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

Target ID:

DLG3A

Aliases:

KIAA1232MRXMRX90NE-DlgNEDLGSAP102

Deposition Date:

Monday 14th August 2006

Families:

Linkers

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

2I1N

Authors:

A.P.TURNBULL,C.PHILLIPS,G.BUNKOCZI,J.DEBRECZENI,E.UGOCHUKWU,A.C.W.PIKE,F.GORREC,C.UMEANO,J.ELKINS,G.BERRIDGE,P.SAVITSKY,O.GILEADI,F.VON DELFT,J.WEIGELT,A.EDWARDS,C.ARROWSMITH,M.SUNDSTROM,D.DOYLE,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2I1N

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 that are organised as shown below.

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: First PDZ domain of the human discs, large homolog 3 protein.

PDB:2I1N

Revision

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

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfq*smFKYEEIVLERGNSGLGFSIAGGIDNPHVPDDPGIFITKIIPGGAAAMDGRLGVNDCVLRVNEVDVSEVVHSRAVEALKEAGPVVRLVVRRRQPPPEETSV
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.

Purification

Procedure

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:Expected 10918.3; recorded 12602.49.
Crystallization:Crystals grew from a 2:1 mixture of DLG3-to-reservoir (1.0 M LiSO4, 0.5 M TMAO).
NMR Spectroscopy:
Data Collection:Resolution: 1.1 Å; X-ray source: Synchrotron SLS -X10, single wavelength.
Data Processing:

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.

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.

Masuko N, Makino K, Kuwahara H, Fukunaga K, Sudo T, Araki N, Yamamoto H, Yamada Y, Miyamoto E, Saya H. (1999). Interaction of NE-dlg/ SAP 102, a neuronal and endocrine tissue-specific membrane-associated guanylate kinase protein, with calmodulin and PSD -95/ SAP 90. A possible regulatory role in molecular clustering at synaptic sites. J. Biol. Chem. 274: 5782-5790.

Migaud M, Charlesworth P, Dempster M, Webster LC, Watabe AM, Makhinson M, He Y, Ramsay MF, Morris RG, Morrison JH, O'Dell TJ, Grant SG. (1998). Enhanced long-term potentiation and impaired learning in mice with mutant postsynaptic density-95 protein. Nature.396: 433-439.

Smith SA, Holik P, Stevens J, Mazoyer S, Melis R, Williams B, White R, Albertsen H. (1996). Isolation of a gene (DLG3) encoding a second member of the discs-large family on chromosome 17q12-q21. Genomics. 31: 145-150.

Tarpey P, Parnau J, Blow M, Woffendin H, Bignell G, Cox C, Cox J, Davies H, Edkins S, Holden S, Korny A, Mallya U, Moon J, O'Meara S, Parker A, Stephens P, Stevens C, Teague J, Donnelly A, Mangelsdorf M, Mulley J, Partington M, Turner G, Stevenson R, Schwartz C, Young I, Easton D, Bobrow M, Futreal PA, Stratton MR, Gecz J, Wooster R, Raymond FL. (2004). Mutations in the DLG3 gene cause nonsyndromic X-linked mental retardation. Am. J. Hum. Genet. 75: 318-324.