSWLIVDKNNRYKSIDGSDETKANWMRYVVISREEREQNLLAFQHSERIYFRACRDIRPGEWLRVWYSEDYMKRLHSMSQETIHRNLARGEKRLQREKSEQVLDNPEDLRGPIHLSVLRQGK
Vector:pET28a-MHL

Growth

Medium:Terrific Broth (TB) medium
Antibiotics:50 μg/ml of kanamycin
Procedure:PRDM11 was expressed in E.coli BL21 (DE3) V2RpRARE in Terrific Broth (TB) medium in the presence of 50 μg/ml of kanamycin. Cell were grown at 37 °C to an OD₆₀₀ of 1.5 and induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM, and incubated overnight at 15 °C.

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 fractions containing PRDM11 were pooled and TEV protease was added to the fractions and incubated at 4 degrees Celsius overnight. 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 10.2 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 degrees Celsius. For the purification the cell paste was thawed and resuspended in lysis buffer with protease inhibitor (0.1 mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:38.8 mg/ml
Ligand
MassSpec:Expected MW is 27434.9 Da
Measured MW is 27436.347 Da
Crystallization:Purified PRDM11 (10.1 mg/mL) was crystallized using hanging drop vapor diffusion method at 20 °C by mixing 1 µl of the protein solution with 1 µl of the reservoir solution containing 20 % PEG3350, 0.2 M KCl.
NMR Spectroscopy:
Data Collection:
Data Processing: