Human E3 ubiquitin ligase

Target ID:

NRDP1

Aliases:

MGC45228NRDP1SBBI03

Deposition Date:

Monday 13th February 2006

Families:

DiabetesUbiquitin Related BiologyUbiquitylation - E3 Ligases

Related Diseases:

SignallingDiabetes

Structure type:

apo

Download Coordinates:

2FZP

Authors:

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

Site:

Toronto

ICM Datapack:

ICM Datapack

2FZP

tabs

Structure Details

The RING finger E3 ligase NRDP1 catalyzes the ubiquitylation of epidermal growth factor receptors, which leads to clathrin mediated internalization and proteasomal degradation. 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 catalytic domain, coiled-coil domains, and a rhodanese domain. We report the high-resolution crystal structure of the NRDP1 carboxy-terminal domain, which is sufficient to interact with USP8 and which we have named the USP8 interaction domain. The NRDP1 USP8 interaction domain forms a novel protein fold, composed of a central beta sheet surround by alpha helices.

Materials and Methods

NRDP1

PDB:2FZP

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:37588860
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal His-tag with integrated thrombin-cleavage site MGSSHHHHHHSSGLVPRGS
Host:E.coli BL21 (DE3)

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsTIEYNEILEWVNSLQPARVTRWGGMISTPDAVLQAVIKRSLVESGCPASIVNELIENAHERSWPQGLATLETRQMNRRYYENYVAKRIPGKQAVVVMACENQHMGDDMVQEPGLVMIFAHGVEEI
Vector:p28a-LIC

Growth

Medium:
Antibiotics:
Procedure:NRDP1 was expressed in E. coli BL21 (DE3) grown in Terrific Broth (TB) in the presence of 50 µg/ml of kanamycin at 37 degC 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 15ºC. The culture was centrifuged and the cell pellets were collected and stored at -80 degC.

Purification

Procedure
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 (10mM Tris-HCl pH 8.0, 0.5 M NaCl, 5% glycerol, 10 mM imidazole, 1 mM ß-mercaptoethanol), wash buffer B (same as wash buffer A but containing 0.05% Tween with protease 20) 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 pellet was resuspended in lysis buffer (10 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 5% glycerol, 2 mM imidazole, 1 mM ß-mercaptoethanol) inhibitor (0.1mM phenylmethyl sulfonyl fluoride, PMSF) and lysed using Microfluidizer. The lysate was cleared by centrifugation.
Concentration:20 mg/mL
Ligand
MassSpec:
Crystallization:Purified NRDP1 was crystallized using the hanging drop vapor diffusion method. Crystals grew when the protein (20 mg/mL) was mixed with the reservoir solution in a 1:1 volume ratio, and the drop was equilibrated against a reservoir solution containing 1.8M ammonium sulphate, 0.2M sodium chloride, 0.1M Hepes, 1mM DTT, pH 7.0.
NMR Spectroscopy:
Data Collection:
Data Processing: