Human WD40 protein CIAO1

Target ID:

WDR39

Deposition Date:

Friday 19th December 2008

Families:

Miscellaneous

Structure type:

apo

Download Coordinates:

3FM0

Authors:

Dong A, Ravichandran M, Crombet L, Cossar D, Edwards AM, Arrowsmith CH, Weigelt J, Bountra C, Bochkarev A, Min J, Ouyang H

Site:

Toronto

3FM0

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.

WDR39 is a 7-WD40-repeat protein, which is demonstrated to interact with WT1, a Wilms' tumor suppressor. It is shown that interactions of WDR39 with WT1 results in a change in the mobility of a WT1 and DNA complex in vitro and causes a dose-dependent decrease in WT1-mediated transcriptional activation. The functional importance of WDR39 is further supported by the observation that its putative yeast homolog is an essential gene. These findings suggest that WDR39 is an important cellular regulator whose functions may include modulation of transcription mediated by WT1.[1]

We determined the crystal structure of WDR39 at 1.7A. The structure shows that the full-length WDR39 folds into a seven-blade β-propeller structure, and the seven blades are symmetrically arranged around a pseudosymmetry axis and a narrow channel runs through the middle of the β-propeller structure, which is very similar to the WDR5 structure we determined previously.[2]

Materials and Methods

CIAO1

PDB:3FM0

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_004795
Entry Clone Source:MGC
SGC Clone Accession:AU6-E9 (BC001395)
Tag:C-terminal tag: HHHHHH
Host:BL21 (DE3)_V2R

Construct

Prelude:
Sequence:MKDSLVLLGRVPAHPDSRCWFLAWNPAGTLLASCGGDRRIRIWGTEGDSWICKSVLSEGHQRTVRKVAWSPCGNYLASASFDATTCIWKKNQDDFECVTTLEGHENEVKSVAWAPSGNLLATCSRDKSVWVWEVDEEDEYECVSVLNSHTQDVKHVVWHPSQELLASASYDDTVKLYREEEDDWVCCATLEGHESTVWSLAFDPSGQRLASCSDDRTVRIWRQYLPGNEQGVACSGSDPSWKCICTLSGFHSRTIYDIAWCQLTGALATACGDDAIRVFQEDPNSDPQQPTFSLTAHLHQAHSQDVNCVAWNPKEPGLLASCSDDGEVAFWKYQRPEGLhhhhhh
Vector:pET28a-lic

Growth

Medium:
Antibiotics:
Procedure:A 250 ml flask containing LB (Sigma L7658) supplemented with 50 ug/ ml kanamycin (BioShop Canada KAN 201) was inoculated from a glycerol stock of the bacteria. The flask was shaken overnight (16 hours) at 250 rpm at 37 degC.

Using the Lex system, a 2L bottle (VWR 89000-242) containing 1800 ml of TB (Sigma T0918) supplemented with 1.5% glycerol, 50 ug/ ml kanamycin and 600 ul antifoam 204 (Sigma A-8311) was inoculated with 50 ml overnight LB culture, and incubated at 37 degC. The temperature of the media was reduced to 15 degC one hour prior to induction and induced at OD600 = 6 with 100 uM isopropyl-thio-β-D-galactopyranoside (BioShop Canada IPT 001). Cultures were aerated overnight (16 hours) at 15 degC, and cell pellets collected by centrifugation and frozen at -80 degC.

Purification

Procedure
IMAC: Unclarified lysate was mixed with 2-3 mL of Ni-NTA superflow Resin (Qiagen) per 40 mL lysate. The mixture was incubated with mixing for at least 45 minutes at 4 degC. The mixture was than loaded onto an empty comlum (BioRad) and washed with 100 ml wash buffer. Samples were eluted from the resin by exposure to 2-3 column volumes (approx. 10-15 mL) of elution buffer. Concentration of eluted protein was estimated by OD280.

Gel filtration chromatography: An XK 26x65 column (GE Healthcare) packed with HighLoad Superdex 75 resin (GE Healthcare) was pre-equilibrated with gel filtration buffer for 1.5 column volumes using an AKTA explorer (GE Healthcare) at a flow rate of 2.5mL/min. The Eluted sample from the IMAC step (approx. 15 mL) was loaded onto the column at 1.5 mL/min, and 2mL fractions were collected into 96-well plates (VWR 40002-012) using peak fractionation protocols). Fractions observed by a UV absorption chromatogram to contain the protein were pooled.

Extraction

Procedure
Frozen cell pellet contained in bags (Beckman 369256) obtained from 2L of culture were thawed by soaking in warm water. Each cell pellet was resuspended in 25-40 mL lysis buffer and homogenized using an Ultra-Turrax T8 homogenizer (IKA Works) at maximal setting for 30-60 seconds per pellet. Cell lysis was accomplished by sonication (Virtis408912, Virsonic) on ice: the sonication protocol was 10 sec pulse at half-maximal frequency (5.0), 10 second rest, for 10 minutes total sonication time per pellet.
Concentration:Purified proteins were concentrated using 15 mL concentrators with a 5,000 molecular weight cut-off (Amicon Ultra-15, UFC900524, Millipore) at 3750 rpm, typically resulting in a final concentration around 30 mg/mL.
Ligand
MassSpec:
Crystallization:Diffraction quality crystals were grown using the following protocol: 25% PEG 3350, 0.1 M (NH₄)₂SO₄, 0.1M Bis-Tris, pH6.0, 8% 2,2,2-Trifluro Ethanol, vapor diffusion, sitting drop, temperature 297k.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Johnstone, R.W. et al. Ciao 1 is a novel WD40 protein that interacts with the tumor suppressor protein WT1. J Biol Chem 273, 10880-7 (1998).
Schuetz, A. et al. Structural basis for molecular recognition and presentation of histone H3 by WDR5. Embo J 25, 4245-52 (2006).