Human ubiquitin specific protease 2 in complex with Ubiquitin

Target ID:

USP02

Aliases:

UBP41USP9

Deposition Date:

Monday 11th September 2006

Families:

Deubiquitinating Enzymes - USPUbiquitin Related Biology

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

2IBI

Authors:

J.R.WALKER,G.V.AVVAKUMOV,G.BERNSTEIN,S.XUE,P.J.FINERTY JR.,F.MACKENZIE,J.WEIGELT,M.SUNDSTROM,C.H.ARROWSMITH,A.M.EDWARDS,A.BOCHKAREV,S.DHE-PAGANON,STRUCTURAL GENOMICSCONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2IBI

tabs

Structure Details

Ubiquitin Specific Protease 2 (USP2) is a cysteine-type isopeptidase specific for ubiquitin conjugates. It has been implicated in mediating the proliferative effects of parathyroid hormone on osteoblasts and other bone cells 1 and because it is significantly overexpressed in prostate tumor cells, USP2 and its catalytic activity is implicated in the anabolic effects of testosterone 2. A candidate substrate of USP2 in these cells is the Fatty Acid Synthase (FAS), which itself has also been implicated in prostate cancer 2. And, USP2 has an antiapoptotic effect in cultured cells 3. USP2 might therefore be a candidate target for prostate cancer.

We solved a 2.2 Angstrom resolution crystal structure of the catalytic domain of human USP2 with covalently bound ubiquitin. The overall model quality is excellent with an Rwork/Rfree of 0.19/0.26. The structure reveals the tripartite structure with ubiquitin grasped by the palm/fingers region and the carboxy-terminal tail of ubiquitin occupying the P1-P4 tunnel, formed by blocking loops 1 and 2. Notably, electron density of the covalent bond between the catalytic cystein (Cys276) and the terminal ubiquitin residues (G76) is clearly observed. This structure is currently a good representation of the transition state of USP2-catalyzed deubiquitylation. The exact conformation of the catalytic triad, the oxyanion hole, and the tunnel residues will be important in evaluating the mechanism of the reaction as well as the mechanism of future pharmacophores.

To generate this complex structure, the intein expression system was used to generate the ubiquityl-ethylbromine reagent. A cDNA encoding residues 1 to 75 of human ubiquitin was inserted into pTBY2 plasmid (New England Biolabs) and expressed as an intein fusion protein that was purified as ubiquityl(1-75)-2-mercaptoethanesulfonic acid thiol ester and then converted into 2-[ubiquityl(1-75)]-aminoethylbromine (note: bromine not bromide) as described in 4. The latter compound was incubated with USP2 in order to produce a covalent complex in which ubiquitin(1-75) is conjugated, through an aminoethyl spacer mimicking Gly76 of the full-length ubiquitin, to the sulfur atom of the usp2 catalytic Cys residue. Subsequent size-exclusion purification is conducted to remove unreacted molecules.

Materials and Methods

USP2

PDB:2IBI

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:usp02.BC002854.MGC.AU22C2.pOTB7
SGC Clone Accession:usp02.251.605:E472A:K473A , plate GBC003:G10
Tag:N-terminal histag: MGSSHHHHHHSSGLVPR*GS
Host:E. coli BL21 (DE3) Gold

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgs*SSPGRDGMNSKSAQGLAGLRNLGNTCFMNSILQCLSNTRELRDYCLQRLYMRDLHHGSNAHTALVEEFAKLIQTIWTSSPNDVVSPSEFKTQIQRYAPRFVGYNQQDAQEFLRFLLDGLHNEVNRVTLRPKSNPENLDHLPDDEKGRQMWRKYLEREDSRIGDLFVGQLKSSLTCTDCGYCSTVFDPFWDLSLPIAKRGYPEVTLMDCMRLFTKEDVLDGDAAPTCCRCRGRKRCIKKFSIQRFPKILVLHLKRFSESRIRTSKLTTFVNFPLRDLDLREFASENTNHAVYNLYAVSNHSGTTMGGHYTAYCRSPGTGEWHTFNDSSVTPMSSSQVRTSDAYLLFYELASPPSRM

covalently conjugated through Cys276 thiol group with

MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRG-amidoethyl
Vector:pET28a-LIC

Growth

Medium:TB
Antibiotics:
Procedure:The protein 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 (12,000 x g, 15 minutes) and the cell pellets were collected and stored at -80ºC.

Purification

Procedure
Column 1: Econo-Column (Bio-Rad 732-1010)
Column 2: HighLoad Superdex 200 (GE Healthcare)

Immobilized-metal Affinity Chromatography: To the clarified supernatant, 3 mL of 50% suspension of TALON metal-affinity resin (BD Bioscience) in the Lysis buffer was added. The tubes were incubated for 1 h in refrigerator with constant stirring, centrifuged at 4°C for 2 min at 1,000 rpm in SX4750 rotor using Allegra X-12R centrifuge (Beckman Coulter) and the supernatant was discarded. The affinity resin pellets were suspended in 10 mL Lysis buffer and transferred into an Econo-Column (Bio-Rad 732-1010). The buffer was allowed to drain and discarded. The settled gel was washed with 10 mL Wash buffer A followed by 10 mL Wash buffer B and 30 mL Wash buffer A again. The protein was then eluted with 6 ml Elution buffer.

Size-exclusion Chromatography: This step was performed using an AKTA Purifier system (GE Healthcare). The protein sample was loaded onto an XK16x65 column packed with HighLoad Superdex 200 (GE Healthcare) and equilibrated with Gel Filtration buffer. Elution was performed with the same buffer at a flow-rate of 1.5 mL/min, and 3-ml fractions were collected. Fractions corresponding to the major peak on the chromatogram were combined and analyzed by SDS-PAGE and LC/MS. Protein concentration was determined by measuring UV absorbance of the combined fractions at 280 nm.

Protein Modification with Suicide Substrate: Purified Usp2 mutant was incubated with 2x molar excess of ubiquitin(1-75)-bromoethylamide prepared according to K.D. Wilkinson et al. (Meth. Enzymol., 2005, 339B, 37-50) for 1 h at room temperature (21°C). An aliquot was taken for LC/MS analysis to confirm completeness of the covalent modification of the catalytic cysteine residue. The covalent complex was purified by gel-filtration chromatography as above and concentrated by ultrafiltration.

Extraction

Procedure
The cell pellet from a 2 L culture was resuspended in 50 ml Lysis buffer, lysed using a Microfluidizer at 18,000 PSI, and cleared by centrifugation at 40,000 x g for 30 min.
Concentration:Purified protein was concentrated using an ultrafiltration concentrator (Amicon Ultra-15 10,000 MWCO, UFC901024, Millipore) to a final concentration of approx. 5 mg/mL for determination of enzymatic activity, biophysical characterization and modification with suicide substrate.
Ligand
MassSpec:
Crystallization:Purified covalent complex was crystallized using the hanging drop vapor diffusion method at 20 ºC. Crystals grew when the protein (1 mg/mL) was mixed with the reservoir solution in a 1:1 volume ratio, and the drop was equilibrated against the reservoir solution containing 22% PEG1500, 0.1 M bicine, pH 9, 0.2 M NaCl, 1 mM DTT.
NMR Spectroscopy:
Data Collection:Data for the diffracting crystal was collected at 2.2 Å on the Rigaku FRE Superbright diffractometer at 1.54 Å.
Data Processing:

References

Miles, R. R., Sluka, J. P., Halladay, D. L., Santerre, R. F., Hale, L. V., Bloem, L., Patanjali, S. R., Galvin, R. J., Ma, L., Hock, J. M., and Onyia, J. E.; J.Cell Biochem.; Parathyroid hormone (hPTH 1-38) stimulates the expression of UBP41, an ubiquitin-specific protease, in bone; 2002; v85; p229-242
Graner, E., Tang, D., Rossi, S., Baron, A., Migita, T., Weinstein, L. J., Lechpammer, M., Huesken, D., Zimmermann, J., Signoretti, S., and Loda, M.; Cancer Cell; The isopeptidase USP2a regulates the stability of fatty acid synthase in prostate cancer; 2004; v5; p253-261
Priolo, C., Tang, D., Brahamandan, M., Benassi, B., Sicinska, E., Ogino, S., Farsetti, A., Porrello, A., Finn, S., Zimmermann, J., Febbo, P., and Loda, M.; Cancer Res.; The Isopeptidase USP2a Protects Human Prostate Cancer from Apoptosis; 9-1-2006; v66; p8625-8632
Wilkinson, K. D., Gan-Erdene, T., and Kolli, N.; Methods Enzymol.; Derivitization of the C-Terminus of Ubiquitin and Ubiquitin-like Proteins Using Intein Chemistry: Methods and Uses; 2005; v399; p37-51