Human ubiquitin specific protease 8 - E3 ligase complex

Target ID:

USP08

Aliases:

FLJ34456HumORF8KIAA0055MGC129718UBPY

Deposition Date:

Thursday 4th May 2006

Families:

Deubiquitinating Enzymes - USPUbiquitin Related Biology

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

2GWF

Authors:

J.R.WALKER,G.V.AVVAKUMOV,S.XUE,E.M.NEWMAN,C.BUTLER-COLE,P.J.FINERTY JR.,J.WEIGELT,M.SUNDSTROM,C.H.ARROWSMITH,A.M.EDWARDS,A.BOCHKAREV,S.DHE-PAGANON,STRUCTURAL GENOMICSCONSORTIUM (SGC)

Site:

Toronto

2GWF

tabs

Structure Details

The RING finger E3 ligase Nrdp1 catalyzes the ubiquitylation of epidermal growth factor receptors and the antiapoptotic protein IAP/BRUCE. The autoubiquitylation of Nrdp1 contributes to its own degradation by the proteasomal system, but this activity is countered by the association of Nrdp1 with the deubiquitylase ubiquitin specific protease 8 (Usp8/UBPy). Usp8 contains multiple domains, including a rhodanese domain, which was shown to be necessary for stable interaction with Nrdp1.We report the high-resolution crystal structure of the Usp8 rhodanese domain in complex with the Nrdp1 carboxy-terminal domain. Nrdp1 interacts with a conserved peptide loop of the rhodanese domain. The consensus sequence of this loop is found in other Nrdp1 targets, suggesting a common method of interaction.

Materials and Methods

Human NRDP1 + USP8 complex

PDB:2GWF

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:
SGC Clone Accession:NRDP1: nrdp1.193.317; plate SDC046:F7; USP8: usp08.0181.0319, plate SDC051:H2
Tag:N-terminal his tag with integrated thrombin protease site: MGSSHHHHHHSSGLVPR*GS
Host:

Construct

Prelude:
Sequence:USP8:
mgsshhhhhhssglvprgsGAITAKELYTMMTDKNISLIIMDARRMQDYQDSCILHSLSVPEEAISPGVTASWIEAHLPDDSKDTWKKRGNVEYVVLLDWFSSAKDLQIGTTLRSLKDALFKWESKTVLRNEPLVLEGGYENWLLCYPQYTTNAKVT
NRDP1:
MGSSGLVPRGSTIEYNEILEWVNSLQPARVTRWGGMISTPDAVLQAVIKRSLVESGCPASIVNELIENAHERSWPQGLATLETRQMNRRYYENYVAKRIPGKQAVVVMACENQHMGDDMVQEPGLVMIFAHGVEEI
Vector:p28a-LIC

Growth

Medium:TB
Antibiotics:
Procedure:The proteins were expressed independently in E. coli BL21 (DE3) grown in Terrific Broth (TB) in the presence of 50 microG/ml of kanamycin at 37degC to an OD600 of 7.5. Cells were then induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 0.05 mM, and incubated overnight at 15degC. The culture was centrifuged and the cell pellets were collected and stored at -80degC.

Purification

Procedure
Column 1: TALON metal-affinity resin column (BD Biosciences)
Column 2: HighLoad 16/60 Superdex 200 column (GE Healthcare, Amersham)

The cleared lysate was loaded onto a TALON metal-affinity resin column from BD Biosciences at 4degC. The column was washed with wash buffer A, wash buffer B and again wash buffer A, and the protein was eluted with 10 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 5% glycerol, 200 mM imidazole, 1 mM ß-mercaptoethanol. The protein was further purified by gel filtration on a HighLoad 16/60 Superdex 200 column (GE Healthcare, Amersham) equilibrated with 20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 5% glycerol, 2mM dithiothreitol and concentrated by ultrafiltration.

Extraction

Procedure
The cell pellets were resuspended in lysis buffer and inhibitor (0.1mM phenylmethyl sulfonyl fluoride, PMSF) and lysed using Microfluidizer. The lysate was cleared by centrifugation.
Concentration:
Ligand
MassSpec:
Crystallization:The proteins were mixed in 1:1 ratio and crystallized by means of hanging drop vapor diffusion under the following conditions:12% PEG5000 MME, 0.1 M bis-tris, 1 MM DTT, pH 6.5, 298 K.
NMR Spectroscopy:
Data Collection:
Data Processing: