Crystal Structure of USP7 Ubiquitin-like Domains with an ICP0 Peptide Reveals a Novel Mechanism Used by Viral and Cellular Proteins to Target USP7

Herpes simplex virus-1 immediate-early protein ICP0 activates viral genes during early stages of infection, affects cellular levels of multiple host proteins and is crucial for effective lytic infection. Being a RING-type E3 ligase prone to auto-ubiquitination, ICP0 relies on human deubiquitinating enzyme USP7 for protection against 26S proteasomal mediated degradation. USP7 is involved in apoptosis, epigenetics, cell proliferation and is targeted by several herpesviruses. Several USP7 partners, including ICP0, GMPS, and UHRF1, interact through its C-terminal domain (CTD), which contains five ubiquitin-like (Ubl) structures. Despite the fact that USP7 has emerged as a drug target for cancer therapy, structural details of USP7 regulation and the molecular mechanism of interaction at its CTD have remained elusive. Here, we mapped the binding site between an ICP0 peptide and USP7 and determined the crystal structure of the first three Ubl domains bound to the ICP0 peptide, which showed that ICP0 binds to a loop on Ubl2. Sequences similar to the USP7-binding site in ICP0 were identified in GMPS and UHRF1 and shown to bind USP7-CTD through Ubl2. In addition, co-immunoprecipitation assays in human cells comparing binding to USP7 with and without a Ubl2 mutation, confirmed the importance of the Ubl2 binding pocket for binding ICP0, GMPS and UHRF1. Therefore we have identified a novel mechanism of USP7 recognition that is used by both viral and cellular proteins. Our structural information was used to generate a model of near full-length USP7, showing the relative position of the ICP0/GMPS/UHRF1 binding pocket and the structural basis by which it could regulate enzymatic activity.     

Ubiquitin-specific protease 4 is inhibited by its ubiquitin-like domain

USP4 is a member of the ubiquitin-specific protease (USP) family of deubiquitinating enzymes that has a role in spliceosome regulation. Here, we show that the crystal structure of the minimal catalytic domain of USP4 has the conserved USP-like fold with its typical ubiquitin-binding site. A ubiquitin-like (Ubl) domain inserted into the catalytic domain has autoregulatory function. This Ubl domain can bind to the catalytic domain and compete with the ubiquitin substrate, partially inhibiting USP4 activity against different substrates. Interestingly, other USPs, such as USP39, could relieve this inhibition.     

Structure of the p53 binding domain of HAUSP/USP7 bound to Epstein-Barr nuclear antigen 1 implications for EBV-mediated immortalization

USP7/HAUSP is a key regulator of p53 and Mdm2 and is targeted by the Epstein-Barr nuclear antigen 1 (EBNA1) protein of Epstein-Barr virus (EBV). We have determined the crystal structure of the p53 binding domain of USP7 alone and bound to an EBNA1 peptide. This domain is an eight-stranded beta sandwich similar to the TRAF-C domains of TNF-receptor associated factors, although the mode of peptide binding differs significantly from previously observed TRAF-peptide interactions in the sequence (DPGEGPS) and the conformation of the bound peptide. NMR chemical shift analyses of USP7 bound by EBNA1 and p53 indicated that p53 binds the same pocket as EBNA1 but makes less extensive contacts with USP7. Functional studies indicated that EBNA1 binding to USP7 can protect cells from apoptotic challenge by lowering p53 levels. The data provide a structural and conceptual framework for understanding how EBNA1 might contribute to the survival of Epstein-Barr virus-infected cells.     

Structural determinants for the binding of ubiquitin-like domains to the proteasome

HHR23A, a protein implicated in nucleotide excision repair, belongs to a class of proteins containing both a ubiquitin-like (Ubl) domain and one or more ubiquitin-associated (UBA) domains, suggesting a role in the ubiquitin-proteasome pathway as well. The Ubl domain binds with high affinity to the second ubiquitin-interacting motif (UIM) of the S5a subunit of the proteasome. Here we present the solution structures of the HHR23A Ubl domain, the second UIM of S5a (UIM-2), and the Ubl:S5a-UIM-2 complex. The HHR23A Ubl domain is structurally similar to ubiquitin. The S5a UIM forms an alpha-helix with an unexpected hairpin loop that contributes to the binding interface with Ubl. The molecular determinants of the Ubl-proteasome interaction are revealed by analysis of the structures, chemical shift mapping, mutant binding studies and sequence conservation.     

Protein interaction domains of the ubiquitin-specific protease, USP7/HAUSP

USP7 or HAUSP is a ubiquitin-specific protease in human cells that regulates the turnover of p53 and is bound by at least two viral proteins, the ICP0 protein of herpes simplex type 1 and the EBNA1 protein of Epstein-Barr virus. We have overexpressed and purified USP7 and shown that the purified protein is monomeric and is active for cleaving both a linear ubiquitin substrate and conjugated ubiquitin on EBNA1. Using partial proteolysis of USP7 coupled with matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we showed that USP7 comprises four structural domains; an N-terminal domain known to bind p53, a catalytic domain, and two C-terminal domains. By passing a mixture of USP7 domains over EBNA1 and ICP0 affinity columns, we showed that the N-terminal p53 binding domain was also responsible for the EBNA1 interaction, while the ICP0 binding domain mapped to a C-terminal domain between amino acids 599-801. Tryptophan fluorescence assays showed that an EBNA1 peptide mapping to residues 395-450 was sufficient to bind the USP7 N-terminal domain and did so with a dissociation constant of 0.9-2 microM, whereas p53 peptides spanning the USP7-binding region gave dissociation constants of 9-17 microM in the same assay. In keeping with these relative affinities, gel filtration analyses of the complexes showed that the EBNA1 peptide efficiently competed with the p53 peptide for USP7 binding, suggesting that EBNA1 could affect p53 function in vivo by competing for USP7.     

The herpesvirus associated ubiquitin specific protease, USP7, is a negative regulator of PML proteins and PML nuclear bodies

The PML tumor suppressor is the founding component of the multiprotein nuclear structures known as PML nuclear bodies (PML-NBs), which control several cellular functions including apoptosis and antiviral effects. The ubiquitin specific protease USP7 (also called HAUSP) is known to associate with PML-NBs and to be a tight binding partner of two herpesvirus proteins that disrupt PML NBs. Here we investigated whether USP7 itself regulates PML-NBs. Silencing of USP7 was found to increase the number of PML-NBs, to increase the levels of PML protein and to inhibit PML polyubiquitylation in nasopharyngeal carcinoma cells. This effect of USP7 was independent of p53 as PML loss was observed in p53-null cells. PML-NBs disruption was induced by USP7 overexpression independently of its catalytic activity and was induced by either of the protein interaction domains of USP7, each of which localized to PML-NBs. USP7 also disrupted NBs formed from some single PML isoforms, most notably isoforms I and IV. CK2α and RNF4, which are known regulators of PML, were dispensable for USP7-associated PML-NB disruption. The results are consistent with a novel model of PML regulation where a deubiquitylase disrupts PML-NBs through recruitment of another cellular protein(s) to PML NBs, independently of its catalytic activity.     

X-ray Structural and Functional Studies of the Three Tandemly Linked Domains of Non-structural Protein 3 (nsp3) from Murine Hepatitis Virus Reveal Conserved Functions

Murine hepatitis virus (MHV) has long served as a model system for the study of coronaviruses. Non-structural protein 3 (nsp3) is the largest nsp in the coronavirus genome, and it contains multiple functional domains that are required for coronavirus replication. Despite the numerous functional studies on MHV and its nsp3 domain, the structure of only one domain in nsp3, the small ubiquitin-like domain 1 (Ubl1), has been determined. We report here the x-ray structure of three tandemly linked domains of MHV nsp3, including the papain-like protease 2 (PLP2) catalytic domain, the ubiquitin-like domain 2 (Ubl2), and a third domain that we call the DPUP (domain preceding Ubl2 and PLP2) domain. DPUP has close structural similarity to the severe acute respiratory syndrome coronavirus unique domain C (SUD-C), suggesting that this domain may not be unique to the severe acute respiratory syndrome coronavirus. The PLP2 catalytic domain was found to have both deubiquitinating and deISGylating isopeptidase activities in addition to proteolytic activity. A computationally derived model of MHV PLP2 bound to ubiquitin was generated, and the potential interactions between ubiquitin and PLP2 were probed by site-directed mutagenesis. These studies extend substantially our structural knowledge of MHV nsp3, providing a platform for further investigation of the role of nsp3 domains in MHV viral replication.     

VWA domain of S5a restricts the ability to bind ubiquitin and Ubl to the 26S proteasome

The 26S proteasome recognizes a vast number of ubiquitin-dependent degradation signals linked to various substrates. This recognition is mediated mainly by the stoichiometric proteasomal resident ubiquitin receptors S5a and Rpn13, which harbor ubiquitin-binding domains. Regulatory steps in substrate binding, processing, and subsequent downstream proteolytic events by these receptors are poorly understood. Here we demonstrate that mammalian S5a is present in proteasome-bound and free states. S5a is required for efficient proteasomal degradation of polyubiquitinated substrates and the recruitment of ubiquitin-like (Ubl) harboring proteins; however, S5a-mediated ubiquitin and Ubl binding occurs only on the proteasome itself. We identify the VWA domain of S5a as a domain that limits ubiquitin and Ubl binding to occur only upon proteasomal association. Multiubiquitination events within the VWA domain can further regulate S5a association. Our results provide a molecular explanation to how ubiquitin and Ubl binding to S5a is restricted to the 26S proteasome.     

Structural insights into E1-catalyzed ubiquitin activation and transfer to conjugating enzymes

Ubiquitin (Ub) and ubiquitin-like proteins (Ubls) are conjugated to their targets by specific cascades involving three classes of enzymes, E1, E2, and E3. Each E1 adenylates the C terminus of its cognate Ubl, forms a E1 approximately Ubl thioester intermediate, and ultimately generates a thioester-linked E2 approximately Ubl product. We have determined the crystal structure of yeast Uba1, revealing a modular architecture with individual domains primarily mediating these specific activities. The negatively charged C-terminal ubiquitin-fold domain (UFD) is primed for binding of E2s and recognizes their positively charged first alpha helix via electrostatic interactions. In addition, a mobile loop from the domain harboring the E1 catalytic cysteine contributes to E2 binding. Significant, experimentally observed motions in the UFD around a hinge in the linker connecting this domain to the rest of the enzyme suggest a conformation-dependent mechanism for the transthioesterification function of Uba1; however, this mechanism clearly differs from that of other E1 enzymes.     

Deubiquitination of Chfr, a checkpoint protein, by USP7/HAUSP regulates its stability and activity

Chfr, a mitotic stress checkpoint, plays an important role in cell cycle progression, tumor suppression and the processes that require the E3 ubiquitin ligase activity mediated by the RING finger domain. Chfr stimulates the formation of polyubiquitin chains by ub-conjugating enzymes, and induces the proteasome-dependent degradation of a number of cellular proteins including Plk1 and Aurora A. In this study, we identified USP7 (also known as HAUSP), which is a member of a family of proteins that cleave polyubiquitin chains and/or ubiquitin precursors, as an interacting protein with Chfr by immunoaffinity purification and mass spectrometry, and their interaction greatly increases the stability of Chfr. In fact, USP7 can remove ubiquitin moiety from the autoubiquitinated Chfr both in vivo and in vitro, which results in the accumulation of Chfr in the cell. Thus, our finding suggests that USP7-mediated deubiquitination of Chfr leads to its accumulation, which might be a key regulatory step for Chfr activation and that USP7 may play an important role in the regulation of Chfr-mediated cellular processes including cell cycle progression and tumor suppression.     

The role of UBL domains in ubiquitin-specific proteases

Ubiquitin conjugation and deconjugation provides a powerful signalling system to change the fate of its target enzymes. Ubiquitination levels are organized through a balance between ubiquitinating E1, E2 and E3 enzymes and deubiquitination by DUBs (deubiquitinating enzymes). These enzymes are tightly regulated to control their activity. In the present article, we discuss the different ways in which DUBs of the USP (ubiquitin-specific protease) family are regulated by internal domains with a UBL (ubiquitin-like) fold. The UBL domain in USP14 is important for its localization at the proteasome, which enhances catalysis. In contrast, a UBL domain in USP4 binds to the catalytic domain and competes with ubiquitin binding. In this process, the UBL domain mimics ubiquitin and partially inhibits catalysis. In USP7, there are five consecutive UBL domains, of which the last two affect catalytic activity. Surprisingly, they do not act like ubiquitin and activate catalysis rather than inhibiting it. These C-terminal UBL domains promote a conformational change that allows ubiquitin binding and organizes the catalytic centre. Thus it seems that UBL domains have different functions in different USPs. Other proteins can modulate the roles of UBL domains in USP4 and USP7. On one hand, the inhibition of USP4 can be relieved when the UBL is sequestered by another USP. On the other, the activation of USP7 is increased, when the UBL-activated state is stabilized by allosteric binding of GMP synthetase. Altogether, UBL domains appear to be able to regulate catalytic activity in USPs, but they can use widely different mechanisms of action, in which they may, as in USP4, or may not, as in USP7, use the direct resemblance to ubiquitin.     

