human dishevelled 2

Target ID:

DVL2A

Aliases:

DVL2

Deposition Date:

Thursday 27th September 2007

Families:

Linkers

Structure type:

apo

Download Coordinates:

2REY

Authors:

Papagrigoriou, E., Gileadi, C., Elkins, J., Cooper, C., Ugochukwu, E., Turnbull, A., Pike, A. C. W., Gileadi, O., von Delft, F., Sundstrom, M., Arrowsmith, C. H., Weigelt, J., Edwards, A., Doyle, D.

Site:

Oxford

2REY

tabs

Structure Details

PDZ domains function as protein-protein interaction modules. The best characterised interaction of the PDZ domain is with the C-terminal 4-5 residues of the target protein that binds in an extended fashion between the betaB strand and the alphaB helix of the PDZ domain. In many cases the protein that contains the PDZ domain also possesses additional PDZ domains or interaction modules. DVL2 is one such protein which contains a Dax or DIX domain, a DEP domain, a dishevelled domain plus a PDZ domain.

The Wnt signalling pathway is important in embryogenesis and cancer. Some of the proteins involved in this signalling pathway include LDL-related protein receptor, frizzled, axin, GSK, Daam1, Rac, JNK, PKC, CamK2, beta-catenin and dishevelled. These proteins function via three independent pathways known as the canonical, non-canonical or planar cell polarity pathway and the Wnt-Ca2+ pathway. However in all three pathways a common intermediatory protein is Dishevelled (DVL).

The DVL family members interact with VANG2, a protein that when mutated results in the neural tube defect disease craniorachischisis (Torban et al, 2004). DVL2 is also part of the 'interactome' that is part of inhertied neurodegenerative disorders (Lim et al, 2006).

Here we present the crystal structure of DVL2 in complex with the C-terminal peptide -Glu-Thr-Ser-Val-COO- derived from VANGL2.

Materials and Methods

DVL2

PDB:2REY

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|4758216
Entry Clone Source:MGC
SGC Clone Accession:IMAGE:3852554
Tag:N-terminal, TEV cleavable hexahistidine tag
Host:BL-21(DE3)-R3-Rosetta

Construct

Prelude:
Sequence:SMSLNIITVTLNMEKYNFLGISIVGQSNERGDGGIYIGSIMKGGAVAADGRIEPGDMLLQVNDMNFENMSNDDAVRVLRDIVHKPGPIVLTVAKCWETSV
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:Transformation: The construct DNA was transformed into homemade chemically competent cells of the expression strain by a standard heat shock procedure.Glycerol stock prepataion: A number of colonies from the transformation were used to innoculate 1 ml of LB media containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol, which was placed in a 37°C shaker overnight. The next day glycerol stocks were prepared from this overnight culture.Expression: 10 ml of a thawed glycerol stock was used to innoculate 40 ml of TB media containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol, which was placed in a 37°C shaker overnight. The next day this starter culture was used to innoculate 2x 1L of TB media (18 ml starter culture into each) containing 50 µg/ml kanamycin. After 4.5 hours, the temperature was reduced to 22°C. The incubation was continued for 1 hour. At OD~3, the cells were induced by the addition of 0.5 mM IPTG. The expression was continued overnight (~ 18 hours).Cell harvest: Cells were spun at 6238x g for 15 mins at 4°C. The cell pellets were placed in a -80°C freezer.

Purification

Procedure
Column 1: HisTrap 1ml.The clarified cell extract was passed through the column at a flow rate of 0.8 ml/min. The column was then washed with Wash Buffer until a stable UV baseline was achieved. The protein was eluted with 5 ml of Elution Buffer.Column 2: Gel filtration. Hiload S200 16/60 - 120 ml volume.The gel filtration column was pre-equilibrated with Gel Filtration Buffer. The HisTrap eluant was loaded on the gel filtration column automatically after the HisTrap elution at a flow rate of 1.2 ml/min. Eluted proteins were collected in 1.8 ml fractions. The fractions containing protein were identified on a coomasie blue stained gel.TEV protease digestion: The gel filtration fractions containing DVL2A were pooled and 100 ml of TEV protease solution (~1 mg/ml) was added to 6 mg protein. The digestion was left overnight at 4°C.Rebinding of impurities to Ni-NTA: The protein was mixed with Ni-cellulose resin (0.4 ml, pre-equilibrated into Gel Filtration Buffer) at 4°C for 60 minutes. The resin was spun down and the supernatant was filtered through a 0.2 mM filter and collected.

Extraction

Procedure
Cell Lysis: Two liter-culture pellets were resuspended in lysis buffer. They were passed 4 times through an Emulsiflex C5 high-pressure homogeniser, collecting a final volume of approximately 90 ml. PEI was added to a final concentration of 0.25 % and the cell debris and precipitated DNA were spun down at 45000x g, 90 min (Beckman JA 18 17500 rpm). ). The supernatant was filtered through a 0.2 uM filter.
Concentration:The TEV protease cleaved DVL2A was concentrated to 17.4 mg/ml,distributed into 30 ml aliquots and frozen at -80°C.
Ligand
MassSpec:Measured: 10813.3; Expected: 10813.4.
Crystallization:Crystals grew from a 2:1 ratio of protein to precipitant solution (0.2M (NH4)(ac); 0.1M BIS-TRIS pH 5.5; 25% PEG 3350), using the vapour diffusion method.
NMR Spectroscopy:
Data Collection:Data was collected to a resolution of 1.55 Å.
Data Processing:

References

Lim J, Hao T, Shaw C, Patel AJ, Szabo G, Rual JF, Fisk CJ, Li N, Smolyar A, Hill DE, Barabasi AL, Vidal M, Zoghbi HY. (2006). A protein-protein interaction network for human inherited ataxias and disorders of Purkinje cell degeneration. Cell. 125: 801-814.

Torban E, Wang HJ, Groulx N, Gros P. (2004). Independent mutations in mouse Vangl2 that cause neural tube defects in looptail mice impair interaction with members of the Dishevelled family. J. Biol. Chem. 279: 52703-52713.