Tax1 (human T-cell leukemia virus type I) binding protein 3

Target ID:

TAX1BP3A

Aliases:

TAX1BP3TIP-1

Deposition Date:

Wednesday 30th July 2008

Families:

Linkers

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

2VZ5

Authors:

J.W.MURRAY,N.SHAFQAT,W.YUE,E.PILKA,,C.JOHANNSSON,E.SALAH,C.COOPER,J.M.ELKINS,A.C.PIKE,A.ROOS,P.FILIPPAKOPOULOS,F.VONDELFT,M.WICKSTROEM,C.BOUNTRA,A.M.EDWARDS,C.H.ARROWSMITH,U.OPPERMANN

Site:

Oxford

2VZ5

tabs

Structure Details

Tax-interacting protein-1, TIP-1 is a ubiquitously expressed protein that has originally been identified as one of the protein interacting with the C-terminus of the HTLV-1 Tax oncoprotein (Hampson et al., 2004).

TIP-1 consists almost entirely of a single PDZ domain with no other obvious protein interaction module and has been suggested to act as a general negative regulator of PDZ scaffolding competing with other PDZ containing proteins for binding to specific target proteins. TIP-1 antagonizes the retention function of the mLin-7/CASK complex by competing for interaction with Kir 2.3 resulting in internalization of Kir 2.3 channels (Alewine et al., 2007). TIP-1 has also been implicated in regulating the canonical Wnt β-catenin signaling pathway. Overexpression of TIP-1 reduces β -catenin dependent transcription and decreases proliferation and anchorage-independent growth of colorectal cancer cells. In zebrafish TIP-1 induces filopodia growth and is important for gastrulation movements during development (Besser et al., 2007).

We have determined the structure of the PDZ domain of human TAX1BP3 at 1.8 Å resolution.

Materials and Methods

Entry Clone Source: MGC

Entry Clone Accession: IMAGE:4069168

SGC Construct ID: TAX1BP3A-c001

GenBank GI number: gi|11993943

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

Tags and additions: N-terminal His-tag with TEV protease cleavage site (*)

Final protein sequence (tag sequence in lowercase):

mhhhhhhssgvdlgtenlyfqs*mVQRVE
IHKLRQGENLILGFSIGGGIDQDPSQNPF
SEDKTDKGIYVTRVSEGGPAEIAGLQIGD
KIMQVNGWDMTMVTHDQARKRLTKRSEEV
VRLLVTRQSLQKAVQQSMLSETEV

Host: BL21(DE3)-R3 pRARE2

Growth medium, induction protocol: 10ul of a glycerol stock was inoculated into 5ml of TB medium (supplemented with 50ug/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 1.4. 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 200 16/60 HiLoad (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 degree for 48 hours.

TEV clean up: The TEV cleaved protein was applied to a 1 ml Ni-NTA column, already equilibrated 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.

Sample Concentration: The protein sample (in buffer; 10 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP) was concentrated to1.5 mg/ml using Vivaspin 10K concentrators and stored at -80C.

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

Crystallization: Crystals were grown by vapour diffusion in sitting drops at 4°C. A sitting drop consisting of 150 nl protein and 75 nl well solution was equilibrated against well solution containing 10% PEG 3350, 150mM Zinc acetate pH 3.8. Crystals were cryo protected in 25% ethylene glycol and flash-cooled in liquid nitrogen.

Data Collection, Resolution: 1.8 Å , X-ray source: Synchrotron SLS-X10SA, single wavelength.

References

Alewine, C., Kim, B.Y., Hegde, V., and Welling, P.A. (2007). Lin-7 targets the Kir 2.3 channel on the basolateral membrane via a L27 domain interaction with CASK. American journal of physiology 293, C1733-1741.

Besser, J., Leito, J.T., van der Meer, D.L., and Bagowski, C.P. (2007). Tip-1 induces filopodia growth and is important for gastrulation movements during zebrafish development. Development, growth & differentiation 49, 205-214.

Hampson, L., Li, C., Oliver, A.W., Kitchener, H.C., and Hampson, I.N. (2004). The PDZ protein Tip-1 is a gain of function target of theHPV16 E6 oncoprotein. International journal of oncology 25, 1249-1256.