Human Ubiquitin Conjugating Enzyme E2 UEV1

Target ID:

UBC62

Aliases:

CROC-1BKua-UEVUBE2V1

Deposition Date:

Tuesday 28th June 2005

Families:

Ubiquitin Related BiologyUbiquitylation - E2 Conjugate Enzymes

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

2A4D

Authors:

J.R.WALKER,G.V.AVVAKUMOV,S.XUE,E.M.NEWMAN,F.MACKENZIE,J.WEIGELT,M.SUNDSTROM,C.ARROWSMITH,A.EDWARDS,A.BOCHKAREV,S.DHE-PAGANON,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Toronto

2A4D

tabs

Structure Details

DNA damage induces a cascade of cellular events that include the activation of DNA repair enzymes as well as inhibition of DNA replication. In yeast, DNA replication is inhibited by ubiquitylation of the essential DNA polymerase processivity factor, PCNA. Two prominent steps are necessary:

monoubiquitylation of PCNA Lys-164 by E3 (RAD18)/E2(UBC-RAD6);
subsequent extension and chain formation through ubiquitin's K63 by E3(RAD5)/E2 (UBC-13 and UBC-MMS2).

It has been established that the E2 pair, UBC-13 and UBC-MMS2, form a stable heterodimeric complex competent to catalyze unanchored K63-linked polyubiquitin chains from free ubiquitin in vitro; MMS2, which does not have the reactive cysteine in its active, acts instead as an E3 ligase.

Although absent of any ubiquitin molecules, the structure of the heterodimer suggests an elegant mechanism: if ubiquitin binds in canonical sites, the nucleophilic amino group of K63 from the MMS2-associated ubiquitin is poised to attack the thiolester bond of the covalently linked UBC13-associated ubiquitin. A confounding issue is that both E3's, RAD5 and MMS2, are required for chain elongation, throwing into question the proposed mechanism of MMS2 mediated ligation.

UEV1 is an additional human UBC conjugase that does not contain a reactive cysteine. The amino acid sequence of UEV1 suggests that the canonical active site, where the vast majority of differences with MMS2 occur, may play an important role in the molecular function of these E3-like UBC's. UEV1 may play biological roles similar to MMS2, and the high resolution crystallographic structure of UEV1 may provide further clues to the mechanism of its ligase activity and specificity.

Materials and Methods

UEV1

PDB:2A4D

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi:40806189
Entry Clone Source:MGC
SGC Clone Accession:ubc62.230.370; SDC021:E1
Tag:N-terminal His-tag with integrated thrombin-cleavage site MGSSHHHHHHSSGLVPRGS.
Host:E.coliBL21 (DE3)

Construct

Prelude:
Sequence:MGSSHHHHHHSSGLVPRGSTGVKVPRNFRLLEELEEGQKGVGDGTVSWGLEDDEDMTLTRWTGMIIGPPRTIYENRIYSLKIECGPKYPEAPPFVRFVTKINMNGVNSSNGVVDPRAISVLAKWQNSYSIKVVLQELRRLMMSKENMKLPQPPEGQCYSN
Vector:p28a-LIC

Growth

Medium:TB
Antibiotics:
Procedure:UEV1 was expressed in E. coli BL21 (DE3) grown in Terrific Broth (TB) in the presence of 50 µg/ml of kanamycin at 37ºC 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 -80ºC.

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 (DTT) 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:
Ligand
MassSpec:
Crystallization:Purified UEV1 was crystallized using the hanging drop vapor diffusion method. Crystals grew when the protein (12 mg/mL) was mixed with the reservoir solution in a 1:1 volume ratio, and the drop was equilibrated against a reservoir solution containing 32% PEG1500, 0.2 M NaCl, 1 MM DTT, 0.1 M Na Cacodylate at pH 6.5 in 293K temperature.
NMR Spectroscopy:
Data Collection:
Data Processing: