Human ubiquitin-conjugating enzyme E2Q

Target ID:

ubc64

Aliases:

GTAPNICE-5PRO3094UBE2Q

Deposition Date:

Friday 29th June 2007

Families:

Ubiquitin Related BiologyUbiquitylation - E2 Conjugate Enzymes

Related Diseases:

Cancer

Structure type:

apo

Download Coordinates:

2QGX

Authors:

Neculai D, Avvakumov GV, Xue S, Walker JR, Mackenzie F, Weigelt J, Arrowsmith CH, Edwards AM, Bochkarev A, Sicheri F, Dhe-Paganon S

Site:

Toronto

ICM Datapack:

ICM Datapack

2QGX

tabs

Structure Details

The biological roles of the related human E2s, UBE2Q1 and UBE2Q2, are unknown, but they have been implicated in epidermal differentiation (Marenholz et al., 2001), cell cycle progression and mitosis (Banerjee et al., 2007), endometrial implantation (Melner et al., 2006), cytoskeletal function (Seghatoleslam et al., 2006), and neuromuscular function in worms (Seghatoleslam et al., 2006). These E2 family members uniquely contain amino terminal RWD domains.

We have determined the high resolution structure of the E2 domain of UBE2Q1, showing the canonical E2 fold with an active site cysteine surrounded by hydophobic residues (Tyr96, Leu98, Tyr155, Leu158). The carboxy-terminal helix, like its UBE2Q2 paralog, sits atop of the cysteine, potentially providing a scaffold for substrate specificity.

Materials and Methods

UBE2Q1

PDB:2QGX

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_060052
Entry Clone Source:ubc64.GSC.ubc64.221.422.pUC57
SGC Clone Accession:ubc64.248.414; plate SDC45G03
Tag:mgsshhhhhhssglvpr*gs
Host:BL21 (DE3)

Construct

Prelude:
Sequence:mgsshhhhhhssglvpr*GSGSVQVTDRLMKELRDIYRSQSFKGGNYAVELVNDSLYDWNVKLLKVDQDSALHNDLQILKEKEGADFILLNFSFKDNFPFDPPFVRVVSPVLSGGYVLGGGAICMELLTKQGWSSAYSIESVIMQISATLVKGKARVQFGANKSQYSLTRAQQSYKSLVQIHEKNGW
Vector:p28a-LIC-Thrombin

Growth

Medium:TB
Antibiotics:
Procedure:The protein 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. Protein expression was then induced with 0.05 mM isopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15ºC. The culture was centrifuged (12,000 x g, 15 min) and cell pellets collected and stored at -80ºC.

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 from a 2 liter culture was loaded onto 3 ml TALON metal-affinity resin column (BD Biosciences) at 4ºC. The column was washed with 10 ml Wash buffer A, 10 ml Wash buffer B and then 30 ml Wash buffer A, and the protein was eluted with 6 ml Elution buffer.

His-tags was removed by incubation of the protein with 1 U thrombin per mg protein (overnight at 4 °C), and the protein was further purified by gel filtration on a HighLoad 16/60 Superdex 200 column (GE Healthcare, Amersham) equilibrated with Gel Filtration buffer and concentrated by ultrafiltration using Amicon Ultra centrifugal filter with 10kD cutoff.

Extraction

Procedure
The cell pellet from a 2 liter culture was resuspended in 50 ml Lysis buffer and lysed using Microfluidizer at 18,000 psi. The lysate was cleared by centrifugation (40,000 x g, 30 minutes).
Concentration:
Ligand
MassSpec:
Crystallization:Crystals were grown in hanging drops by mixing 2 microL protein solution (10 mg/ml) with 2 microL well solution (22% PEG4000, 0.1 M Tris-Cl, pH7.5, 0.12 M MgCl2, 1 mM DTT) at 21°C. For cryoprotection, the crystals were soaked in well solution supplemented with 20% glycerol.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Banerjee,S., Brooks,W.S., and Crawford,D.F. (2007). Inactivation of the ubiquitin conjugating enzyme UBE2Q2 causes a prophase arrest and enhanced apoptosis in response to microtubule inhibiting agents. Oncogene.
Marenholz,I., Zirra,M., Fischer,D.F., Backendorf,C., Ziegler,A., and Mischke,D. (2001). Identification of human epidermal differentiation complex (EDC)-encoded genes by subtractive hybridization of entire YACs to a gridded keratinocyte cDNA library. Genome Res. 11, 341-355.
Melner,M.H., Haas,A.L., Klein,J.M., Brash,A.R., Boeglin,W.E., Nagdas,S.K., Winfrey,V.P., and Olson,G.E. (2006). Demonstration of ubiquitin thiolester formation of UBE2Q2 (UBCi), a novel ubiquitin-conjugating enzyme with implantation site-specific expression. Biol. Reprod. 75, 395-406.
Seghatoleslam,A., Zambrano,A., Millon,R., Ganguli,G., Argentini,M., Cromer,A., Abecassis,J., and Wasylyk,B. (2006). Analysis of a novel human gene, LOC92912, over-expressed in hypopharyngeal tumours. Biochem. Biophys. Res. Commun. 339, 422-429.