Human WD repeat domain 5

Target ID:

WDR5

Aliases:

BIG-3SWD3

Deposition Date:

Saturday 10th June 2006

Families:

Miscellaneous

Related Diseases:

Chromatin and epigenetics

Structure type:

apo

Download Coordinates:

2H9P

Authors:

J.R.MIN,A.SCHUETZ,A.ALLALI-HASSANI,F.MARTIN,P.LOPPNAU,M.VEDADI,J.WEIGELT,M.SUNDSTROM,A.M.EDWARDS,C.H.ARROWSMITH,A.BOCHKAREV,A.N.PLOTNIKOV,STRUCTURAL GENOMICS CONSORTIUM(SGC)

Site:

Toronto

2H9P

tabs

Structure Details

The WD40-repeat proteins are a large family of proteins found in all eukaryotes and implicated in very diverse functions such as signal transduction, vesicular trafficking, cytoskeletal assembly, cell cycle control, apoptosis, and transcription regulation. The common function of these WD40-repeat proteins is serving as a rigid scaffold for protein-protein interaction, and coordinating downstream events, such as ubiquitination and lysine methylation.

Recently it has been shown that a common component of MLL1, MLL2, and Set1 H3 K4 methyltransferase complexes, the WD40-repeat protein WDR5, directly associates with K4-dimethyalted H3 in vitro and in vivo. WDR5 is required for binding of the methyltransferase complex to the K4-dimethylated H3 tail as well as for global H3 K4 trimethylation and HOX gene activation in verterbrates.

Here we present the crystal structure of human WDR5 protein in complex with trimethylated H3K4 peptide. The structure provides important insights into substrate specificity.

Materials and Methods

WDR5-Histone 3

PDB:2H9P

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_060058
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal his tag with integrated thrombin protease site MGSSHHHHHHSSGLVPRGS
Host:E. coli BL21 (DE3) Codon Plus RIL (Stratagen)

Construct

Prelude:
Sequence:gsMATEEKKPETEAARAQPTPSSSATQSKPTPVKPNYALKFTLAGHTKAVSSVKFSPNGEWLASSSADKLIKIWGAYDGKFEKTISGHKLGISDVAWSSDSNLLVSASDDKTLKIWDVSSGKCLKTLKGHSNYVFCCNFNPQSNLIVSGSFDESVRIWDVKTGKCLKTLPAHSDPVSAVHFNRDGSLIVSSSYDGLCRIWDTASGQCLKTLIDDDNPPVSFVKFSPNGKYILAATLDNTLKLWDYSKGKCLKTYTGHKNEKYCIFANFSVTGGKWIVSGSEDNLVYIWNLQTKEIVQKLQGHTDVVISTACHPTENIIASAALENDKTIKLWKSDC
Vector:p28aLIC

Growth

Medium:
Antibiotics:
Procedure:WDR5 was expressed in E. coli BL21 (DE3) Codon Plus RIL in Terrific Broth (TB) in the presence of 50 µg/ml of kanamycin. Cell were grown at 37 oC to an OD600 of 1.5 and induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM, and incubated overnight at 12 degC.

Purification

Procedure
Column 1: 5 ml HiTrap Chelating column (Amersham Biosciences)
Column 2: Superdex200 column (26x60) (Amersham Biosciences)

The crude extract was cleared by centrifugation. 5 mM imidazole was added to the lysate. The sample was loaded onto 5 ml HiTrap Chelating column (Amersham Biosciences), charged with Ni2+. The column was washed with 10 CV of 20 mM Hepes-NaOH buffer, pH 7.5, containing 500 mM NaCl and 50 mM imidazole, and the protein was eluted with elution buffer. 10 mM DTT was added to WDR5 containing fractions. The protein was loaded on Superdex200 column (26x60) (Amersham Biosciences), equilibrated with 20 mM Hepes-NaOH buffer, pH 7.5, and 500 mM NaCl, at flow rate 4 ml/min. Thrombin (Sigma) was added to combined fractions containing WDR5 and incubated overnight at 4°C. Since the cleavage was not complete the sample was further purified to homogeneity by another affinity chromatography on Ni HisTrap (1 mL) (Amersham Biosciences). Purification yield is 98 mg of 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 (0.1 mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 18,000 psi.
Concentration:28.9 mg/ml
Ligand
MassSpec:expected mass = 36732.6 Da, measured mass = 36733.4 Da
Crystallization:Purified WDR5 was crystallized using the sitting drop vapor diffusion method at 18 °C by mixing 1 µl of 10 mg/mL protein solution with 1 µl of the reservoir solution containing 20 % PEG 5000 MME and 0.1 M BisTris pH 6.5.
NMR Spectroscopy:
Data Collection:
Data Processing: