Human ubiquitin specific protease 8, catalytic domain, in complex with the ubiquitin variant UBV8.2

Target ID:

USP08

Aliases:

FLJ34456HumORF8KIAA0055MGC129718UBPY

Deposition Date:

Thursday 20th May 2010

Families:

Deubiquitinating Enzymes - USPUbiquitin Related Biology

Related Diseases:

Signalling

Structure type:

protein-protein

Download Coordinates:

3N3K

Authors:

Walker JR, Avvakumov GV, Xue S, Li Y, Allali-Hassani A, Lam R, Ernst A, Sidhu S, Weigelt J, Bountra C, Arrowsmith CH, Edwards AM, Bochkarev A, Dhe-Paganon S

Site:

Toronto

ICM Datapack:

ICM Datapack

3N3K

tabs

Structure Details

Deubiquitylases catalyze the hydrolysis of the isopeptide amide bond between the C-terminal carboxyl group of ubiquitin and the epsilon amino group of a lysine residue of the acceptor protein. USP8 has been implicated in ErbB receptor down-regulation1 and may be involved in the pathophysiology of breast cancer. It contains multiple targeting domains and motifs that mediate binding to E3 ligases, substrate proteins, or adaptor molecules. We have previously solved the structure of the unliganded catalytic domain (2GFO).

In the structure presented here, USP8 forms a tight, but non-covalent complex with a ubiquitin mutant that has been created by phage-display approach and selected as a high-affinity, specific inhibitor of this protease. It provides a structural basis for the understanding of the mechanism of action of this new type of inhibitors. In this particular ubiquitin variant, 12 residues have been mutated: Q2R, F4V, T9M, K11R, T14I, Q62H, K63N, E64H, T66A, H68Y, V70L and R72K. Compared to the structure of the apoprotein (2GFO) the zinc finger has opened up by nearly eight angstroms to allow access of the ubiquitin variant to the substrate binding site. Blocking loop BL2, has also shifted slightly outwards (2.7 Å) to accommodate the C-terminus of the ubiquitin variant, although the terminal residues do not extend into the cleft leading towards the catalytic site.

Materials and Methods

USP8

PDB:3N3K

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:usp08.NM_005154.ORI.AB1489_E06.vec
Entry Clone Source:OriGene
SGC Clone Accession:
Tag:N-terminal Tag: MGSSHHHHHHSSGLVPRGS
Host:Bacterial cells

Construct

Prelude:
Sequence:PTVTPTVNRENKPTCYPKAEISRLSASQIRNLNPVFGGSGPALTGLRNLGNTCYMNSILQCLCNAPHLADYFNRNCYQDDINRSNLLGHKGEVAEEFGIIMKALWTGQYRYISPKDFKITIGKINDQFAGYSQQDSQELLLFLMDGLHEDLNKADNRKRYKEENNDHLDDFKAAEHAWQKHKQLNESIIVALFQGQFKSTVQCLTCHKKSRTFEAFMYLSLPLASTSKCTLQDCLRLFSKEEKLTDNNRFYCSHCRARRDSLKKIEIWKLPPVLLVHLKRFSYDGRWKQKLQTSVDFPLENLDLSQYVIGPKNNLKKYNLFSVSNHYGGLDGGHYTAYCKNAARQRWFKFDDHEVSDISVSSVKSSAAYILFYTSLG
Vector:pET28a-LIC

Growth

Medium:TB (Sigma T0918) supplemented with 150 mM glycerol, 100 µM Kanamycin and 600 µl antifoam 204 (Sigma A-8311).
Antibiotics:
Procedure:Competent BL21 (DE3) cells (Invitrogen C6000-03) were transformed and grown using the LEX system (Harbinger BEC) at 37°C in 2L bottles (VWR 89000-242) containing 1800 ml of TB (Sigma T0918) supplemented with 150 mM glycerol, 100 µM Kanamycin and 600 µl antifoam 204 (Sigma A-8311). When OD(600) ~ 6 was reached, the temperature was reduced to 15°C, and one hour later protein expression was induced with 100 µM IPTG (BioShop IPT001) and the culture was incubated overnight (16 hours) at 15°C. Cell pellets were collected by centrifugation (12,227 x g, 20 min), frozen and stored at -80°C.

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. The protein was further purified by gel filtration on a HighLoad 16/60 Superdex 200 column (GE Healthcare) using Gel Filtration Buffer. Fractions containing protein corresponding to the USP8 peak were pooled and concentrated by ultrafiltration to the final protein concentration of 7.6 mg/ml. The yield of the protein was 3 mg per litre bacterial culture.

Extraction

Procedure
After resuspension in 30 mL per litre bacterial culture of Lysis Buffer, cells were lysed using a Microfluidics M110-EH microfluidizer at 18,000 psi.
Concentration:
Ligand
MassSpec:
Crystallization:USP8 solution (50 µl, 7.6 mg/ml) was mixed with Ubv.8.2 (30 µl, 3.5 mg/ml), which resulted in USP8 : inhibitor molar ratio of 1:1. Before setting crystallization plate, the mixture was incubated for 1 h at ambient temperature (294 K) followed by incubation for 16 h at 281 K. Crystals of the USP8-inhibitor complex were grown at 291 K using the hanging drop method by mixing equal volumes of the above complex solution and Crystallization Buffer (24% polyethyleneglycol 3350, 0.1 M bis-Tris, pH 6.0, 0.2 M ammonium acetate and 0.5 mM dithiothreitol). The crystals were cryoprotected by immersion in the Crystallization Buffer mixed (1:1, v/v) with cryoprotecting mixture that consisted of 20% (w/v) sucrose, 4% (w/v) glucose, 18% (v/v) glycerol and 18% (v/v) ethylene glycol in water and placed in liquid nitrogen.
NMR Spectroscopy:
Data Collection:Diffraction data from a crystal of the USP8 catalytic domain in complex with Ubv.8.2 inhibitor was collected on a MAR-300 detector at the Canadian Light Source beamline CMCF 08ID-1. The data set was integrated and scaled using the HKL2000 program suite. The structure was solved by molecular replacement techniques using the program PHASER and search model PDB entry 2GFO and 3MTN. Iterative model building using the graphics program Coot and refinement package REFMAC5 led to a model with an R factor of 17.8 (R_free 24.2%) for data between 35-2.6 Å.
Data Processing:

References

Mizuno, E. et al. Regulation of epidermal growth factor receptor down-regulation by UBPY-mediated deubiquitination at endosomes. Molecular Biology of the Cell. (2005). 16, 5163-5174.