Human Ubiquilin 3

Target ID:

UBH26

Aliases:

TUP-1

Deposition Date:

Tuesday 1st February 2005

Families:

Ubiquitin Related Biology

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

1YQB

Authors:

J.R.WALKER,S.XUE,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

1YQB

tabs

Structure Details

Cell-cycle progression is determined by a set of serine/threonine protein kinases that are themselves regulated by the cyclin family of proteins. The concentration of cyclins in the cell, hence their rates of degradation and synthesis, during different phases of the cell cycle, regulate the cyclin-dependent kinases.

Degradation of cyclins occurs through the classical proteasomal pathway, where E3 ligases catalyze ubiquitination of cyclins at their amino termini. An additional layer of complexity is provided by inhibitory proteins that prevent degradation of cyclins. These include ubiquilins, which bind cyclins also at their amino termini, competing with and inhibiting E3 ligase activity. Ubiquilins bind cyclins through their amino terminal ubiquitin-like domains.

We have recently determined the high resolution structure of the ubiquitin-like domain of human ubiquilin-3, a 655 residue protein exclusively expressed in testis. The fold of ubiquilin more closely resembles ubiquitin than sumo, and the electrostatic surface potential of the polar face is again more similar to ubiquitin than sumo. The relatively basic face of ubiquilin-3 could mediate interaction with a 20 residue region of cyclin A1 implicated in ubiquilin binding. This region is acidic, occurring between the destruction box at the very amino terminus and the first cyclin domain.

Materials and Methods

UBQLN3

PDB:1YQB

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi 22477824
Entry Clone Source:MGC
SGC Clone Accession:ubh26.019.099; plate SDC008:B6
Tag:N-terminal His-tag with integrated thrombin-cleavage site MGSSHHHHHHSSGLVPRGS.
Host:E.coli BL21 (DE3)

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsPHLIKVTVKTPKDKEDFSVTDTCTIQQLKEEISQRFKAHPDQLVLIFAGKILKDPDSLAQCGVRDGLTVHLVIKRQHRAMG
Vector:p28a-LIC

Growth

Medium:
Antibiotics:
Procedure:Ubiquilin 3 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:36 mg/mL
Ligand
MassSpec:
Crystallization:Purified ubiquilin 3 was crystallized using the sitting drop vapor diffusion method. Crystals grew when the protein (18 mg/mL) was mixed with the reservoir solution in a 1:1 volume ratio, and the drop was equilibrated against a reservoir solution containing 2M ammonium sulfate, 0.1 M bis-Tris, pH 6.5.
NMR Spectroscopy:
Data Collection:
Data Processing: