Human ubiquitin specific peptidase 5, second ZnF domain, in complex with inhibitor DAT194

Target ID:

USP05

Deposition Date:

Thursday 20th December 2018

Families:

Deubiquitinating Enzymes - USPUbiquitin Related Biology

Related Diseases:

Signalling and cancer

Structure type:

protein-ligand

Download Coordinates:

6NFT

Authors:

Harding RJ, Mann MK, Tempel W, Bountra C, Arrowmsmith CM, Edwards AM, Schapira M

Site:

Toronto

6NFT

tabs

Structure Details

USP5 is a ubiquitin-specific cysteine protease which disassembles unanchored polyubiquitin chains to recycle free mono-ubiquitin¹. USP5 is an 835 amino acid protein that has been associated with tumorigenesis^2–6, neurodegeneration^7–9, viral infections^10,11, DNA damage repair¹² and stress granules¹³. The domain architecture of USP5 consists of a zinc finger ubiquitin-binding domain (ZnF-UBD) spanning residues 173-283, a functional catalytic domain, two ubiquitin associated domains (UBA), and a cryptic zinc finger ubiquitin-binding domain (nUBP)¹⁴. The ZnF-UBD recognizes the C-terminal di-glycine motif of ubiquitin and has been associated with allosteric modulation of USP5 activity^1,15,16 and alternatively with substrate recognition and positioning¹⁴. This co-crystal structure is one of the first of the USP5 ZnF-UBD with a ligand bound in the ubiquitin-binding pocket.

(1) Wilkinson, K. D.; Tashayev, V. L.; O’Connor, L. B.; Larsen, C. N.; Kasperek, E.; Pickart, C. M. Metabolism of the Polyubiquitin Degradation Signal: Structure, Mechanism, and Role of Isopeptidase T. Biochemistry 1995. https://doi.org/10.1021/bi00044a032.

(2) Dayal, S.; Sparks, A.; Jacob, J.; Allende-Vega, N.; Lane, D. P.; Saville, M. K. Suppression of the Deubiquitinating Enzyme USP5 Causes the Accumulation of Unanchored Polyubiquitin and the Activation of P53. J. Biol. Chem. 2009. https://doi.org/10.1074/jbc.M805871200.

(3) Liu, Y.; Wang, W. M.; Zou, L. Y.; Li, L.; Feng, L.; Pan, M. Z.; Lv, M. Y.; Cao, Y.; Wang, H.; Kung, H. F.; et al. Ubiquitin Specific Peptidase 5 Mediates Histidine-Rich Protein Hpn Induced Cell Apoptosis in Hepatocellular Carcinoma through P14-P53 Signaling. Proteomics 2017. https://doi.org/10.1002/pmic.201600350.

(4) Kaistha, B. P.; Krattenmacher, A.; Fredebohm, J.; Schmidt, H.; Behrens, D.; Widder, M.; Hackert, T.; Strobel, O.; Hoheisel, J. D.; Gress, T. M.; et al. The Deubiquitinating Enzyme USP5 Promotes Pancreatic Cancer via Modulating Cell Cycle Regulators. Oncotarget 2017.

(5) Wang, S.; Juan, J.; Zhang, Z.; Du, Y.; Xu, Y.; Tong, J.; Cao, B.; Moran, M. F.; Zeng, Y.; Mao, X. Inhibition of the Deubiquitinase USP5 Leads to C-Maf Protein Degradation and Myeloma Cell Apoptosis. Cell Death Dis. 2017. https://doi.org/10.1038/cddis.2017.450.

(6) Ma, X.; Qi, W.; Pan, H.; Yang, F.; Deng, J. Overexpression of USP5 Contributes to Tumorigenesis in Non-Small Cell Lung Cancer via the Stabilization of β-Catenin Protein. Am. J. Cancer Res. 2018.

(7) García-Caballero, A.; Gadotti, V. M.; Stemkowski, P.; Weiss, N.; Souza, I. A.; Hodgkinson, V.; Bladen, C.; Chen, L.; Hamid, J.; Pizzoccaro, A.; et al. The Deubiquitinating Enzyme USP5 Modulates Neuropathic and Inflammatory Pain by Enhancing Cav3.2 Channel Activity. Neuron 2014. https://doi.org/10.1016/j.neuron.2014.07.036.

(8) Garcia-Caballero, A.; Gadotti, V. M.; Chen, L.; Zamponi, G. W. A Cell-Permeant Peptide Corresponding to the CUBP Domain of USP5 Reverses Inflammatory and Neuropathic Pain. Mol. Pain 2016. https://doi.org/10.1177/1744806916642444.

(9) Stemkowski, P. L.; Garcia-Caballero, A.; Gadotti, V. M.; M’Dahoma, S.; Chen, L.; Souza, I. A.; Zamponi, G. W. Identification of Interleukin-1 Beta as a Key Mediator in the Upregulation of Cav3.2–USP5 Interactions in the Pain Pathway. Mol. Pain 2017. https://doi.org/10.1177/1744806917724698.

(10) Liu, Q.; Wu, Y.; Qin, Y.; Hu, J.; Xie, W.; Xiao-Feng Qin, F.; Cui, J. Broad and Diverse Mechanisms Used by Deubiquitinase Family Members in Regulating the Type i Interferon Signaling Pathway during Antiviral Responses. Sci. Adv. 2018. https://doi.org/10.1126/sciadv.aar2824.

(11) Ovaa, H.; Kessler, B. M.; Rolen, U.; Galardy, P. J.; Ploegh, H. L.; Masucci, M. G. Activity-Based Ubiquitin-Specific Protease (USP) Profiling of Virus-Infected and Malignant Human Cells. Proc. Natl. Acad. Sci. 2004. https://doi.org/10.1073/pnas.0308411100.

(12) Nakajima, S.; Lan, L.; Wei, L.; Hsieh, C. L.; Rapić-Otrin, V.; Yasui, A.; Levine, A. S. Ubiquitin-Specific Protease 5 Is Required for the Efficient Repair of DNA Double-Strand Breaks. PLoS One 2014. https://doi.org/10.1371/journal.pone.0084899.

(13) Xie, X.; Matsumoto, S.; Endo, A.; Fukushima, T.; Kawahara, H.; Saeki, Y.; Komada, M. Deubiquitylases USP5 and USP13 Are Recruited to and Regulate Heat-Induced Stress Granules through Their Deubiquitylating Activities. J. Cell Sci. 2018. https://doi.org/10.1242/jcs.210856.

(14) Avvakumov, G. V.; Walker, J. R.; Xue, S.; Allali-Hassani, A.; Asinas, A.; Nair, U. B.; Fang, X.; Zuo, X.; Wang, Y. X.; Wilkinson, K. D.; et al. Two ZnF-UBP Domains in Isopeptidase T (USP5). Biochemistry 2012. https://doi.org/10.1021/bi200854q.

(15) Reyes-Turcu, F. E.; Horton, J. R.; Mullally, J. E.; Heroux, A.; Cheng, X.; Wilkinson, K. D. The Ubiquitin Binding Domain ZnF UBP Recognizes the C-Terminal Diglycine Motif of Unanchored Ubiquitin. Cell 2006. https://doi.org/10.1016/j.cell.2006.02.038.

(16) Dang, L. C.; Melandri, F. D.; Stein, R. L. Kinetic and Mechanistic Studies on the Hydrolysis of Ubiquitin C- Terminal 7-Amido-4-Methylcoumarin by Deubiquitinating Enzymes. Biochemistry 1998. https://doi.org/10.1021/bi9723360.

Materials and Methods

PDB: 6NFT

Entry Clone Accession:

Entry Clone Source:

SGC Clone Accession: SDC132-E06

Tag: N-terminal tag: MHHHHHHSSGRENLYFQG

Host: BL21 Codon Plus RIL

Vector: pet28-MHL

Sequence: gGEVRQVSKHAFSLKQLDNPARIPPCGWKCSKCDMRENLWLNLTDGSILCGRRYFDGSGGNNHAVEHYRETGYPLAVKLGTITPDGADVYSYDEDDMVLDP
SLAEHLSHFGIDMLKMQKTD

Growth:

Medium: M9

Antibiotic: 50 µg/mL Kanamycin, 34 µg/mL chloramphenicol

Shaker: Cultures were grown in M9 media supplemented with 50 µM ZnCl₂, and grown at 37⁰C until OD₆₀₀ of 0.6 and induced with 0.5 mM IPTG at 15⁰C for overnight growth. Cell pellets were collected by centrifugation: 6000 RPM for 10 min (JLA8.1000), frozen and stored at -80⁰C.

Purification:

Buffers: Buffer A: 50 mM Tris pH 8, 150 mM NaCl, 1 mM TCEP; Buffer B: 50 mM Tris pH 8, 150 mM NaCl, 1 mM TCEP, 15 mM imidazole; Buffer C: 50 mM Tris pH 8, 150 mM NaCl, 1 mM TCEP, 300 mM imidazole

Procedure: Clarified lysate rocked with 5 mL Ni-NTA resin (Qiagen) for 1 hour at 4˚C. Beads washed with 100 mL Buffer A, then 200 mL Buffer B before elution with 2x15 mL Buffer C. Eluent supplemented with TEV and dialysed with snakeskin MWCO 3500 against 1 L Buffer A for 2 hours then fresh 2 L Buffer A overnight. Cleavage of protein was verified with SDS-PAGE analysis. Cleaved protein incubated with 5 mL Ni resin and rocked for 1 hour at 4˚C. The beads were eluted with Buffer C, concentrated to 5 mL and run on S75 16/60. The protein was concentrated (Amicon MWCO 10,000) to 8 mg/mL. The yield of the protein was approximately 2 mg per litre of bacterial culture.

Extraction:

Cells were re-suspended in 400 mL of Buffer A and supplemented with benzonase and 1x protease inhibitors and lysed by sonication for 10 mins at 5” on 7” off at 85W output power. Lysates were clarified by centrifugation: 15,000 RPM for 1 hour (JLA 16.250).

Mass Spec: 13517.29 g/mol

Structure Determination:

Ligand: (4-oxoquinazolin-3(4H)-yl)acetic acid

Concentration: protein: 12 mg/mL

Crystallization: Crystals of the complex were grown at 291 K using vapor diffusion sitting drop by mixing equal volumes of 1:2.5 protein (12 mg/mL, 1.1% DMSO (v/v)): ligand and crystallization buffer (2 M sodium/potassium phosphate pH 7.0). The crystals were cryoprotected in 25% ethylene glycol (v/v) and cryo cooled in liquid nitrogen.

Data Collection & Processing: X-ray diffraction data was collected at 100 K at a Rigaku FR-E Superbright copper source. Diffraction data were processed with the XDS and AIMLESS, using the Xia2 interfaces and were solved by direct refinement of protein chains A and B of the isomorphous PDB entry 2G43.