Human ubiquitin-conjugating enzyme UBCH6

Target ID:

ubc47

Aliases:

UBCH6

Deposition Date:

Thursday 17th January 2008

Families:

Ubiquitin Related BiologyUbiquitylation - E2 Conjugate Enzymes

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

3BZH

Authors:

Walker JR, Avvakumov GV, Xue S, Li Y, Weigelt J, Arrowsmith CH, Edwards AM, Bochkarev A, Dhe-Paganon S

Site:

Toronto

ICM Datapack:

ICM Datapack

3BZH

tabs

Structure Details

The modification of proteins with ubiquitin is an important cellular mechanism for targeting abnormal or short-lived proteins for degradation. Ubiquitination involves at least three classes of enzymes: ubiquitin-activating enzymes, or E1s, ubiquitin-conjugating enzymes, or E2s, and ubiquitin-protein ligases, or E3s. This gene encodes a member of the E2 ubiquitin-conjugating enzyme family. Two alternatively spliced transcript variants encoding distinct isoforms have been found for this gene.[1]

The crystal structure of UBE2E1 reveals a canonical E2 fold, composed of a four-stranded, anti-parallel curled β-sheet surrounded on three sides by α-helical segments. Most of the amino-terminal 40 residues are disordered, except for a short stretch of residues (21-27) that loosely interact with a neighboring molecule. The catalytic cysteine is in a shallow pocket, surrounded by the conserved active site residues Asn123, Ser146, and Leu165. Lys136, implicated in auto-ISGylation of the closely-related UBE2E2 [Takeuchi, 2005], is disordered in UBE2E1. The amino-terminal α1 is extended by two turns and mediates symmetric, head-to-head contact with an adjacent molecule, potentially representing a biologically active unit.

Materials and Methods

UBE2E1

PDB:3BZH

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_997236
Entry Clone Source:ubc47.NM_003341.EXT.janosch_ube2e1.pETkan
SGC Clone Accession:ubc47.001.193.129A11 (SDC129A11)
Tag:N-terminal: MHHHHHHSSGRENLYFQ*G (removed after metal-affinity chromatography)
Host:BL21 (DE3)

Construct

Prelude:
Sequence:mhhhhhhssgrenlyfq*gMSDDDSRASTSSSSSSSSNQQTEKETNTPKKKESKVSMSKNSKLLSTSAKRIQKELADITLDPPPNCSAGPKGDNIYEWRSTILGPPGSVYEGGVFFLDITFTPEYPFKPPKVTFRTRIYHCNINSQGVICLDILKDNWSPALTISKVLLSICSLLTDCNPADPLVGSIATQYMTNRAEHDRMARQWTKRYAT
Vector:pET28-MHL

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 induced 0.05 mM isopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15ºC. The culture was centrifuged (12,000 x g, 15 minutes) and cell pellets collected and stored at -80ºC.

Purification

Procedure
The cleared lysate was loaded onto a 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 10 mL Wash buffer A. The protein was eluted with 6 mL Elution buffer.

His-Tag was removed by incubation with TEV protease (10 μg/ mg protein) overnight at 4°C.

The protein was further purified by gel filtration on a HighLoad 16/60 Superdex 200 column (GE Healthcare, Amersham) equilibrated with Gel Filtration buffer. Fractions containing protein (analyzed by SDS-PAGE) were pooled and concentrated by ultrafiltration using an Amicon Ultra centrifugal filter with 10 kD cutoff to a final concentration of 30 mg/ml.

Protein yield was 4.5 mg per liter bacterial culture.

Extraction

Procedure
The cell pellet from a 2 L culture was resuspended in 50 ml Lysis buffer, lysed using a Microfluidizer at 18,000 PSI, and cleared by centrifugation at 40,000 x g for 30 min.
Concentration:15 mg/ml
Ligand
MassSpec:Mass-spectroscopy by LCMS showed that the product was pure and of correct molecular weight
Crystallization:Crystals of the UBE2E1 were grown at 298 K using the hanging drop method by mixing 1 volume of 15 mg/ml protein with 1 volume of well solution consisting of 2.2 M ammonium sulfate, 0.1 M bis-Tris-propane, pH 9.0 and 1 mM dithiothreitol. The crystals were cryoprotected by Paratone N/paraffin oil mixture (1:1).
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Takeuchi, T., Iwahara, S., Saeki, Y., Sasajima, H., and Yokosawa, H. (2005). Link between the Ubiquitin Conjugation System and the ISG15 Conjugation System: ISG15 Conjugation to the UbcH6 Ubiquitin E2 Enzyme. J.Biochem.(Tokyo) 138, 711-719.