Hungtingtin Interacting Protein 2

Target ID:

UBC45

Aliases:

DKFZp564C1216DKFZp686J24237E2-25KHIP2HYPGLIG

Deposition Date:

Wednesday 19th January 2005

Families:

Ubiquitin Related BiologyUbiquitylation - E2 Conjugate Enzymes

Related Diseases:

Neurobiology

Structure type:

apo

Download Coordinates:

1YLA

Authors:

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

Site:

Toronto

ICM Datapack:

ICM Datapack

1YLA

tabs

Structure Details

Huntington's disease is an autosomal dominant neurodegenerative disorder characterised by death of cortical and striatal neurons and caused by an expanded polyglutamine tract at the amino terminus of the protein huntingtin. Although the pathogenic mechanism is still unknown, important disease phenotypes have been discovered, such as neuronal intranuclear inclusions of mutant huntingtin and the presence of specific aggregate-interacting proteins.

Two important huntingtin-associated proteins are ubiquitin and, via yeast-two-hybrid experiments and in-vitro pull-downs, the ubiquitin-conjugating enzyme E2-25, also known as huntingtin interacting protein 2 (HIP2). Huntingtin is an extremely large protein of about 3000 amino acids, widely and ubiquitously expressed, and part of a protein network that regulates membrane trafficking.

HIP2 presumably contributes to the ubiquitination of huntingtin, possibly through an E3-ligase independant and dependent manner. We have determined the high resolution structure of HIP2. In addition to the canonical ubiquitin conjugating (UBC) fold, our data reveals the structure and orientation of the carboxy-terminal ubiquitin associated domain (UBA) (residues 159-194). This helical bundle forms extensive interactions with the alpha-3 helix (139-153) of the core UBC domain; aromatic residues (residues 80,148,152,162,188) from both the UBC and UBA dominate at the interface. The UBA subdomain is spatially removed from both the reactive cysteine (Cys92) and from the putative E3-ligase binding surface (residues 4,8,11,67,68,102,103). The ubiquitin associated domain, which in other proteins has been shown to function as a ubiquitin binding module, may provide an additional site for ubiquitin binding, potentially directing specificity for polyubiquitylation reactions.

Materials and Methods

HIP2 (E2-25)

PDB:1YLA

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi 30048149
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal His-tag with integrated thrombin-cleavage site MGSSHHHHHHSSGLVPRGS
Host:E.coli BL21 (DE3)

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsMANIAVQRIKREFKEVLKSEETSKNQIKVDLVDENFTELRGEIAGPPDTPYEGGRYQLEIKIPETYPFNPPKVRFITKIWHPNISSVTGAICLDILKDQWAAAMTLRTVLLSLQALLAAAEPDDPQDAVVANQYKQNPEMFKQTARLWAHVYAGAPVSSPEYTKKIENLCAMGFDRNAVIVALSSKSWDVETATELLLSN
Vector:p28a-LIC

Growth

Medium:
Antibiotics:
Procedure:UBC E2-25K 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 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:
Ligand
MassSpec:
Crystallization:Purified UBC E2-25K was crystallized using the hanging drop vapor diffusion method. Crystals grew when the protein (20 mg/mL) was mixed with the reservoir solution in a 1:1 volume ratio, and the drop was equilibrated against a reservoir solution containing 18% PEG 8000, 0.1M calcium acetate, 0.1M sodium cacodylate, pH 6.6.
NMR Spectroscopy:
Data Collection:
Data Processing: