Third PDZ domain of the human discs, large homolog 2 protein

Target ID:

DLG2A

Aliases:

DKFZp781D1854DKFZp781E0954FLJ37266MGC131811PSD-93

Deposition Date:

Wednesday 21st June 2006

Families:

Linkers

Related Diseases:

SignallingNeurobiology

Structure type:

apo

Download Coordinates:

2HE2

Authors:

A.P.TURNBULL,C.PHILLIPS,G.BERRIDGE,P.SAVITSKY,C.E.A.SMEE,E.PAPAGRIGORIOU,J.DEBRECZENI,F.GORREC,J.M.ELKINS,F.VONDELFT,J.WEIGELT,A.EDWARDS,C.ARROWSMITH,M.SUNDSTROM,D.DOYLE,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2HE2

tabs

Structure Details

DLG2, also known as PSD-93 or Chapsyn-110, is a member of the MAGUK (members of the membrane-associated guanylate kinase family) multi-domain protein family. It contains three disparate domains including a guanylate kinase domain, an SH3 domain and three PDZ domains that are organised as follows:

All three type of domains are known to play important roles in protein-protein interactions pointing to the role that this family of proteins play within the cell. The proteins that DLG2 are known to interact with include: inward rectifying K+ channels (Leonoudakis et al, 2004); Ca2+-ATPase (DeMarco & Strehler, 2001); ionotrophic NMDA receptors (Niethammer et al, 1996); voltage-dependent K+ channel Kv1.4 (Kim et al, 1995); and the glutamate receptor delta2 (Roche et al, 1999).

One of the best characterised interactions is with the PDZ domains and their target proteins. The interaction occurs between the C-terminal four amino acids of the target protein and the PDZ domain. Heteromultimerisation of the DLG2 with the related protein PSD-95 results in the formation of a scaffold that brings together the target proteins (Kim et al, 1996). This is an important process in the formation clustering of ion channels, transporters and signalling proteins at the postsynaptic sites where DLG2 is located.

The NMDA receptors, which bind to PSD-93, is involved in pain sensation (Petrenko et al, 2003). Activation of the NMDA receptors cause an increase in a cells response to pain. Disruption of the complexes formed by DLG2 significantly reduces induced chronic pain (Zhang et al, 2003; Tao et al, 2003). Thus DLG2 is a potential target for the treatment of persistent pain.

Here we present the struture of the third PDZ domains of DLG2 in complex with a mode I PDZ binding motif the sequence of which is Glu-Thr-Ser-Val-COO-. The structure also resolves a helix C-terminal to the standard PDZ domain.

Materials and Methods

DLG2

PDB:2HE2

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:DLG2A_s001
Entry Clone Source:Origene
SGC Clone Accession:
Tag:N-terminal hexahistidine tag before TEV cleavage site C-terminal PDZ recognition motif (ETSV).
Host:

Construct

Prelude:sm residues are a result of the construct design
Sequence:sm EPRKVVLHKGSTGLGFNIVGGEDGEGI FVSFILAGGPADLSGELQRGDQILSVNGI DLRGASHEQAAAALKGAGQTVTIIAQYQP EDYARFEAKIHETSV
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:Transformed 90 µl competent BL-21 (DE3) phage resistant cells with 2 µl of the plasmid DNA and plated out onto LB plate plus 50 µg/ml kanamycin and 34µg/ml chloramphenicol. The next day colonies were picked out into 50 ml LB + 50 µg/ml kanamycin. 15 ml of the culture was used to innoculate 1 litre of TB + 50 µg/ml kanamycin and 34µg/ml chloramphenicol. When the OD600 reached 1.4 the incubator temperature was dropped to 25°C. Protein production was induced with the addition of 1mM IPTG one hour after the temperature shift. After 4 hours cells were harvested by centrifugation at 4000 rpm for 15 minutes resuspended in Lysis Buffer before storage in the -80°C freezer.

Purification

Procedure
Column 1 : Ni²⁺-NTA column purification
1 ml of 50 % Ni2+-NTA slurry was added to a clean empty 10 mm diameter gravity column. The resin was equilibrated with 10 ml Lysis/Binding buffer (see above). The supernatant was allowed to drip through the resin twice. The resin was then washed with 50 mls of Lysis Buffer and 30 mls of Wash Buffer. Finally the protein was eluted with 15 mls of Elute buffer with 2 ml fractions collected into eppendorf tubes.

Column 2: Gel filtration, Hiload 16/60, S75 16/60 - 120 ml
The eluted fractions from the Ni-affinity Histrap columns were loaded on the gel filtration column in GF buffer at 1.0 ml/min. Eluted proteins were collected in 1 ml fractions. The fractions containing protein were identified on a Coomassie blue stained SDS - PAGE gel.

Enzymatic treatment : Added 100 µl of the home produced TEV protease to fractions containing protein and left at 4°C overnight. The following steps were carried out to remove the cleaved products and TEV protease. Place 200 µl of 50 % Ni-NTA agarose in a 1.5 ml eppendorf tubes, add 1 ml of 50 mM HEPES pH 7.5, 100 mM NaCl, 0.5 mM TCEP, 2 mM MgCl2 mix, spin down and remove buffer. Repeat this resin wash step once. Add the TEV treated protein sample to the resin and mix for 30 min. Finally spin down resin and collect the supernatant which contains the cleaved DLG2 PDZ domain. The protein was then concentrated to 4 mg/ml before aliquoted into 50 µl volumes and storing in a -80°C freezer.

Extraction

Procedure
Once the cells were thawed they were broken by several passages through the Avestin C5 high pressure homogeniser. The final broken cells suspension came to an approx. volume of 50 mls. To this 0.15 % of PEI was added to precipitate the DNA before centrifugation at 21,500 rpm for 45 mins at 4°C to remove the precipitated insoluble material.
Concentration:
Ligand
MassSpec:Expected MWt. after his-tag removal: 10665.8; Measured MWt.:10665.
Crystallization:Crystals grew from a 2:1 ratio mix of DLG2-to-reservoir (25.5 % PEG 3350 , 0.17 M (NH4)2SO4, 15 % glycerol)
NMR Spectroscopy:
Data Collection:Resolution: 1.50Å; X-ray source: Synchrontron SLS-X10.
Data Processing:

References

DeMarco, S. J., Strehler, E. E. (2001). Plasma membrane Ca2+-atpase isoforms 2b and 4b interact promiscuously and selectively with members of the membrane-associated guanylate kinase family of PDZ ( PSD 95/Dlg/ZO-1) domain-containing proteins. J. Biol. Chem. 276: 21594-21600.

Leonoudakis, D., Conti, L. R., Anderson , S., Radeke, C. M., McGuire, L. M., Adams , M. E., Froehner, S. C., Yates, J.R. 3rd., Vandenberg, C. A. (2004). Protein trafficking and anchoring complexes revealed by proteomic analysis of inward rectifier potassium channel (Kir2.x)-associated proteins. J. Biol. Chem. 279: 22331-22346.

Kim, E., Cho, K. O., Rothschild, A., Sheng, M. (1996). Heteromultimerization and NMDA receptor-clustering activity of Chapsyn-110, a member of the PSD -95 family of proteins. Neuron. 17: 103-113.

Kim, E., Niethammer, M., Rothschild, A., Jan, Y. N., Sheng, M. (1995). Clustering of Shaker-type K+ channels by interaction with a family of membrane-associated guanylate kinases. Nature 378: 85-88.

Niethammer, M., Kim, E., Sheng, M. (1996). Interaction between the C terminus of NMDA receptor subunits and multiple members of the PSD -95 family of membrane-associated guanylate kinases. J. Neuroscience 16: 2157-2163.

Petrenko, A. B., Yamakura, T., Baba, H., Shimoji, K. (2003). The role of N-methyl-D-aspartate (NMDA) receptors in pain: a review. Anesth. Analg. 97: 1108-1116.

Roche, K. W., Ly, C. D., Petralia, R. S., Wang, Y. X., McGee, A. W., Bredt, D. S., Wenthold, R. J. (1999). Postsynaptic density-93 interacts with the delta2 glutamate receptor subunit at parallel fiber synapses. J. Neurosci. 19: 3926-3934.

Tao, Y. X., Rumbaugh, G., Wang, G. D., Petralia, R. S., Zhao, C., Kauer, F. W., Tao, F., Zhuo, M., Wenthold, R. J., Raja, S. N., Huganir, R. L., Bredt, D. S., Johns, R. A. (2003). Impaired NMDA receptor-mediated postsynaptic function and blunted NMDA receptor-dependent persistent pain in mice lacking postsynaptic density-93 protein. J. Neurosci. 23: 6703-6712.

Zhang, B., Tao, F., Liaw, W. J., Bredt, D. S., Johns, R. A., Tao, Y. X.(2003). Effect of knock down of spinal cord PSD -93/chapsin-110 on persistent pain induced by complete Freund's adjuvant and peripheral nerve injury. Pain 106: 187-196.