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

Target ID:

DLG2A

Aliases:

DKFZp781D1854DKFZp781E0954FLJ37266MGC131811PSD-93

Deposition Date:

Monday 1st August 2005

Families:

Linkers

Related Diseases:

SignallingNeurobiology

Structure type:

apo

Download Coordinates:

2BYG

Authors:

J.M.ELKINS,G.A.SCHOCH,C.E.A.SMEE,G.BERRIDGE,E.SALAH,M.SUNDSTROM,A.EDWARDS,C.ARROWSMITH,J.WEIGELT,D.A.DOYLE

Site:

Oxford

ICM Datapack:

ICM Datapack

2BYG

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 in the figure below the structure image.

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.

Materials and Methods

DLG2

PDB:2BYG

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
Host:BL-21(DE3_R3 phage resistant)

Construct

Prelude:
Sequence:MHHHHHHSSGVDLGTENLYFQSMTVVEIKLFKGPKGLGFSIAGGVGNQHIPGDNSIYVTKIIDGGAAQKDGRLQVGDRLLMVNNYSLEEVTHEEAVAILKNTSEVVYLKVGKPTTIY
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:Transformed 50 µL competent BL21 (DE3) phage resistant cells with 10 µL of the plasmid DNA and plated out onto LB plate plus 50 µg/mL kanamycin. The next day colonies were picked out into fresh deep well blocks containing 1 mL TB + 50 µg/mL kanamycin. These were grown overnight and glycerol stocks prepared by adding 333 µL of 60 % glycerol to 1 mL of cell suspension, mixing and then storing in a -80C freezer.

The glycerol stock was used to innoculate 10 mLs of TB + 50 µg/mL kanamycin which was grown overnight at 37C as a starter culture for a 1 litre growth. The large scale growth was grown at 37C until approximately 30 mins before induction when the temperature was lowered to 25C. Protein production was induced with the addition of 1mM IPTG. The next day cells were harvested by centrifugation at 4000 rpm for 15 minutes. The pellet was then stored in the -80C freezer.

Purification

Procedure
Column 1 : Ni-affinity, HisTrap, 1 mL (GE/Amersham)

Buffers: Affinity binding buffer: 10 mM Imidazole, 300 mM NaCl, 50 mM pH 8.0 NaH₂PO₄ , 0.5 mM TCEP. Affinity wash buffer: 50mM Imidazole, 300mM NaCl, 50mM pH8.0 NaH2PO4 , 0.5mM TCEP. Affinity Elution Buffer: 250mM Imidazole, 300mM NaCl, 50mM pH8.0 NaH2PO4 , 0.5mM TCEP

Procedure: The cell extract was loaded on the column at 0.8 mL/min on an AKTA-express system (GE/Amersham). The column was then washed with 10 column volumes of Affinity Binding buffer, 10 column volumes of Affinity wash buffer, and then eluted with Affinity elution buffer at 0.8 mL/min. The eluted peak of A280 was automatically collected.

Column 2: Gel filtration, Hiload 16/60, S75 16/60 - 120 mL

Buffers: Gel Filtration: 10mM pH7.4 Hepes, 500mM NaCl, 5% glycerol, 0.5 mM TCEP

Procedure: 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.

Concentration: Using a Centricon 10 K cutoff concentrator the DLG2A-p002 pooled fractions was concentrated to 25.8 mg/mL. Concentration was determined from the absorbance at 280 nm.

Extraction

Procedure
Lysis buffer: 10 mM Imidazole, 300mM NaCl, 50mM pH8.0 NaH₂PO₄ 0.5 mM TCEP, 1x complete PI EDTA free tablet/50mL. The pellet (19.52 g) was resuspended with 3x volume of lysis buffer (approximately 50 mL final) by intermitently placing the pellet in a 37C water bath and vortexing. Once resuspended the cells were (1) broken by one passage through the Constant Systems cell breaker; (2) sonicating; (3) DNA precipitation with the addition of PEI to a final oncentration of 0.15% for 30 mins on ice followed by a 17,000 rpm at 4C to remove precipitation; (4) the supernatant was filtered through a GF/0.2 µM serum acrodiscs.
Concentration:
Ligand
MassSpec:
Crystallization:Column 1 : Ni-affinity, HisTrap, 1 mL (GE/Amersham)

Column 2: Gel filtration, Hiload 16/60, S75 16/60 - 120 mL

Buffers: Gel Filtration: 10mM pH7.4 Hepes, 500mM NaCl, 5% glycerol, 0.5 mM TCEP

Procedure: 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.

Concentration: Using a Centricon 10 K cutoff concentrator the DLG2A-p002 pooled fractions was concentrated to 25.8 mg/mL. Concentration was determined from the absorbance at 280 nm.
NMR Spectroscopy:
Data Collection:
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.