Human PDZ domain containing 1 (4th PDZ domain)

Target ID:

PDZK1A

Aliases:

CAP70CLAMPNHERF3PDZD1PDZK1

Deposition Date:

Wednesday 30th April 2008

Families:

Linkers

Related Diseases:

MetabolismSignalling

Structure type:

apo

Download Coordinates:

2VSP

Authors:

W. W. Yue, N. Shafqat, E. S. Pilka, C. Johansson, J. Elkins, A. Roos, J. Murray, F. von Delft,, D. Doyle, A. Edwards, M. Wikstrom, C.H. Arrowsmith, C. Bountra and U. Oppermann

Site:

Oxford

2VSP

tabs

Structure Details

PDZK1 is a protein that consists of 4 tandem PDZ domains. As such it is a scaffold protein that connects plasma membrane proteins and regulatory components, regulating their surface expression in epithelial cells apical domains. It has been proposed to be involved in the coordination of a diverse range of regulatory processes for ion transport and second messenger cascades like regulation of proximal tubular Na(+)-dependent inorganic phosphate or cellular mechanisms associated with multidrug resistance through its interaction with ABCC2 and PDZK1IP1. PDZK1 has also been proposed to potentiate CFTR chloride channel activity and function to connect SCARB1 with the cellular machineries for intracellular cholesterol transport and/or metabolism. It plays a physiologically role in both oligopeptide handling as well as peptide-like drug transport in the human kidney and has been suggested in regulation of tubule function via regulation of phosphate cotransport (Kato et al., 2006). In addition PDZK1 is required for HDL/SR-BI signaling in endothelium and is involved in the maintenance of endothelial monolayer integrity (Zhu et al., 2008).
Overexpression of PDZK1 has been found in several tumors of epithelial origin and estrogen receptor positive breast cancer (Ghosh et al., 2000) and is likely to be associated with drug-resistance phenotype in multiple myeloma (Inoue et al., 2004).

PDZK1 knockout mice do not have a gross anatomical phenotype but have elevated levels of cholesterol due to a defect in membrane expression of the scavenger receptor ((Kocher et al., 2003b) (Kocher et al., 2003a)). When these mice are challenged with a high phosphate diet, they show decreased NaPi-IIa expression and increased renal phosphate excretion compared with the wild-type animals. (Capuano et al., 2005).

Here we present the structure of the PDZ domain of human PDZ containing 1.

Materials and Methods

Entry Clone Source: MGC

Entry Clone Accession: IMAGE:2959384

SGC Construct ID: PDZK1A-c025

GenBank GI number: gi|21361142

Vector: pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ]

E.coli strain: BL21(DE3)-R3-pRARE2

Tags and additions: N-terminal His-tag with TEV protease cleavage site (Tag sequence in lowercase)
mhhhhhhssgvdlgtenlyfqsMKPKLCR
LAKGENGYGFHLNAIRGLPGSFIKEVQKG
GPADLAGLEDEDVIIEVNGVNVLDEPYEK
VVDRIQSSGKNVTLLVCGKKAQDTV

Growth medium, induction protocol: 10µl of a glycerol stock was inoculated into 5ml of TB medium (supplemented with 50µg/ml Kanamycin) and cultured at 37°C o/n in a shaking incubator (275 rpm). Next day 0.75 ml of o/n culture was used to inoculate 1 litre of TB medium (2x) and grown at 37°C with vigorous shaking (160 rpm) until the culture reaches an OD₆₀₀ of 1.5. Temperature was reduced to 18°C, and cells were induced with IPTG at a concentration of 0.5 mM, and further cultivated for 16 hrs. Cells were harvested by centrifugation at 6500 rpm for 10 min, and the cell pellet was stored at -20°C until further use.

Extraction buffer, extraction method: Lysis buffer: 500 mM NaCl, 5% Glycerol, 50 mM HEPES pH 7.5, 5 mM Imidazole Complete® protease inhibitors (Roche, 1 tbl/50 ml). Frozen cell pellets were thawed and resuspended in a total volume of 30-40 ml of lysis buffer, and disrupted by using Avestin C-5 microfluidizer, and a supernatant containing the target protein was obtained by centrifugation at 21,000 (rpm) for 45 minutes.

Column 1: Ni-Sepharose 6 Fast Flow

Buffers: Lysis buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 5 mM Imidazole; Wash buffer: 50 mM Tris-HCl pH 7.5, 500 mM NaCl, 5% Glycerol, 30 mM Imidazole; Elution buffer:50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 250 mM Imidazole. note: All the buffers contain 0.5mM TCEP.

Procedure: The column was packed with 2 ml of Ni-Sepharose 6 Fast Flow slurry and equilibrated with 15 ml of binding buffer. The supernatant was loaded onto the column and the column was washed with 20 ml of binding buffer and then 20 ml of washing buffer. The protein was eluted with 10 ml of elution buffer.

Column 2: SuperDex S-75 (GE/Amersham)

Buffer: 10 mM HEPES, pH 7.5, 500 mM NaCl, 5 % glycerol, 0.5 mM TCEP.

Procedure: The eluted protein from the Ni-affinity column was loaded on the gel filtration column in GF buffer at 1.0 ml/min on an AKTA Purifier system. Eluted proteins were collected in 1 ml fractions.

Enzymatic treatment: TEV cleaved.

Column 3: Ni-NTA (TEV clean up)

Buffer: 10 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP

Procedure: Total 10 mgs of protein was cleaved with 600 ug of TEV protease at 4°C for 48 hours.

TEV clean up: The TEV cleaved protein was applied to a 1 ml Ni-NTA column, already equalibrated with gel filtration buffer (10 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP). The flow through form the column was collected. The eluate from the column was monitored by SDS gel analysis.

Concentration: The PDZK1A protein (in buffer; 10 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP) was concentrated to 3.0 mg/ml using Vivaspin 5K concentrators and stored at -80°C.

Mass spec characterization: Corresponds to theoretical mass, as determined by ESI-TOF MS.

Crystallization: PDZK1A was crystallized at 20°C using the sitting-drop vapour diffusion method. Diffraction quality crystals were obtained by mixing 100nl of protein solution (3.0 mg/ml) with 50nl reservoir solution (0.1M Na/K-PO₄ pH6.2, 0.20M NaCl and 50% PEG300).

Data Collection: Crystals were flash frozen in liquid nitrogen. Diffraction data were collected to 2.2 Å on beam-line X10SA at the Swiss light source (SLS); Resolution: 2.2 Å

References

Capuano, P., Bacic, D., Stange, G., Hernando, N., Kaissling, B., Pal, R., Kocher, O., Biber, J., Wagner, C.A., and Murer, H. (2005). Expression and regulation of the renal Na/phosphate cotransporter NaPi-IIa in a mouse model deficient for the PDZ protein PDZK1. Pflugers Arch 449, 392-402.

Ghosh, M.G., Thompson, D.A., and Weigel, R.J. (2000). PDZK1 and GREB1 are estrogen-regulated genes expressed in hormone-responsive breast cancer. Cancer research 60, 6367-6375.

Inoue, J., Otsuki, T., Hirasawa, A., Imoto, I., Matsuo, Y., Shimizu, S., Taniwaki, M., and Inazawa, J. (2004). Overexpression of PDZK1 within the 1q12-q22 amplicon is likely to be associated with drug-resistance phenotype in multiple myeloma. The American journal of pathology 165, 71-81.

Kato, Y., Watanabe, C., and Tsuji, A. (2006). Regulation of drug transporters by PDZ adaptor proteins and nuclear receptors. Eur J Pharm Sci 27, 487-500.

Kocher, O., Pal, R., Roberts, M., Cirovic, C., and Gilchrist, A. (2003a). Targeted disruption of the PDZK1 gene by homologous recombination. Molecular and cellular biology 23, 1175-1180.

Kocher, O., Yesilaltay, A., Cirovic, C., Pal, R., Rigotti, A., and Krieger, M. (2003b). Targeted disruption of the PDZK1 gene in mice causes tissue-specific depletion of the high density lipoprotein receptor scavenger receptor class B type I and altered lipoprotein metabolism. The Journal of biological chemistry 278, 52820-52825.

Zhu, W., Saddar, S., Seetharam, D., Chambliss, K.L., Longoria, C., Silver, D.L., Yuhanna, I.S., Shaul, P.W., and Mineo, C. (2008). The scavenger receptor class B type I adaptor protein PDZK1 maintains endothelial monolayer integrity. Circulation research 102, 480-487.