Human multiple PDZ domain protein (13th domain)

Target ID:

MPDZA

Aliases:

DKFZp781P216FLJ25909FLJ34626FLJ90240MUPP1

Deposition Date:

Wednesday 11th January 2006

Families:

Linkers

Related Diseases:

NeurobiologyCancer

Structure type:

apo

Download Coordinates:

2FNE

Authors:

E.PAPAGRIGORIOU,G.BERRIDGE,C.JOHANSSON,S.COLEBROOK,E.SALAH,N.BURGESS,C.SMEE,P.SAVITSKY,J.BRAY,G.SCHOCH,C.PHILLIPS,C.GILEADI,M.SOUNDARAJAN,X.YANG,J.ELKINS,F.GORREC,A.TURNBULL,A.EDWARDS,C.ARROWSMITH,J.WEIGELT,M.SUNDSTROM,D.DOYLE,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2FNE

tabs

Structure Details

Epithelial tight junctions perform a barrier role controlling the movement of water, solutes and immune cells through the paracellular pathway as well as defining the polarity of a cell.
How tightly these functions are controlled depends on the composition of proteins at the tight junction.
Some of the major protein families that form the "backbone" of the barrier are the zonula occludens (ZO) and claudins.
MPDZ is a protein that interacts with various members of these families helping to form and regulate the tight junction structure (Turksen & Troy, 2004).

MPDZ (MUPP-1) is a large protein of 2042 amino acids that contains 13 individual PDZ domains (Ullmer et al, 1998). These domains bind to the C-terminal 4-5 residues of the target protein, localising the proteins to defined regions. The large number of PDZ domains in MPDZ allows the protein to form many connections with several different proteins thus helping to form the large protein scaffold assemblies found at tight junctions.

Here we present the crystal structure of the13th PDZ domain of MPDZ.
This PDZ domain was crystallised by adding a C-terminal extension that matched the expected binding motif.
This allowed the C-terminus of one domain to reach over and bind to the ligand binding site of another PDZ domain, initiating the protein-protein interactions that eventually formed the crystal.

Two proteins have been identified that interact with the 13th PDZ domain of MPDZ.
The first is TAPP1 (Kimber et al, 2002) which binds specifically to phosphatidylinositol-3,4-diphosphate and may function as a recruitment point for other proteins to the plasma membrane.
The second is another protein that is found at the tight junctions of polarised epithelial cell.
This protein is the coxsackievirus and adenovirus receptor which mediates cell attachment and infection by coxsackie B viruses and by a number of adenoviruses (Coyne et al, 2002).

Materials and Methods

MPDZ-13

PDB:2FNE

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:MPDZA-s001
Entry Clone Source:Origene
SGC Clone Accession:
Tag:N-terminal hexahistidine tag before TEV cleavage site. C-terminal PDZ recognition motif
Host:BL-21(DE3)R3 phage resistant

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfqsmPQCKSITLERGPDGLGFSIVGGYGSPHGDLPIYVKTVFAKGAASEDGRLKRGDQIIAVNGQSLEGVTHEEAVAILKRTKGTVTLMVLSSDETSV

Lowercase characters highlight the N-terminal hexahistidine tag and TEV cleavage recognition site.
ETSV: C-terminal extension containing PDZ recognition motif.
Vector:pNIC28-BSA4

Growth

Medium:
Antibiotics:
Procedure:Transformed 50 µL competent BL-21 (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 m l of 60 % glycerol to 1 mL of cell suspension, mixing and then storing in a -80°C freezer.

Purification

Procedure
Column 1 : Ni-affinity, HisTrap, 1 mL (GE/Amersham)
Buffers: Affinity binding buffer: 10mM Imidazole, 300mM NaCl, 50mM pH8.0 NaH2PO4, 0.5mM 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, 0.5mM 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 MPDZA-p069 pooled fractions was concentrated to 7.3 mg/mL. Concentration was determined from the absorbance at 280 nm.

Extraction

Procedure
Lysis buffer: 10mM Imidazole, 300mM NaCl, 50mM pH8.0 NaH2PO4, 0.5mM TCEP, 1x complete PI EDTA free tablet/50mLs. The pellet (19.52 gms) was resuspended with 3x volume of lysis buffer (approximately 50 mLs final) by intermitently placing the pellet in a 37°C 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 4°C 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)
Buffers: Affinity binding buffer: 10mM Imidazole, 300mM NaCl, 50mM pH8.0 NaH2PO4, 0.5mM 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.

Concentration : Using a Centricon 10 K cutoff concentrator the MPDZA-p069 pooled fractions was concentrated to 7.3 mg/mL. Concentration was determined from the absorbance at 280 nm.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Coyne CB, Voelker T, Pichla SL, Bergelson JM. (2004) The coxsackievirus and adenovirus receptor interacts with the multi-PDZ domain protein-1 (MUPP-1) within the tight junction. J. Biol. Chem. 279: 48079-48084.

Kimber WA, Trinkle-Mulcahy L, Cheung PC, Deak M, Marsden LJ, Kieloch A, Watt S, Javier RT, Gray A, Downes CP, Lucocq JM, Alessi DR. (2002) Evidence that the tandem-pleckstrin-homology-domain-containing protein TAPP1 interacts with Ptd(3,4)P2 and the multi-PDZ-domain-containing protein MUPP1 in vivo. Biochem. J. 361: 525-536.