Second PDZ domain of the human discs, large homolog 3 protein

Target ID:

DLG3A

Aliases:

KIAA1232MRXMRX90NE-DlgNEDLGSAP102

Deposition Date:

Thursday 15th December 2005

Families:

Linkers

Related Diseases:

SignallingNeurobiology

Structure type:

apo

Download Coordinates:

2FE5

Authors:

E.UGOCHUKWU,C.PHILLIPS,G.SCHOCH,G.BERRIDGE,E.SALAH,S.COLEBROOK,C.SMEE,P.SAVITSKY,J.BRAY,J.ELKINS,D.DOYLE,G.BUNKOCZI,J.DEBRECZENI,A.TURNBULL,F.GORREC,F.VON DELFT,M.SUNDSTROM,C.ARROWSMITH,J.WEIGEL,A.EDWARDS,STRUCTURALGENOMICS CONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2FE5

tabs

Structure Details

DLG3, also known as SAP102, is a member of the MAGUK (members of the membrane-associated guanylate kinase family) multi-domain protein family (Smith et al, 1996).
It contains three disparate domains including a guanylate kinase domain, an SH3 domain and three PDZ domains.
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.

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 DLG3 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 cell junctions where DLG3 is located.

Major sites where several members of the MAGUK family members including DLG3 are found are the neuronal junctions (Masuko et al, 1998). Mutations of DLG3 have been identified in patients with non-syndromic (only cognitive impairment associated with the clinical features) X-linked mental retardation (Tarpey et al, 2004). The clinical features of this mutation maybe due to the loss of clustering of the SAP102 target proteins which includes the NMDA receptor, proteins that play a critical role in learning (Ehrlich & Malinow, 2004; Migaud et al, 1998).

Materials and Methods

DLG3-PDZ domain

PDB:2FE5

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:DLG3A-s001
Entry Clone Source:Origene
SGC Clone Accession:
Tag:N-terminal hexahistidine tag
Host:BL-21(DE3)R3 phage resistant strain

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfq*smTIMEVNLLKGPKGLGFSIAGGIGNQHIPGDNSIYITKIIEGGAAQKDGRLQIGDRLLAVNNTNLQDVRHEEAVASLKNTSDMVYLKVAKPGS
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:Freshly transformed E. coli cells was used to inoculate 40 ml of LB containing 50 µg/ml kanamycin for overnight growth. The following day, 10 mls of this starter was used to inoculate 1 litre of TB plus 50 µg/ml kanamycin. When OD600 reached ~1.6 the temperature was shifted down from 37°C to 25°C for 1 hour before induction with the addition of 1 mM IPTG. Protein expression was allowed to carry on for a futher 4 hours before harvest. The cells were harvested by centrifugation, resuspended in lysis buffer before storing in a -80°C freezer.
Lysis/Binding Buffer: 50 mM Hepes pH 8.0, 500 mM NaCl, 5 % glycerol, 5 mM imidazole pH 8.0, 0.5 mM TCEP.

Purification

Procedure
Column 1 : Wash Buffer: 50 mM Tris pH 8.0, 500 mM NaCl, 5 % Glycerol, 25 mM Imidazole pH 8.0, 0.5 mM TCEP; Elute Buffer: 50 mM Hepes pH 8.0, 500 mM NaCl, 5 % Glycerol, 250 mM Imidazole pH 8.0, 0.5 mM TCEP; GF Buffer: 50 mM Tris pH 8.5, 500 mM NaCl, 0.5 mM TCEP.
Procedure: The supernatent was filtered through a 1.2 micron and then a 0.2 micron syringe filter. 4 mls of a 50 % Ni2+-NTA slurry was added to a clean 15 mm diameter gravity column. The resin was equilibrated with 5 column volumes (10 ml) Lysis/Binding buffer. The supernatant was allowed to drip through the resin twice. The resin was then washed with 5 column volumes of Lysis/Binding Buffer and 25 column volumes of Wash Buffer. Finally the protein was eluted with 6 column volumes of Elute buffer and the eluate collected in 2ml fractions into eppendorf tubes.
Enzymatic treatment : HIS- TAG removal. To improve the purity of the sample the hexahistidine tag was cleaved and the cleavage products plus the contaminants rebound to a Ni2+-resin thus allowing the cleaved DLG3A sample to run through the column.

The DLG3A-p032 fractions were pooled and cleaved overnight with 100 µl of 2.8 mg/ml of in-house produced TEV protease. 500 µl of 50 % Ni-sepharose (Amersham) was added and the sample inverted repeatedly on a rotator for 60 minutes at 4°C to remove the His tag with TEV and other contaminants. The protein was concentrated to a final concentration of 143 mg/ml before storing at -80°C.

Extraction

Procedure
To the cell pellet (approx. 80 mls) was added 20 mls of Lysis/Binding buffer and PMSF was added to a final concentration of 1.0 mM. Cell breakage: 4 passes through the Emulsiflex C5 high pressure homogeniser. Total vol: 100 mls (estimate).PEI was added to a final concentration of 0.05 % to precipitate the DNA . Centrifuge for 40 mins at 18000 rpm and 4°C to remove cell debris. Discard pellet.
Concentration:
Ligand
MassSpec:
Crystallization:Column 1 : Wash Buffer: 50 mM Tris pH 8.0, 500 mM NaCl, 5 % Glycerol, 25 mM Imidazole pH 8.0, 0.5 mM TCEP; Elute Buffer: 50 mM Hepes pH 8.0, 500 mM NaCl, 5 % Glycerol, 250 mM Imidazole pH 8.0, 0.5 mM TCEP; GF Buffer: 50 mM Tris pH 8.5, 500 mM NaCl, 0.5 mM TCEP.
Procedure: The supernatent was filtered through a 1.2 micron and then a 0.2 micron syringe filter. 4 mls of a 50 % Ni2+-NTA slurry was added to a clean 15 mm diameter gravity column. The resin was equilibrated with 5 column volumes (10 ml) Lysis/Binding buffer. The supernatant was allowed to drip through the resin twice. The resin was then washed with 5 column volumes of Lysis/Binding Buffer and 25 column volumes of Wash Buffer. Finally the protein was eluted with 6 column volumes of Elute buffer and the eluate collected in 2ml fractions into eppendorf tubes.
Enzymatic treatment : HIS- TAG removal. To improve the purity of the sample the hexahistidine tag was cleaved and the cleavage products plus the contaminants rebound to a Ni2+-resin thus allowing the cleaved DLG3A sample to run through the column.

References

Ehrlich I, Malinow R. (2004). Postsynaptic density 95 controls AMPA receptor incorporation during long-term potentiation and experience-driven synaptic plasticity. J. Neurosci. 24: 916-927.