Human tumor susceptibility gene 101, coiled-coil domain

Target ID:

UBC80

Deposition Date:

Monday 31st August 2009

Families:

Ubiquitin Related BiologyUbiquitylation - E2 Conjugate Enzymes

Related Diseases:

Cancer

Structure type:

apo

Download Coordinates:

3IV1

Authors:

Neculai D, Avvakumov GV, Wernimont AK, Xue S, Walker JR, Li Y, Weigelt J, Bountra C, Arrowsmith CH, Edwards AM, Bochkarev A, Dhe-Paganon S

Site:

Toronto

3IV1

tabs

Structure Details

TSG101, the protein encoded by tumor susceptibility gene 101 (tsg101)[1] stimulates normal proliferation and growth by regulating the level of proteins that are important for controlling the cell cycle[2-5]. TSG101 is a part of endosomal sorting complex required for transport I (ESCRT-I), which mediate protein sorting, MVB biogenesis, tumour suppression and viral budding[6]. Two of the most central negative regulators of the cell cycle, p53 and p21, accumulate in cells and tissues deficient in Tsg101[4, 7]. Recent reports have implied that Tsg101 reduces the level of p53 by stimulating its ubiquitin-dependent proteosomal degradation mediated by the ubiquitin ligase Mdm2[4]. The UEV domain of Tsg101 binds and stabilizes the monoubiquitinated form of Mdm2 that might prevent the protein from being further polyubiquitinated and degraded in the cytoplasm. Tsg101 also functions as a transcriptional regulator of steroid hormone receptors[8, 9]. In addition to the N-terminal UVE domain, TSG101contains a coiled-coil domain that is required for both the transcription suppressive and the tumor suppressive functions of TSG101[10]. This domain was found to interact with stathmin, a cytosolic phosphoprotein implicated in tumorigenesis[1]. Here we present the crystal structure of the coiled-coil domain of human TSG101.

Materials and Methods

TSG101

PDB:3IV1

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:LIFESEQ2072052.OBS.IHS1380-97652701.pINCY
Entry Clone Source:OpenBiosystems
SGC Clone Accession:ubc80.0229.0304.172D02 (SDC172D02)
Tag:N-termlnal tag: MGSSHHHHHHSSGLVPRGS
Host:BL21 (DE3)

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsSLISAVSDKLRWRMKEEMDRAQAELNALKRTEEDLKKGHQKLEEMVTRLDQEVAEVDKNIELLKKKDEELSSALEK
Vector:pET28a-LIC

Growth

Medium:
Antibiotics:
Procedure:Competent BL21 (DE3) cells (Invitrogen C6000-03) were transformed and grown using the LEX system (Harbinger BEC) at 37 degC in 2L bottles (VWR 89000-242). In order to obtain the selenomethionyl derivative of the TSG101 domain, the cells were grown in M9 medium supplemented with supplemented with 150 mM glycerol, 100 µg/ml Kanamycin and 600 µl antifoam 204 (Sigma A-8311) using a M9 SeMET High-Yield growth media kit package (Medicilon MD045004-50L) according to manufacturer's instruction. When OD600 0.8 - 1.2 was reached, the temperature was reduced to 20 degC, and one hour later protein expression was induced with 100 µM IPTG (BioShop IPT001) and the culture was incubated overnight (16 hours) at 20 degC. Cell pellets were collected by centrifugation (12,227 x g, 20 min), frozen and stored at -80 degC.

Purification

Procedure
The cleared lysate was loaded onto a 3 mL TALON metal-affinity resin column (Clontech Laboratories 635504) at 4ºC. The column was washed with 10 mL Wash Buffer A, 10 mL Wash Buffer B and 10 mL Wash Buffer A. The protein was eluted with 6 mL Elution Buffer. The protein was further purified by gel filtration on a HighLoad 16/60 Superdex 200 column (GE Healthcare 17-1069-01) using Gel Filtration Buffer. Fractions containing SeMet TSG101 were pooled and concentrated by ultrafiltration. The N-terminal His-tag was removed by overnight incubation of the protein with thrombin (1 unit/mg protein) at 4°C, and final protein purification was achieved by anion-exchange chromatography on a 5-ml HiTrapQ HP column (GE Healthcare 17-1154-01) using Ion-Exchange Buffer A and a 0 - 0.5 M NaCl gradient created by mixing Buffer A with Ion-Exchange Buffer B. The yield of the protein was 6 mg per liter of bacterial culture; its purity (SDS-PAGE) was about 98 %. Molecular weight of the product (9181.2, LC/MS) indicated that all 3 methionyls were substituted with selenomethionyls.

Extraction

Procedure
After resuspension in 30 mL per liter bacterial culture of Lysis Buffer, cells were lysed using a Microfluidics M110-EH microfluidizer at 18,000 psi.
Concentration:
Ligand
MassSpec:
Crystallization:Crystals of the selenomethionyl derivative of the TSG101 domain were grown at 291 K using the hanging drop method by mixing equal volumes of protein solution (14 mg/ml) and Crystallization Buffer (2.4 M ammonium sulfate, 0.1 M bis-Tris, pH 5.5 and 1 mM dithiothreitol). The crystals were cryoprotected by immersion in well solution mixed in an 1 : 1 ratio with water solution of and placed in liquid nitrogen.
NMR Spectroscopy:
Data Collection:Diffraction data from a crystal of the selenomethionyl derivative of the TSG101 domain were collected at beamline APS-19ID of the GM/CA-CAT at the Advanced Photon Source (Argonne National Laboratory). The data set was integrated and scaled using the XDS program package.
Data Processing:The structure was solved using programs SHELXD and SHELXE. Iterative model building using the graphics program COOT and refinement package BUSTER led to a model with an R factor of 18.8 % (Rfree 21.8 %) for data between 50 and 2.59 Å.

References

Li, L. and Cohen, S.N. (1996) tsg101: A novel tumor susceptibility gene isolated by controlled homozygous functional knockout of allelic loci in mammalian cells. Cell 85, 319-329.
Carstens, M.J. et al. (2004) Cell cycle arrest and cell death are controlled by p53-dependent and p53-independent mechanisms in Tsg101-deficient cells. J. Biol. Chem. 279, 35984-35994.
Krempler, A. et al. (2002) Targeted deletion of the Tsg101 gene results in cell cycle arrest at G1/S and p53-independent cell death. J. Biol. Chem. 277, 43216-43223.
Li, L. et al. (2001) ATSG101/MDM2 regulatory loop modulatesMDM2 degradation and MDM2/p53 feedback control. Proc. Natl. Acad. Sci. U. S. A. 98, 1619-1624.
Oh, H. et al. (2002) Negative regulation of cell growth and differentiation by TSG101 through association with p21(Cip1/WAF1). Proc. Natl. Acad. Sci. U. S. A. 99, 5430-5435.
Slagsvold, et al.(2006) Endosomal and non-endosomal functions of ESCRT proteins. Trends Cell Biol.16, 317-326.
Ruland, J. et al. (2001) p53 accumulation, defective cell proliferation, and early embryonic lethality in mice lacking tsg101. Proc. Natl. Acad. Sci. U. S. A. 98, 1859-1864.
Burgdorf, S. et al. (2004) TSG101 interacts with apoptosis-antagonizing transcription factor and enhances androgen receptor-mediated transcription by promoting its monoubiquitination. J. Biol. Chem. 279, 17524-17534.
Sun, Z. et al. (1999) Tumor susceptibility gene 101 protein represses androgen receptor transactivation and interacts with p300. Cancer 86, 689-696.
Watanabe, M. et al. (1998) A putative tumor suppressor, TSG101, acts as a transcriptional suppressor through its coiled-coil domain. Biochem. Biophys. Res. Commun.245, 900-905.