High incidence of ubiquitin-like domains in human ubiquitin-specific proteases

Ubiquitin-specific proteases (USPs) emerge as key regulators of numerous cellular processes and account for the bulk of human deubiquitinating enzymes (DUBs). Their modular structure, mostly annotated by sequence homology, is believed to determine substrate recognition and subcellular localization. Currently, a large proportion of known human USP sequences are not annotated either structurally or functionally, including regions both within and flanking their catalytic cores. To extend the current understanding of human USPs, we applied consensus fold recognition to the unannotated content of the human USP family. The most interesting discovery was the marked presence of reliably predicted ubiquitin-like (UBL) domains in this family of enzymes. The UBL domain thus appears to be the most frequently occurring domain in the human USP family, after the characteristic catalytic domain. The presence of multiple UBL domains per USP protein, as well as of UBL domains embedded in the USP catalytic core, add to the structural complexity currently recognized for many DUBs. Possible functional roles of the newly uncovered UBL domains of human USPs, including proteasome binding, and substrate and protein target specificities, are discussed.     

Annexin-1 regulated by HAUSP is essential for UV-induced damage response

DNA damage can occur through diverse stimulations such as toxins, drugs, and environmental factors. To respond to DNA damage, mammalian cells induce DNA damage response (DDR). DDR signal activates a rapid signal transduction pathway, regulating the cell fate based on the damaged cell condition. Moreover, serious damaged cells have to be eliminated by the macrophage to maintain homeostasis. Because the DDR induces genomic instability followed by tumor formation, targeting the DDR signaling can be applied for the cancer therapy. Herpes virus-associated ubiquitin-specific protease (HAUSP/USP7) is one of the well-known deubiquitinating enzymes (DUBs) owing to its relevance with Mdm2-p53 complex. The involvement of HAUSP in DDR through p53 led us to investigate novel substrates for HAUSP, which is related to DDR or apoptosis. As a result, we identified annexin-1 (ANXA1) as one of the putative substrates for HAUSP. ANXA1 has numerous roles in cellular systems including anti-inflammation, damage response, and apoptosis. Several studies have demonstrated that ANXA1 can be modified in a post-translational manner by processes such as phosphorylation, SUMOylation, and ubiquitination. In addition, DNA damage gives various functions to ANXA1 such as stress response or cleavage-mediated apoptotic cell clearance. In the current study, our proteomic analysis using two-dimensional electrophoresis, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) and nano LC-MS/MS, and immunoprecipitation revealed that ANXA1 binds to HAUSP through its HAUSP-binding motif (P/AXXS), and the cleavage and damage-responsive functions of ANXA1 upon UV-induced DNA damage may be followed by HAUSP-mediated deubiquitination of ANXA1. Intriguingly, the UV-induced damage responses via HAUSP-ANXA1 interaction in HeLa cells were different from the responses shown in the Jurkat cells, suggesting that their change of roles may depend on the cell types.Most proteins follow the ubiquitin-proteasome pathway (UPP) to degradation; this involves successive enzymatic activities of the E1, E2, and E3 enzymes. In addition to proteasomal degradation, the proteins obtain or alter their functions through mono- or polyubiquitination.¹ Thus, the ‘ubiquitin tag'' is considered as an important feature for intracellular homeostasis. Deubiquitination is a reversible process against ubiquitination that detaches ubiquitin molecules from ubiquitinated proteins, and the process of deubiquitination is mediated by specific enzymes called deubiquitinating enzymes (DUBs). To date, almost ~100 DUBs have been identified, and they are involved in various cellular functions through their capability by which they deubiquitinate and thereby stabilize or alter the functions of their target proteins.² DUBs are composed of at least six subfamilies: ubiquitin-specific proteases (USPs), ubiquitin C-terminal hydrolases (UCHs), ovarian tumor (OTU), Machado-Josephin domain papain-like cysteine proteases (MJDs), JAB1/MPN/Mov34 metalloenzyme (JAMM) domain zinc-dependent metalloprotease family, and monocyte chemotactic protein-induced proteases (MCPIPs).³ In addition, DUBs share specific regions including Cys, Asp/Asn, and His boxes for their deubiquitinating activities.⁴ The USP family has the most number among DUBs (~58 USPs),⁵ and many studies have demonstrated that human USPs have important roles in a broad range of cellular systems.⁶ In particular, their involvement in cell proliferation, signal transduction, and apoptosis emphasizes that abnormal or deregulated functions of USPs can be related to severe diseases including immune disorders and cancers.^{2, 6, 7} Accordingly, USPs have been widely targeted for the therapy of several diseases; however, a clear understanding of the molecular details underlining USPs and other DUBs has not yet been obtained.HAUSP, also known as USP7, is a member of the USP family of DUBs. The importance of HAUSP in cells was demonstrated by its ability to specifically recognize and deubiquitinate both the tumor suppressor p53 and Mdm2, a p53-specific E3 ligase. In the normal state, HAUSP specifically binds to and deubiquitinates Mdm2, thereby stabilizing Mdm2 and subsequently inducing the proteasomal degradation of p53 through Mdm2 activity. Upon DNA damage, HAUSP is dephosphorylated by PPM1G. In this state, the deubiquitinating activity of HAUSP for Mdm2 decreases and HAUSP prefers p53 for its substrate instead of Mdm2. Such altered affinity of HAUSP to p53 leads to DNA repair and tumor-suppressive functions of p53.^{8, 9, 10} In addition to Mdm2 and p53, further studies have revealed that HAUSP can regulate various substrates, including ataxin-1, Chfr, claspin, Daxx, FOXO4, histone H2B, PTEN, NF-κB, Tip60, UbE2E1, and UVSSA.² These findings suggest that HAUSP has diverse roles in the cell through the regulation of different substrates and other additional proteins. In a present study, we performed two-dimensional gel electrophoresis (2-DE) and other proteomics-based experiments using HeLa cells to identify putative substrates regulated by HAUSP. We found several putative substrates, some of which are known to be involved in apoptosis or DNA damage response (DDR). Annexin a1, also known as ANXA1 and lipocortin 1, was also found as a putative binding partner for HAUSP, suggesting that ANXA1 may possibly be regulated by HAUSP-mediated deubiquitination.Annexins consist of 13 annexin members and have four conserved repeated domains, which are responsible for Ca²⁺ and phospholipid binding. In most annexins, the conserved annexin domains enable them to bind the phospholipid of the membranes in a Ca²⁺-dependent manner, resulting in subsequent activities such as membrane trafficking, signal transduction, and exocytosis.¹¹ However, major differences of annexins derive from their unique N-terminal regions. The N-terminus of each annexin member, which is responsible for specific functions, varies.¹² ANXA1, the first member of the annexin superfamily, is a 37-kDa protein abundant in cells. Like other annexin proteins, ANXA1 binds to phospholipid in the presence of Ca²⁺.¹³ The biological functions of ANXA1 are extensively studied: anti-inflammatory mediator,^{14, 15} relationship with tumorigenesis,¹⁶ DDR,^{17, 18} and involvement in apoptosis and apoptotic cell clearance.^{19, 20} Another important feature of ANXA1 activity is the cleavage of the N-terminal region of ANXA1. When DNA damage or stress occurs, ANXA1 is cleaved by several proteases, resulting in the generation of the N-terminal fragment (Ac2-26) and cleaved form of ANXA1 (33 kDa). Importantly, both the full-length ANXA1 and Ac2-26 can be translocated to the cell membrane and induce apoptotic cell clearance by recruiting monocytes via chemoattraction.²⁰ Thus, the ANXA1 cleavage process is considered essential for cell phagocytosis, as also revealed in neutrophil apoptosis and phagocytosis during inflammation.¹⁴ Otherwise, in response to cell damage, ANXA1 functions as a stress protein or a protective protein for DNA damage, resulting in nuclear localization of ANXA1.^{18, 21, 22} Overall, it is evident that ANXA1 participates in various cellular responses.In the current study, we have identified ANXA1 as a novel substrate for HAUSP. HAUSP can bind to, deubiquitinate, and co-localize with ANXA1. Surprisingly, upon UV-induced DNA damage, the binding and the deubiquitinating activity of HAUSP to ANXA1 are increased. In addition, ANXA1 in HAUSP-deficient cells showed different localization and altered expression level and cleavage. Moreover, HAUSP-mediated regulation of ANXA1 shown in HeLa cells was different from the one in Jurkat cells. We found that apoptosis and transmigrative ratio of monocytes in HAUSP-depleted Jurkat cells coincides with the regulation of ANXA1 protein level and cleavage. Taken together, we suggest that ANXA1 functions of UV-induced DDR are regulated by the deubiquitinating activity of HAUSP.     

The E3 Ubiquitin Ligase Parkin Is Recruited to the 26 S Proteasome via the Proteasomal Ubiquitin Receptor Rpn13

Mutations in the Park2 gene, encoding the RING-HECT hybrid E3 ubiquitin ligase parkin, are responsible for a common familial form of Parkinson disease. By mono- and polyubiquitinating target proteins, parkin regulates various cellular processes, including degradation of proteins within the 26 S proteasome, a large multimeric degradation machine. In our attempt to further elucidate the function of parkin, we have identified the proteasomal ubiquitin receptor Rpn13/ADRM1 as a parkin-interacting protein. We show that the N-terminal ubiquitin-like (Ubl) domain of parkin binds directly to the pleckstrin-like receptor for ubiquitin (Pru) domain within Rpn13. Using mutational analysis and NMR, we find that Pru binding involves the hydrophobic patch surrounding Ile-44 in the parkin Ubl, a region that is highly conserved between ubiquitin and Ubl domains. However, compared with ubiquitin, the parkin Ubl exhibits greater than 10-fold higher affinity for the Pru domain. Moreover, knockdown of Rpn13 in cells increases parkin levels and abrogates parkin recruitment to the 26 S proteasome, establishing Rpn13 as the major proteasomal receptor for parkin. In contrast, silencing Rpn13 did not impair parkin recruitment to mitochondria or parkin-mediated mitophagy upon carbonyl cyanide m-chlorophenyl hydrazone-induced mitochondrial depolarization. However, it did delay the clearance of mitochondrial proteins (TIM23, TIM44, and TOM20) and enhance parkin autoubiquitination. Taken together, these findings implicate Rpn13 in linking parkin to the 26 S proteasome and regulating the clearance of mitochondrial proteins during mitophagy.     

The Multifaceted Roles of USP7: New Therapeutic Opportunities

The deubiquitylating enzyme USP7 (HAUSP) sits at a critical node regulating the activities of numerous proteins broadly characterized as tumor suppressors, DNA repair proteins, immune responders, viral proteins, and epigenetic modulators. Aberrant USP7 activity may promote oncogenesis and viral disease making it a compelling target for therapeutic intervention. Disclosed drug discovery programs have identified inhibitors of USP7 such as P005091 with cellular proof of concept and anti-proliferative activity in cancer models. Taken together, USP7 inhibitors hold promise as a new strategy for the treatment of disease.     

Structural and functional studies of USP20 ZnF‐UBP domain by NMR

Deubiquitinase USP20/VDU2 has been demonstrated to play important roles in multiple cellular processes by controlling the life span of substrate proteins including hypoxia‐inducible factor HIF1α, and so forth. USP20 contains four distinct structural domains including the N‐terminal zinc‐finger ubiquitin binding domain (ZnF‐UBP), the catalytic domain (USP domain), and two tandem DUSP domains, and none of the structures for these four domains has been solved. Meanwhile, except for the ZnF‐UBP domain, the biological functions for USP20's catalytic domain and tandem DUSP domains have been at least partially clarified. Here in this study, we determined the solution structure of USP20 ZnF‐UBP domain and investigated its binding properties with mono‐ubiquitin and poly‐ubiquitin (K48‐linked di‐ubiquitin) by using NMR and molecular modeling techniques. USP20's ZnF‐UBP domain forms a spherically shaped fold consisting of a central β‐sheet with either one α‐helix or two α‐helices packed on each side of the sheet. However, although having formed a canonical core structure essential for ubiquitin recognition, USP20 ZnF‐UBP presents weak ubiquitin binding capacity. The structural basis for understanding USP20 ZnF‐UBP's ubiquitin binding capacity was revealed by NMR data‐driven docking. Although the electrostatic interactions between D264 of USP5 (E87 in USP20 ZnF‐UBP) and R74 of ubiquitin are kept, the loss of the extensive interactions formed between ubiquitin's di‐glycine motif and the conserved and non‐conserved residues of USP20 ZnF‐UBP domain (W41, E55, and Y84) causes a significant decrease in its binding affinity to ubiquitin. Our findings indicate that USP20 ZnF‐UBP domain might have a physiological role unrelated to its ubiquitin binding capacity.