Human Ubiquitin-Conjugating Enzyme of Unknown Function

Target ID:

UBC53

Aliases:

MGC35130RP4-636O23.1

Deposition Date:

Friday 4th February 2005

Families:

Ubiquitin Related BiologyUbiquitylation - E2 Conjugate Enzymes

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

1YRV

Authors:

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

Site:

Toronto

ICM Datapack:

ICM Datapack

1YRV

tabs

Structure Details

MGC35130 is one of the most divergent members of the E2 family, containing a minimum number of conserved residues. All conserved amino acids except the nucleophilic cysteine are core residues. The domain fold of MGC35130 is clearly that of the ubiquitin conjugase family, but while the amino terminal secondary structural elements align very well with other members, the carboxyl terminal helices (helix-A3 and A4) are positioned significantly away from the conserved position. The large side chain of Tyrosine-140 contributes to the packing of helix-A4, and may be responsible for the tertiary structure divergence.

The human genome contains numerous genes encoding ubiquitin-like proteins as well as about forty genes for ubiquitin-conjugating proteins. One of the more intriguing questions concerns specificity, namely, which ubiquitin homolog binds to which conjugating protein(s). As an approach to solving this problem, we aim to determine the structures of all human ubiquitin-conjugating proteins.

With the notion that conjugases interact with their corresponding ubiquitin homologs through a mechanism similar to yeast UBC1 (Hamilton et. al., 2001), the ubiquitin binding surface of MGC35130 is expected to be composed of the loop connecting B2 and B3, helix-A2, and the coiled regions just prior to and following helix-A2. Consistent with other ubiquitin conjugating proteins, this surface is relatively flat. The solvent exposed surface of helix-A2, which contributes the largest surface area for interaction with ubiquitin, contains three aliphatic side chains, an asparigine, a serine, and tyrosine that could contribute to direct interactions with corresponding ubiquitin homologs. The coiled regions prior to and following helix-A2 contain glutamates and aspartates that may act as general acids to the ubiquitination and deubiquitination reaction. The amino terminal coiled region, prior to helix-A2, contains a lysine residue that could complement an acid group of the corresponding ubiquitin homolog. In addition, the loop connecting strand-B2 and B3 contains a glutamic acid that could complement a basic residue on corresponding ubiquitin homologs.

Materials and Methods

MGC35130

PDB:1YRV

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi 20988674
Entry Clone Source:MGC
SGC Clone Accession:ubc53.001.150; plate SDC006:D3
Tag:
Host:E.coli BL21 (DE3)

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsMHGRAYLLLHRDFCDLKENNYKGITAKPVSEDMMEWEVEIEGLQNSVWQGLVFQLTIHFTSEYNYAPPVVKFITIPFHPNVDPHTGQPCIDFLDNPEKWNTNYTLSSILLALQVMLSNPVLENPVNLEAARILVKDESLYRTILRLFNRP
Vector:

Growth

Medium:
Antibiotics:
Procedure:MGC35130 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:48 mg/mL
Ligand
MassSpec:
Crystallization:Purified MGC35130 was crystallized using the hanging drop vapor diffusion method. Crystals grew when the protein (12 mg/mL) was mixed with the reservoir solution in a 1:1 volume ratio, and the drop was equilibrated against a reservoir solution containing 11% PEG MME 5000, 5% Tacsimate, 15mM DTT, 0.1M HEPES, pH 7.4.
NMR Spectroscopy:
Data Collection:
Data Processing: