Active site alanine mutations convert deubiquitinases into high‐affinity ubiquitin‐binding proteins
2018; Springer Nature; Volume: 19; Issue: 10 Linguagem: Inglês
10.15252/embr.201745680
ISSN1469-3178
AutoresMarie E. Morrow, Michael T. Morgan, Marcello Clerici, Kateřina Growková, Ming Yan, David Komander, Titia K. Sixma, Michal Šimíček, Cynthia Wolberger,
Tópico(s)Cancer-related Molecular Pathways
ResumoArticle27 August 2018Open Access Transparent process Active site alanine mutations convert deubiquitinases into high-affinity ubiquitin-binding proteins Marie E Morrow Marie E Morrow Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA Search for more papers by this author Michael T Morgan Michael T Morgan Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA Search for more papers by this author Marcello Clerici Marcello Clerici Division of Biochemistry and Oncode Institute, Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Katerina Growkova Katerina Growkova Faculty of Medicine, University of Ostrava, Ostrava, Czech Republic Search for more papers by this author Ming Yan Ming Yan Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA Search for more papers by this author David Komander David Komander orcid.org/0000-0002-8092-4320 Medical Research Council Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Titia K Sixma Titia K Sixma Division of Biochemistry and Oncode Institute, Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Michal Simicek Michal Simicek Faculty of Medicine, University of Ostrava, Ostrava, Czech Republic Medical Research Council Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Cynthia Wolberger Corresponding Author Cynthia Wolberger [email protected] orcid.org/0000-0001-8578-2969 Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA Search for more papers by this author Marie E Morrow Marie E Morrow Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA Search for more papers by this author Michael T Morgan Michael T Morgan Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA Search for more papers by this author Marcello Clerici Marcello Clerici Division of Biochemistry and Oncode Institute, Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Katerina Growkova Katerina Growkova Faculty of Medicine, University of Ostrava, Ostrava, Czech Republic Search for more papers by this author Ming Yan Ming Yan Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA Search for more papers by this author David Komander David Komander orcid.org/0000-0002-8092-4320 Medical Research Council Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Titia K Sixma Titia K Sixma Division of Biochemistry and Oncode Institute, Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Michal Simicek Michal Simicek Faculty of Medicine, University of Ostrava, Ostrava, Czech Republic Medical Research Council Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Cynthia Wolberger Corresponding Author Cynthia Wolberger [email protected] orcid.org/0000-0001-8578-2969 Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA Search for more papers by this author Author Information Marie E Morrow1, Michael T Morgan1, Marcello Clerici2,5, Katerina Growkova3, Ming Yan1, David Komander4, Titia K Sixma2, Michal Simicek3,4 and Cynthia Wolberger *,1 1Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA 2Division of Biochemistry and Oncode Institute, Netherlands Cancer Institute, Amsterdam, The Netherlands 3Faculty of Medicine, University of Ostrava, Ostrava, Czech Republic 4Medical Research Council Laboratory of Molecular Biology, Cambridge, UK 5Present address: Department of Biochemistry, University of Zurich, Zurich, Switzerland *Corresponding author. Tel: +1 410 955 0728; E-mail: [email protected] EMBO Reports (2018)19:e45680https://doi.org/10.15252/embr.201745680 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract A common strategy for exploring the biological roles of deubiquitinating enzymes (DUBs) in different pathways is to study the effects of replacing the wild-type DUB with a catalytically inactive mutant in cells. We report here that a commonly studied DUB mutation, in which the catalytic cysteine is replaced with alanine, can dramatically increase the affinity of some DUBs for ubiquitin. Overexpression of these tight-binding mutants thus has the potential to sequester cellular pools of monoubiquitin and ubiquitin chains. As a result, cells expressing these mutants may display unpredictable dominant negative physiological effects that are not related to loss of DUB activity. The structure of the SAGA DUB module bound to free ubiquitin reveals the structural basis for the 30-fold higher affinity of Ubp8C146A for ubiquitin. We show that an alternative option, substituting the active site cysteine with arginine, can inactivate DUBs while also decreasing the affinity for ubiquitin. Synopsis Mutation of deubiquitinating enzyme (DUB) active site cysteine to alanine dramatically increases the affinity of some DUBs for mono- and polyubiquitin chains. Expression of these alanine mutant DUBs in cells could therefore give rise to spurious or dominant negative effects due to the potential of these DUBs to protect polyubiquitin from cleavage and sequester ubiquitin. Mutation of DUB active site cysteines to alanine relieves steric hindrance, which allows tighter binding of ubiquitin's C-terminus. In vitro, C-to-A mutations lead to a 10–150-fold increase in binding affinity of DUBs for monoubiquitin or polyubiquitin. In cells, overexpressed C-to-A mutant DUBs act as high affinity ubiquitin binding domains, thereby sequestering ubiquitinated proteins from deubiquitination, which leads to a dramatic accumulation of polyubiquitin and ubiquitinated substrates. We recommend the use of arginine active site mutations to avoid undesired dominant negative effects in cellular studies of inactive DUBs. Introduction Deubiquitinating enzymes (DUBs) play fundamental roles in ubiquitin signaling through their ability to remove ubiquitin from target proteins and disassemble polyubiquitin chains 1. These enzymes cleave the isopeptide linkage between the C-terminus of ubiquitin and the substrate lysine or, in some cases, the peptide bond between ubiquitin and a substrate protein N-terminus. The human genome encodes more than 90 DUBs 2, 3, which can be grouped into families based on their fold: ubiquitin-specific protease (USP), ubiquitin carboxyl-terminal hydrolase (UCH), ovarian tumor family (OTU), Machado–Joseph domain (MJD) family, and JAMM/MPN domain (JAMM), as well as the recently discovered MINDY and ZUFSP families 4-6. Studies of deletions as well as disease-causing mutations have revealed specific functions for individual DUBs in biological processes including proteasomal degradation, protein trafficking, transcription, DNA repair, infection, and inflammation 7. The involvement of DUBs in a variety of oncogenic 8, inflammation, and neurodegenerative pathways 9 has made these enzymes attractive targets for drug discovery 10. A common approach to determining the role of a particular DUB in cellular pathways is to knock down expression of the endogenous DUB and express a catalytically inactive version of the enzyme 11-13. With the exception of the JAMM domain family, which are metalloproteases, all other DUBs are cysteine proteases with a papain-like active site in which the catalytic cysteine is activated by an adjacent histidine 14. Cysteine protease DUBs are typically inactivated by substituting the active site cysteine with another residue. Resulting changes in substrate ubiquitination or downstream signaling pathways in cells expressing the mutant DUB are generally assumed to be due to the absence of deubiquitinating activity, with the notable exceptions of OTUB1, which inhibits E2 enzymes by a mechanism independent of catalytic activity 15-17 and OTUD4, which serves as a scaffold for USP enzymes 18. Whereas serine is the most conservative substitution for the active site cysteine, alanine substitutions are often used to avoid the possibility that mutants containing a serine substitution may retain some hydrolase activity. We report here that some active site cysteine-to-alanine substitutions can dramatically increase the affinity of DUBs for either free ubiquitin or polyubiquitin chains. This increase in affinity can confound the interpretation of cell-based experiments, since the mutant DUB is not only incapable of cleaving ubiquitin from substrates but has gained the ability to sequester free ubiquitin and polyubiquitin chains. Altering levels of free ubiquitin has been shown to give rise to off-target effects 19. In addition, these mutant DUBs may stably associate with (poly)ubiquitinated substrates and thereby protect ubiquitin chains from cleavage by other DUBs or prevent interaction with ubiquitin receptors. The effects of such tight-binding DUB mutants thus have the potential to confuse interpretation because of the gain of tight ubiquitin-binding function. We show here that mutating the active site cysteine of human USP4 and yeast Ubp8 to alanine increases the affinity of the DUB for mono- or diubiquitin by 10–150-fold. A similar effect of alanine substitution was previously found for the OTU enzymes, Cezanne 20 and OTULIN 21. The structure of the heterotetrameric SAGA DUB module containing Ubp8C146A bound to free ubiquitin reveals the molecular basis for the increased affinity of monoubiquitin for the mutant enzyme. The alanine substitution alleviates steric hindrance by the active site cysteine sulfhydryl, allowing the C-terminal carboxylate of ubiquitin to form additional hydrogen bonds in the enzyme active site and thus accounting for the high affinity of the mutant enzyme for free ubiquitin. We show that substituting the active site cysteine with arginine in representative USP and OTU DUBs inactivates the enzymes while also disrupting binding to ubiquitin, generating an inert DUB. Based on these findings, we strongly recommend that cell-based and in vivo studies of DUBs avoid the use of active site alanine substitutions and to instead utilize substitutions such as arginine that ablate both enzymatic activity and ubiquitin binding. Results and Discussion Mutation of active site cysteine to alanine increases affinity of the SAGA DUB module for ubiquitin The yeast SAGA complex is a transcriptional coactivator that is involved in transcription of all RNA polymerase II genes 22, 23. Among the SAGA activities are the removal of monoubiquitin from histone H2B, which promotes transcription initiation and elongation 24. The deubiquitinating activity of SAGA resides in a four-protein complex known as the DUB module, which comprises the USP family catalytic subunit, Ubp8, as well as Sgf11, Sus1, and the N-terminal ~100 residues of Sgf73 25, 26. Structural studies of the DUB module complexed with ubiquitin aldehyde 27 and with ubiquitinated nucleosomes 28 have revealed the overall organization of the DUB module and how it interacts with substrate. In addition to its ability to deubiquitinate histone H2B, the DUB module can also cleave a variety of ubiquitin substrates in vitro including ubiquitin-AMC and K48-linked diubiquitin 29. The affinity of the DUB module for ubiquitinated nucleosome has been estimated at around 2 μM 28 and the KM for the model substrate, ubiquitin-AMC, has been estimated at 24 μM 29; however, neither the KM nor binding affinity for other substrates is known. In order to measure the affinity of the DUB module for other substrates using binding assays, we expressed and purified catalytically inactive versions of the DUB module containing Ubp8 with its active site cysteine, C146, substituted with either serine (C146S) or alanine (C146A). The absence of catalytic activity for both mutants was first verified in a ubiquitin-AMC cleavage assay (Fig EV1). We measured the affinity of both mutant complexes for K48-linked diubiquitin using isothermal titration calorimetry (ITC; Fig 1E and F). Whereas DUB module containing Ubp8C146S bound to K48 diubiquitin with a Kd of 4.6 μM, DUB module containing Ubp8C146A bound to K48-linked diubiquitin with a Kd of 0.47 μM, representing 10-fold tighter binding. We also measured the affinity of the reaction product, monoubiquitin, to DUB module containing either wild-type or mutant Ubp8 (Fig 1A–C). Whereas DUB module containing wild-type Ubp8 or Ubp8C146S bound ubiquitin with a Kd of 13.9 μM and 12.8 μM, respectively, the Ubp8C146A mutant bound ~30-fold more tightly to monoubiquitin with a Kd of 0.43 μM. Click here to expand this figure. Figure EV1. Catalytic activity of DUBm-Ubp8 mutantsProgress curve of Ub-AMC cleavage by 125 nM DUBm-Ubp8WT or mutants. The experiment as shown was performed once. The C146A and C146S mutants have been shown to lack any cleavage activity in at least two experiments each performed by other assay methods. Download figure Download PowerPoint Figure 1. Isothermal titration calorimetry assays of SAGA DUB module binding to K48 diubiquitin or monoubiquitin Binding of wild-type DUBm-Ubp8 to monoubiquitin. Binding of DUBmUbp8C146A to monoubiquitin. Binding of DUBm-Ubp8C146S to monoubiquitin. Binding of DUBm-Ubp8C146R to monoubiquitin. Binding of DUBm-Ubp8C146A to K48 diubiquitin. Binding of DUBm-Ubp8C146S to K48 diubiquitin. Data information: Error ranges for Kd values were determined from nonlinear least squares fitting of the data to a one-site binding model. Download figure Download PowerPoint A Ubp8 C146A substitution enables hydrogen bonding with the ubiquitin C-terminus To determine the structural basis for the marked increase in affinity for free ubiquitin when the active site cysteine is substituted with alanine, we solved the crystal structure of the SAGA DUB module containing Ubp8C146A bound to free ubiquitin at a resolution of 2.1 Å (Table 1 and Fig 2A). The overall fold and contacts with ubiquitin are virtually identical to those found in the structure of the wild-type enzyme bound to ubiquitin aldehyde, superimposing all atoms with an RMSD of 0.58 Å 27. The active site of the C146A mutant is virtually identical to that in the wild-type apoenzyme 29, with no significant reordering of residues (Fig 2B) 29. In the Ubp8C146A complex with free ubiquitin, the negatively charged carboxylate of the ubiquitin C-terminal Gly76 forms two hydrogen bonds with backbone amides from Ubp8 residues Thr145 and Ala146, as well as with active site residues, Asn141 and His427 (Figs 2B and EV2). Importantly, the observed position of the ubiquitin C-terminus would not be compatible with the presence of the wild-type active site residue, Cys146, since the sulfhydryl group would clash with the C-terminal residue of ubiquitin, Gly76 (Fig 2C). The multiple hydrogen bonding interactions observed between the C-terminal carboxylate of ubiquitin and Ubp8 can therefore only occur when the active Cys146 is replaced with the smaller alanine side chain, thus explaining the higher affinity of DUB module-Ubp8C146A for free ubiquitin as compared to the wild-type enzyme. Table 1. X-ray crystallographic data and refinement statistics Wavelength (Å) 0.979 Resolution (Å) 2.10 Unique reflections 54,195 Redundancy 5.7 (5.7) Completeness (%) 99.2 (99.7) Average I/σ (I) 13.3 (3.0) R merge 0.093 (0.572) R meas 0.103 (0.630) R pim 0.043 (0.259) CC1/2 0.998 (0.857) CC* 0.999 (0.961) Refinement statistics Space group P212121 Unit cell (Å) a = 78.8, b = 103.2, c = 112.8 Molecules per asymmetric unit 1 Rwork (%) 20.1 Rfree (%) 24.9 Rmsd bonds (Å) 0.0198 Rmsd angles (°) 1.855 Protein atoms 6,302 Zinc ions 8 Average B (Å2) 40.3 Figure 2. X-ray crystal structure of SAGA DUB module mutant DUBm-Ubp8C146A bound to monoubiquitin Overall structure of complex showing Ubp8 (green) with ubiquitin (yellow) bound to the USP domain. Hydrogen bonding contacts between the C-terminal carboxylate of ubiquitin and Ubp8. In blue spheres, van der Waals radii of C146 and A146 in steric proximity of ubiquitin's C-terminal carboxylate (yellow). DUBm-Ubp8WT structure is shown in teal (PDB ID 3MHH) and DUBm-Ubp8C146A is shown in green. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Active site of DUBm-Ubp8C146A bound to monoubiquitinDensity is rendered from the 2Fo–Fc map and contoured at 2σ. Download figure Download PowerPoint Active site cysteine-to-alanine substitution increases the affinity USP4 for ubiquitin Mutating the active site cysteine to alanine has a dramatic effect on the affinity of the human USP family DUB, USP4, for free ubiquitin. USP4 regulates a broad variety of cellular pathways, including TGF-β and NF-κB signaling as well as splicing 26, 30, 31. The affinity of USP4 for free ubiquitin was measured by fluorescence polarization using ubiquitin labeled with an N-terminal fluorophore. As shown in Fig 3, the Kd of ubiquitin for the wild-type enzyme is 92 ± 21 nM, whereas USP4 containing an alanine substituted for the active site cysteine, C311, binds ubiquitin with 0.60 ± 0.17 nM affinity, a ~150-fold difference 32. The pre-steady-state kinetics of ubiquitin dissociation measured by fluorescence polarization in a stopped-flow device shows that the greater affinity of the USP4C311A mutant is due to a dramatic decrease in off-rate (Fig EV3A). Interestingly, ubiquitin dissociation has been shown to be promoted by USP4N-terminal DUSP-Ubl domain and to regulate USP4 activity 32. The increase in affinity for the mutant enzyme is not unique to ubiquitin with a free C-terminus, as ubiquitin conjugated to either an 18-mer peptide or C-terminal fluorophore also binds with similar affinity to USP4C311A (Fig EV3C and D). Since the active sites of USP family DUBs are highly conserved, we speculate that the observed increase in binding affinity is due a relief of steric clash, as is the case for Ubp8. Figure 3. Equilibrium binding of USP4 WT and C311A to TAMRA-labeled monoubiquitinBinding was measured by fluorescence polarization using N-terminally TAMRA-labeled monoubiquitin. The dissociation constants for ubiquitin binding to USP4 WT and C311A are 92 ± 21 nM 32 and 0.60 ± 0.17 nM, respectively. Error bars are s.d. calculated on five measurements per point. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Binding kinetics of USP4 WT and C311A to ubiquitin substrates Dissociation kinetics of USP4 WT and C311A binding to TAMRA-monoubiquitin, measured by stopped-flow fluorescence polarization. Association kinetics for USP4 WT and C311A binding to TAMRA-monoubiquitin. Measured by stopped-flow fluorescence polarization. Equilibrium binding of USP4 C311A to TAMRA-ubiquitin conjugated to lysine. Equilibrium binding of USP4 C311A to TAMRA-ubiquitin conjugated to an 18-mer peptide derived from SMAD4. Data information: The error bars in panels (C) and (D) are s.d. calculated on two measurements per point. Download figure Download PowerPoint Substitution of the catalytic cysteine with arginine disrupts ubiquitin binding in USP and OTU class DUBs We sought to identify alternative active site mutations that would abrogate catalytic activity as well as reduce the affinity of the inactive DUB polyubiquitin chains or ubiquitinated substrates. We reasoned that substituting the active site cysteine with arginine could both inactivate the enzyme and prevent ubiquitin binding because of the bulky nature of the side chain compared to cysteine. To test this hypothesis, we mutated the catalytic cysteine of Ubp8 to arginine and first verified that SAGA DUB module containing the mutant Ubp8C146R protein was inactive in a Ub-AMC cleavage assay (Fig EV1). The affinity of the DUB module containing Ubp8C146R for monoubiquitin as measured by ITC was comparable to that of the wild-type protein (Fig 1D). Substitution of the active cysteine with arginine similarly reduces the affinity for polyubiquitin chains by the OTU family member, OTUD1, which is also a cysteine protease. This DUB preferentially cleaves K63-linked polyubiquitin chains 20, has recently been shown to regulate the nuclear localization and transcriptional coactivator activity of the YAP oncoprotein 33, represses metastasis by deubiquitinating SMAD7 during TGF-β signaling 34, and negatively regulates RIG-I-like receptor (RLR) signaling during viral infection by deubiquitinating Smurf1 35. We measured the affinity of catalytic mutants of OTUD1 for fluorescently labeled K63-linked diubiquitin using a fluorescence polarization assay (Fig 4A). While OTUD1 with an alanine substituted for the active site cysteine (OTUD1C320A) binds K63-linked diubiquitin with a Kd of ~40 μM, an arginine substitution, OTUD1C320R, not only inactivated the enzyme but also completely abolished detectable binding to K63-linked diubiquitin (Fig 4A). Figure 4. Enhanced binding of OTUD1 C320A to K63 diubiquitin in vitro and K63 polyubiquitin chains in cells Equilibrium binding of OTUD1 C320A and C320R to K63-linked diubiquitin was measured by fluorescence polarization using FlAsH-tagged K63-linked diubiquitin in which the proximal ubiquitin was fluorescently labeled. Error bars indicate s.d. and are based on three measurements per data point. One representative experiment of two is shown. Whole cell lysates of HEK293 cells expressing HA-tagged OTUD1 WT, C320R, and C320A were immunoblotted with indicated antibodies. One representative experiment of three is shown. Download figure Download PowerPoint Active site arginine substitutions can overcome artifacts of alanine substitution in cells As mentioned above, active site cysteine-to-alanine substitutions that markedly increase DUB affinity for mono- or polyubiquitin may render these mutants less suitable for physiological studies. Since such cysteine-to-alanine mutants are essentially high-affinity ubiquitin-binding proteins, these DUB mutants have the potential to stabilize modified substrates and preferred chain types by protecting them from digestion by other DUBs or proteases. For example, when OTULIN C129A is expressed in cells, there is a dramatic accumulation of Met1-linked linear polyubiquitin chains that is not seen when a mutant that abrogates ubiquitin binding, L259E, is expressed 21. We hypothesized that substituting the active site cysteine with arginine could be a general approach in cell-based studies to inactivating cysteine protease DUBs while also preventing high-affinity binding to polyubiquitin. To test this idea, we expressed cysteine-to-alanine and cysteine-to-arginine mutants of two DUBs, OTUD1 and USP14, in cells and probed their effects on levels of polyubiquitin. HA-tagged wild-type OTUD1, OTUD1C320A, or OTUD1C320R was expressed in HEK293 cells and whole cell lysates were analyzed by immunoblotting with an antibody specific for K63-polyubiquitin chains. As compared to cells expressing the wild-type protein, cells expressing OTUD1C320A had increased levels of K63-linked polyubiquitin (Fig 4B). By contrast, cells expressing OTUD1C320R did not show enriched levels of K63-linked chains (Fig 4B). We also tested the effects of expressing wild-type and mutant USP14, one of the chain-trimming DUBs that bind to the 26S proteasome 36, 37. HA-tagged USP14 containing the wild-type active site cysteine, Cys114, and C114A and C114R mutants were co-expressed in HEK293 cells along with FLAG-PSMD4, a ubiquitin receptor within the proteasome 38. Proteasome-bound ubiquitinated proteins were co-immunoprecipitated by FLAG-PSMD4 and probed for ubiquitin (Fig 5). Proteasomes with USP14 C114A bind more polyubiquitin chains than USP14 C114R and also retain increased levels of higher molecular weight chains that are unable to be trimmed compared to wild-type USP14 (Fig 5). Both of these results are consistent with the idea that the increase in polyubiquitin chains observed with the cysteine-to-alanine mutants is due to the ability of this mutant to bind to polyubiquitin chains and protect them from cleavage by other DUBs. Our results also validate the benefit of using a Cys to Arg substitution to generate a catalytically inactive DUB that will neither protect nor sequester polyubiquitin chains and ubiquitinated substrates. Figure 5. Binding of USP14 C114A to ubiquitin chains in cellsPolyubiquitin chains were co-immunoprecipitated with FLAG-PSMD4, an ubiquitin receptor for the 26S proteasome, from cells expressing either USP14 wild-type, C114A, or C114R. One representative experiment of two is shown. Download figure Download PowerPoint Implications for cell-based studies of cysteine protease DUBs The surprisingly high affinity for ubiquitin exhibited by DUBs containing alanine substituted for the active site cysteine has important implications for cell-based assays in which catalytically inactive DUBs are expressed. We have found that cysteine-to-alanine substitutions in the USP DUBs, Ubp8 and USP4, dramatically increase their affinity for monoubiquitin (Figs 1 and 3). Similarly, cysteine-to-alanine substitutions in the active sites of Ubp8 and the OTU class DUB, OTUD1, also increase DUB affinity for polyubiquitin (Figs 1 and 4A). The equilibrium dissociation constants of these mutant DUBs are significantly lower than cellular concentrations of their substrates, given the estimated concentration of free ubiquitin in the cell of 4–50 μM 39 and a concentration of polyubiquitin at a fraction of that 40. The mutant DUBs are therefore expected to bind tightly to free ubiquitin and to polyubiquitin chains when expressed in cells. Particularly in experiments where mutant DUBs are overexpressed, there is the risk that cellular consequences ascribed to a lack of catalytic activity in a particular DUB may instead be due to the ability of the mutant DUB to protect polyubiquitin chains from cleavage or to a difference in free ubiquitin available to ubiquitin-conjugating enzymes. The effects of cysteine-to-alanine substitutions shown here for USP and OTU class DUBs are likely to extend to other cysteine protease DUBs. The UCH and MJD classes of cysteine protease DUBs have a conserved active site architecture similar to USP DUBs 14, 41 and could thus form similar interactions with ubiquitin if the active site cysteine was substituted with alanine (Fig EV4A and B). Although the MINDY and ZUFSP DUBs share little homology with the other cysteine protease DUBs 4-6, an alanine substitution would similarly relieve steric clash in the active site, thus potentially increasing the DUB's affinity for ubiquitin (Fig EV4C). These observations support the recommendation that alanine substitutions should be avoided in cell-based and in vivo studies of cysteine protease DUBs. Click here to expand this figure. Figure EV4. Alignment of the active site of Ubp8C146A+monoUb to other cysteine protease DUBs A, B. Alignment of Ubp8C146A+monoUb active site to (A) ubiquitin-bound UCHL1 (PDB ID 3KW5) or (B) ubiquitin-bound Ataxin 3 (PDB ID 3O65). C. The active site of ubiquitin propargyl-bound MINDY (PDB ID 5JQS) shown alone, as it does not align with Ubp8C146A due to a lack of structural conservation. Download figure Download PowerPoint We have presented an alternative to alanine substitutions that is equally effective in abrogating DUB activity while having the advantage of preventing ubiquitin binding. In binding assays of Ubp8 and OTUD1, we show that replacing the active site cysteine with arginine inactivates ubiquitin hydrolase activity while also rendering the enzyme incapable of binding ubiquitin detectably (Figs 1D and 4). We speculate that the ability of arginine substitutions to abolish ubiquitin binding to OTU and USP catalytic domains may be explained by the ability of the arginine side chain to partially occupy the binding site for the C-terminal ubiquitin Gly-Gly in these DUBs. Previous data have indicated that the correct orientation of the ubiquitin C-terminal tail in the DUB S1 site is essential for efficient cleavage 42, 43. An arginine side chain may mimic these interactions in cis, to prevent these important substrate interactions. While we did not test other active site substitutions, it is expected that other amino acids with side chains that are significantly bulkier than cysteine would similarly block ubiquitin binding, in addition to inactivating the enzyme. However, care should be taken to avoid hydrophobic side chains that could cause the protein to aggregate, or side chains that are bulky or beta-branched that could interfere with proper protein folding due to steric clashes with the neighboring protein backbone. Since lysine can be ubiquitinated and is also subject to many other post-translational modifications, this substitution should also be avoided. We recommend that all cell-based and in vivo studies of cysteine protease DUBs avoid alanine substitutions of the active
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