Human Ubiquitin-Conjugating Enzyme Like Protein

Target ID:

UBC44

Aliases:

HSPC150PIG50

Deposition Date:

Thursday 6th January 2005

Families:

Ubiquitin Related BiologyUbiquitylation - E2 Conjugate Enzymes

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

1YH2

Authors:

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

Site:

Toronto

1YH2

tabs

Structure Details

HSPC150 is a ubiquitin conjugating enzyme like protein lacking biological data but important details are apparent at the atomic level.

In addition to the covalent thiolester bond between the carboxyl terminal glycine of ubiquitin and the reactive cysteine of E2, most non-covalent interactions between these two proteins are mediated by the central helix of E2, which is sandwhiched between the two beta sheets, exposing three residues that are important for the interaction. Although our structure of HSPC150 is not complexed with ubiquitin, predictions can be made concerning specificity.

While the catalytic residues are present in HSPC150, including the reactive thiol, Cys-86 and the two potential general acids, Asp-88 and Asp-122, the three solvent exposed residues of the central helix, Thr-106, Leu-113, and Glu-117, are divergent, predicting poor interaction with ubiquitin. HSPC150 also uniquely contains an additional four residues near the active thiol, which forms a tight loop, increasing the concavity of the active site, potentially increasing specificity towards substrates or ubiquitin homologs.

Materials and Methods

HSPC150 protein

PDB:1YH2

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi:33872714
Entry Clone Source:MGC
SGC Clone Accession:ubc44.001.167; plate SDC005:H6
Tag:N-terminal His-tag with integrated thrombin-cleavage site MGSSHHHHHHSSGLVPRGS.
Host:E.coli BL21 (DE3)

Construct

Prelude:
Sequence:gsSGLVPRGSMQRASRLKRELHMLATEPPPGITCWQDKDQMDDLRAQILGGANTPYEKGVFKLEVIIPERYPFEPPQIRFLTPIYHPNIDSAGRICLDVLKLPPKGAWRPSLNIATVLTSIQLLMSEPNPDDPLMADISSEFKYNKPAFLKNARQWTEKHARQKQKADEEEMLDNLP
Vector:p28a-LIC

Growth

Medium:TB
Antibiotics:
Procedure:HSPC150 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 5.5. 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 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) 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) with protease inhibitor (0.1mM phenylmethyl sulfonyl fluoride, PMSF) and lysed using Microfluidizer. The lysate was cleared by centrifugation.
Concentration:17 mg/mL
Ligand
MassSpec:
Crystallization:Purified HSPC150 was crystallized using the sitting drop vapor diffusion method. Crystals grew when the protein (17mg/mL) was mixed with the reservoir solution in a 1:1 volume ratio, and the drop was equilibrated against a reservoir solution containing 4 M sodium formate.
NMR Spectroscopy:
Data Collection:
Data Processing: