Human ubiquitin-conjugating enzyme E2

Target ID:

UBC36

Aliases:

E217KUBC7UBE2G

Deposition Date:

Thursday 1st September 2005

Families:

Ubiquitin Related BiologyUbiquitylation - E2 Conjugate Enzymes

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

2AWF

Authors:

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

Site:

Toronto

ICM Datapack:

ICM Datapack

2AWF

tabs

Structure Details

A subset of ubiquitin conjugating enzymes, including UBE2G1, UBE2G2, UBE2R1, and UBE2R2, are characterized by a unique 12 residue insertion loop between the seventh beta strand and second alpha helix. In order to better understand molecular functions of these proteins, we determined the first high resolution crystal structure of this human subfamily: UBE2G1.

Although the overall fold of UBE2G1 is composed of the canonical three-stranded, anti-parallel curled beta-sheet, there are some glaring absences. A number of secondary structural elements as well as long loop regions are completely disordered, despite being present in the protein. The pair of adjacent, carboxy-terminal alpha helices (alpha-3 and alpha-4) is completely disordered in our crystal structure, including residues Asn-132 to Arg-159. This leaves one end of the curled sheet exposed, which includes a set of aromatic (Trp-79, Phe-55, and tyr-50) and aliphatic residues (Leu-49 and Ile-78).

The loop region connecting alpha-2 and alpha-3 is normally part of the active site, forming one of its walls, containing the putative general acid/base for catalysis. This loop is also disordered in our structure, leaving the active site only partially formed. The distinguishing 12 residue insertion loop (residues 98-106), which is highly negatively charged and contains numerous small side chain residues (proline, serines, and glycines), is also disordered and missing from our structure. Because no significant electron density has been observed for these regions, no residues could be modeled in.

This brings into question the molecular functions of the missing elements. Are they important for specificity of ubiquitin homolog or E3 ligase binding? Are they directly involved in catalysis? Little is known about the cellular role of UBE2G with the exception that it seems to be predominantly expressed in adult muscle.1,2 Complex structures of E2/E3 may help evaluate the role of the flexible elements of UBE2G1.

Materials and Methods

UBE2G1

PDB:2AWF

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:MGC
SGC Clone Accession:ubc36.008.160; plate SDC017:G1
Tag:N-terminal His-tag with integrated thrombin-cleavage site MGSSHHHHHHSSGLVPRGS.
Host:E.coliBL21 (DE3)

Construct

Prelude:
Sequence:MGSSHHHHHHSSGLVPRGSLLLRRQLAELNKNPVEGFSAGLIDDNDLYRWEVLIIGPPDTLYEGGVFKAHLTFPKDYPLRPPKMKFITEIWHPNVDKNGDVCISILHEPGEDKYGYEKPEERWLPIHTVETIMISVISMLADPNGDSPANVDAAKEWREDRNGEFKRKVARC
Vector:p28a-LIC

Growth

Medium:TB
Antibiotics:
Procedure:UBE2G1 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 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 (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 UBE2G1 was crystallized using the hanging drop vapor diffusion method. Crystals grew when the protein was mixed with the reservoir solution in a 1:1 volume ratio, and the drop was equilibrated against a reservoir solution containing 24% PEG3350, 0.2 M Mg acetate, 0.1 M tris, pH 7.5, 5% glycerol in 293K temperature.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

1. Watanabe TK, Kawai A, Fujiwara T, Maekawa H, Hirai Y, Nakamura Y, Takahashi E. Molecular cloning of UBE2G, encoding a human skeletal muscle-specific ubiquitin-conjugating enzyme homologous to UBC7 of C. elegans. Cytogenet Cell Genet. 1996;74(1-2):146-8.

2. Katsanis N, Fisher EM. Identification, expression, and chromosomal localization of ubiquitin conjugating enzyme 7 (UBE2G2), a human homologue of the Saccharomyces cerevisiae ubc7 gene. Genomics. 1998 Jul 1;51(1):128-31.