A 2.2 Å resolution structure of the USP7 catalytic domain in a new space group elaborates upon structural rearrangements resulting from ubiquitin binding

Molland, Katrina L.; Zhou, Qing; Mesecar, Andrew D.

doi:10.1107/s2053230x14002519

Cited by 28 publications

(36 citation statements)

References 20 publications

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“…In the USP7-ubiquitin complex structure (PDB code: 1NBF), we found that the same narrow pocket was occupied by the ubiquitin C- terminus (Supplementary Figure S3). These results suggest that the inhibition mechanism of CDDO-Me may be explained by its displacement of the ubiquitin C terminus while binding with USP7 [25–26]. However, further experimental data, such as crystal structures, are necessary to confirm this docking hypothesis.…”

Section: Resultsmentioning

confidence: 97%

CDDO-Me reveals USP7 as a novel target in ovarian cancer cells

Qin

Wang

et al. 2016

Oncotarget

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Deubiquitinating enzyme USP7 has been involved in the pathogenesis and progression of several cancers. Targeting USP7 is becoming an attractive strategy for cancer therapy. In this study, we identified synthetic triterpenoid C-28 methyl ester of 2-cyano-3, 12-dioxoolen-1, 9-dien-28-oic acid (CDDO-Me) as a novel inhibitor of USP7 but not of other cysteine proteases such as cathepsin B and cathepsin D. CDDO-Me inhibits USP7 activity via a mechanism that is independent of the presence of α, β-unsaturated ketones. Molecular docking studies showed that CDDO-Me fits well in the ubiquitin carboxyl terminus-binding pocket on USP7. Given that CDDO-Me is known to be effective against ovarian cancer cells, we speculated that CDDO-Me may target USP7 in ovarian cancer cells. We demonstrated that ovarian cancer cells have higher USP7 expression than their normal counterparts. Knockdown of USP7 inhibits the proliferation of ovarian cancer cells both in vitro and in vivo. Using the cellular thermal shift assay and the drug affinity responsive target stability assay, we further demonstrated that CDDO-Me directly binds to USP7 in cells, which leads to the decrease of its substrates such as MDM2, MDMX and UHRF1. CDDO-Me suppresses ovarian cancer tumor growth in an xenograft model. In conclusion, we demonstrate that USP7 is a novel target of ovarian cancer cells; targeting USP7 may contribute to the anti-cancer effect of CDDO-Me. The development of novel USP7 selective compounds based on the CDDO-Me-scaffold warrants further investigation.

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Section: Resultsmentioning

confidence: 97%

CDDO-Me reveals USP7 as a novel target in ovarian cancer cells

Qin

Wang

et al. 2016

Oncotarget

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“…The majority of USP catalytic domain structures available to date were determined in complex with either ubiquitin or a covalent inhibitor including USP2 ( 28 ), USP4 ( 24 ), USP5 ( 29 ), USP7 ( 30 ), USP12 ( 31 ), USP18 ( 32 ), USP14 ( 33 ), USP21 ( 34 ), USP30 ( 35 ), USP46 ( 36 ), and CYLD ( 37 ). Structures that do not have a ligand or ubiquitin covalently bound to the catalytic cysteine are available for USP7 (PDB codes 1NB8 , 2F1Z , 4M5X ( 30 , 38 , 39 )), USP14 (PDB code 2AYN ( 33 )), CYLD (PDB code 2VHF ( 40 )), USP8 (PDB code 2GFO ( 27 )), USP18 (PDB code 5CHT ( 32 )), and USP12 (PDB codes 5K1B and 5K16 ( 41 )). Among the latter structures, the USP15 catalytic core is most closely related to USP8 (46% sequence identity) followed by USP12 (26.32% sequence identity), whereas it is more distantly related to USP7 (21.99% sequence identity), USP14 (19.94% sequence identity), and CYLD (11.8% sequence identity).…”

Section: Resultsmentioning

confidence: 99%

“… Comparison with other USP catalytic core structures determined in the absence of ubiquitin. A , crystal structures of USP15 ( blue ) and USP4 (PDB code 2Y6E ( 24 )), USP8 (PDB code 2GFO ( 27 ), USP7 (PDB code 4M5X ( 38 )), USP14 (PDB code 2AYN ( 33 )), and USP12 (PDB code 5K16 ( 41 )) shown in a gray cartoon representation with catalytic triad residues highlighted in green and BL1, SL, and BL2 loop regions each depicted in red. B , superposition of the same structures as seen in A , highlighting the most variable regions in the structures.…”

Section: Resultsmentioning

confidence: 99%

The structure of the deubiquitinase USP15 reveals a misaligned catalytic triad and an open ubiquitin-binding channel

Ward

Gratton

Indrayudha

et al. 2018

Journal of Biological Chemistry

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Ubiquitin-specific protease 15 (USP15) regulates important cellular processes, including transforming growth factor β (TGF-β) signaling, mitophagy, mRNA processing, and innate immune responses; however, structural information on USP15's catalytic domain is currently unavailable. Here, we determined crystal structures of the USP15 catalytic core domain, revealing a canonical USP fold, including a finger, palm, and thumb region. Unlike for the structure of paralog USP4, the catalytic triad is in an inactive configuration with the catalytic cysteine ∼10 Å apart from the catalytic histidine. This conformation is atypical, and a similar misaligned catalytic triad has so far been observed only for USP7, although USP15 and USP7 are differently regulated. Moreover, we found that the active-site loops are flexible, resulting in a largely open ubiquitin tail–binding channel. Comparison of the USP15 and USP4 structures points to a possible activation mechanism. Sequence differences between these two USPs mainly map to the S1′ region likely to confer specificity, whereas the S1 ubiquitin–binding pocket is highly conserved. Isothermal titration calorimetry monoubiquitin- and linear diubiquitin-binding experiments showed significant differences in their thermodynamic profiles, with USP15 displaying a lower affinity for monoubiquitin than USP4. Moreover, we report that USP15 is weakly inhibited by the antineoplastic agent mitoxantrone in vitro. A USP15–mitoxantrone complex structure disclosed that the anthracenedione interacts with the S1′ binding site. Our results reveal first insights into USP15's catalytic domain structure, conformational changes, differences between paralogs, and small-molecule interactions and establish a framework for cellular probe and inhibitor development.

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“…The catalytic domain of USP7 constitutes a well characterized hand like architecture with finger, palm and thumb that is mainly responsible for its catalytic activity. The coordinates for catalytic domain were obtained from PDB-ID 4M5W [ 25 ]. From literature, we found that auxiliary domains are essential for biological function, therefore PDB-ID 5FWI [ 26 ] that contains CD, CH and also three additional domains as UBL123 was also used.…”

Section: Resultsmentioning

confidence: 99%

“…Crystals 4M5W (APO) [ 25 ] and 5FWI (APO) [ 26 ] were obtained from Protein Data Bank The missing residues in 4M5W (L505-R508) and 5FWI (D459-N460 (BL2) and D502-H509) were interpolated by using PLOP algorithm [ 37 ] implemented in Schrodinger suite and the optimum conformation of loop was chosen on the basis of lowest energy. The loop conformations were optimized and then both the structures were minimized.…”

Section: Methodsmentioning

confidence: 99%

Molecular dynamics simulation reveals the possible druggablehot-spotsof USP7

et al. 2018

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The plasticity in Ubiquitin Specific Proteases (USP7) inducing conformational changes at important areas has highlighted an intricate mechanism, by which USP7 is regulated. Given the importance of USP7 in oncogenic pathways and immune-oncology, identification of USP7 inhibitors has attracted considerable interest. Despite substantial efforts, the discovery of deubiquitinases (DUBs) inhibitors, knowledge of their binding site and understanding the possible mechanism of action has proven particularly challenging. We disclose the most likely binding site of P5091 (a potent USP7 inhibitor), which reveal a cryptic allosteric site through extensive computational studies in an inhibitor dependent and independent manner. Overall, these findings demonstrate the tractability and druggability of USP7. Through a series of molecular dynamics simulations and detailed quantitative analysis, a dynamically stable allosteric binding site near catalytic center of the inactive state of USP7 (site partially absent in active state), along with two newly identified sites have been revealed, which opens the avenue for rational structure-guided inhibitor designing in USP7 specific-manner.

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A 2.2 Å resolution structure of the USP7 catalytic domain in a new space group elaborates upon structural rearrangements resulting from ubiquitin binding

Cited by 28 publications

References 20 publications

CDDO-Me reveals USP7 as a novel target in ovarian cancer cells

CDDO-Me reveals USP7 as a novel target in ovarian cancer cells

The structure of the deubiquitinase USP15 reveals a misaligned catalytic triad and an open ubiquitin-binding channel

Molecular dynamics simulation reveals the possible druggablehot-spotsof USP7

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