Human Ubiquitin-Conjugating Enzyme E2E 2

Target ID:

UBC46

Aliases:

FLJ25157UBCH8

Deposition Date:

Monday 6th December 2004

Families:

Ubiquitin Related BiologyUbiquitylation - E2 Conjugate Enzymes

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

1Y6L

Authors:

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

Site:

Toronto

ICM Datapack:

ICM Datapack

1Y6L

tabs

Structure Details

Covalent attachment of ubiquitin to target proteins is a post-translation modification that controls expression, activity, and localization of numerous proteins in the eukaryotic cell.

Ubiquitination involves the sequential actions of three proteins, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin-protein ligases (E3). Polyubiquitinated protein substrates, specifically recognized by the 26S proteasome, an ATP dependant multicatalytic protease, are completely degraded. Consequently, ubiquitination plays important roles in the regulation of cell cycle progression, signal transduction, gene transcription, antigen receptor signaling, immune response and cell differentiation.

Dysfunction of this system has been implicated in a variety of diseases that have common cellular and molecular characteristics including protein aggregation and inclusion body formation. Examples include neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and prion diseases. Innovative therapies can therefore be expected from efforts targeting the ubiquitination system with its rich source of molecular targets. Although little is known about the physiological role of E2-e2, our high resolution structure of E2-e2, one of a number of human members, and homolog of yeast UBC4/5, is an important step towards a better understanding of its specificity and catalytic mechanism.

Materials and Methods

Human UBE2E2

PDB:1Y6L

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi:18490186
Entry Clone Source:MGC
SGC Clone Accession:ubc46.055.201; plate SDC006:C2
Tag:N-terminal His-tag with integrated thrombin-cleavage site MGSSHHHHHHSSGLVPRGS.
Host:E.coli BL21 (DE3)

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsTSAKRIQKELAEITLDPPPNCSAGPKGDNIYEWRSTILGPPGSVYEGGVFFLDITFSPDYPFKPPKVTFRTRIYHCNINSQGVICLDILKDNWSPALTISKVLLSICSLLTDCNPADPLVGSIATQYMTNRAEHDRMARQWTKRYAT
Vector:p28a-LIC

Growth

Medium:
Antibiotics:
Procedure:

Purification

Procedure
The cleared lysate was loaded onto a TALON metal-affinity resin column from BD Biosciences at 4ºC. 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:31 mg/mL
Ligand
MassSpec:
Crystallization:Purified UBC E2E2 was crystallized using the sitting 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 25% PEG 3350, 0.1M bis-Tris, pH 5.5.
NMR Spectroscopy:
Data Collection:
Data Processing: