Human melanoma associated antigen (mutated) 1, PWWP domain, in complex with Arginine

Target ID:

MUM1

Deposition Date:

Wednesday 17th November 2010

Families:

Methyl-lysine readers

Related Diseases:

Chromatin and epigenetics

Structure type:

protein-ligand

Download Coordinates:

3PMI

Authors:

Lam R, Zeng H, Loppnau P, Bountra C, Weigelt J, Arrowsmith CH, Edwards AM, Bochkarev A, Min J, Wu H

Site:

Toronto

3PMI

tabs

Structure Details

Human melanoma associated antigen (mutated) 1 (MUM1) belongs to a family of transcription factors, interferon regulatory factors (IRFs). The IRF family contains at least 10 members. They are widely expressed and can modulate various biological processes including cellular responses to interferon, cell growth, susceptibility to transformation by oncogenes, induction of apoptosis, and the development of the T-cell immune responses. MUM1 is primarily expressed in B cells and activated T-lymphoid cells and is considered to be a critical mediator of lymphoid, myeloid, and dendritic-cell development. In a recent study, this protein has been found to be involved in the DNA damage response pathway by functioning as an architectural component of the chromatin. Upon DNA damage, MUM1 is recruited to the vincinity of DNA breaks by its interaction with 53BP1 and facilitates damage-induced chromatin changes. This function is required for efficient DNA repair and cell survival.

MUM1 contains a PWWP domain which mediates the interaction between MUM1 and nucleosomes. PWWP domain belongs to the Tudor domain 'Royal Family', which consists of Tudor, chromodomain, MBT and PWWP domains. PWWP domains have been found in more than 60 proteins which are involved in the processes of DNA methylation, DNA repair and transcriptional regulation. It is believed to be a chromatin targeting module and the PWWP-mediated chromatin targeting is essential for the protein function during development.

Here we present the crystal structure of the PWWP domain of human MUM1 protein at 2.8 Å resolution.

Materials and Methods

MUM1

PDB:3PMI

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:31652259
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 (Stratagene)

Construct

Prelude:
Sequence:gEPRSFEVGMLVWHKHKKYPFWPAVVKSVRQRDKKASVLYIEGHMNPKMKGFTVSLKSLKHFDCKEKQTLLNQAREDFNQDIGWCVSLITDYRVRLGCGSFAGSFLEYYAADISYPVRKSIQQDVLGTKLPQLSK
Vector:pET28-MHL

Growth

Medium:M9 medium in the presence of 50 μg/ml of kanamycin
Antibiotics:
Procedure:MUM11 was expressed in E.coli BL21 (DE3) V2R pRARE in growth medium. 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, in the presence of 50 mg/L of SeMet 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 dialyzed against buffer containing dialysis buffer. TEV protease was added to combined fractions containing MUM1. 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 13 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:26.3 mg/ml
Ligand
MassSpec:Expected MW is 15572.05 Da
Measured MW is 15572.7 Da
Crystallization:Purified MUM1 was crystallized using sitting drop vapor diffusion method at 20 °C by mixing 1 µl of the protein solution (10.5 mg/mL) with 1 µl of the reservoir solution containing 25% PEG 3,350, 0.1 M ammonium sulfate, 0.1 M HEPES, pH 7.5.
NMR Spectroscopy:
Data Collection:
Data Processing: