The ubiquitin interacting motifs of USP37 act on the proximal Ub of a di-Ub chain to enhance catalytic efficiency

USP37 is a deubiquitinase (DUB) with roles in the regulation of DNA damage repair and the cohesion of sister chromatids during mitosis. USP37 contains a unique insert of three ubiquitin interacting motifs (UIMs) within its catalytic DUB domain. We investigated the role of the three UIMs in the ability of USP37 to cleave di-ubiquitin chains. We found that the third UIM of USP37 recognizes the proximal ubiquitin moiety of K48 di-Ub to potentiate cleavage activity and posit that this mechanism of action may be generalizable to other chain types. In the case of K48-linked ubiquitin chains this potentiation stemmed largely from a dramatic increase in catalytic rate (kcat). We also developed and characterized three ubiquitin variant (UbV) inhibitors that selectively engage distinct binding sites in USP37. In addition to validating the deduced functional roles of the three UIMs in catalysis, the UbVs highlight a novel and effective means to selectively inhibit members of the difficult to drug DUB family.

USP37 has a distinctive domain architecture consisting of an N-terminal PH domain, an interdomain linker and a C-terminal catalytic domain. Located within the catalytic domain is a large insertion of 284 amino acids (hsUSP37) containing three Ub-interacting motifs (UIMs) embedded at a site approximately 30 Å from the catalytic cleft. UIMs are single alpha-helical elements that bind to Ub with modest affinity (0.1-2 mM) 19 . UIMs conform to the consensus sequence e-e-x-x-ϕ-x-x-A-ϕ-x-(ϕ/e)-S-z-x-e, where e is an acidic residue, ϕ is a hydrophobic residue, z is a bulky hydrophobic or polar residue with high aliphatic content, A is alanine, S is serine and x is any residue 20 . UIM binding to Ub is routinely disabled by mutation of the consensus alanine position to glycine or the consensus serine position to alanine. In other DUBs, UIMs have been shown to confer cleavage preference for specific Ub chain types, such as K63-linked chains in the case of OTUD1 and Ataxin-3 or K48-linked Ub chains in the case of USP25 [21][22][23] . Additionally, the UIMs of USP25 and Ataxin-3 have been shown to increase the ubiquitination state of the DUB itself, although the precise mechanism by which this is achieved remains unknown 24,25 .
Previous studies have shown that the UIMs of USP37 play an essential role in Ub cleavage activity and substrate binding properties of USP37. Specifically, A814G and S818A mutations to UIM2 and/or A836G and S840A mutations to UIM3 impaired the ability of USP37 to cleave K48-and K63-linked chains, while V712G and S716A mutations to UIM1 had no discernable effect 26 . Furthermore, while combined mutation of all three UIMs had a marked effect on DUB activity, it had no effect on the cleavage specificity of USP37 towards K48-linked chains over K63-linked chains 14,26 . Lastly, mutation of UIM2 and/or UIM3 perturbed USP37 binding to the cohesin regulator WAPL and to endogenous Ub-protein conjugates 15,26 . While these observations made clear that UIM2 and UIM3 play an important functional role in supporting the DUB activity of USP37, several questions remain unresolved, including: 1) do the individual UIMs impact the ability of USP37 to cleave the 6 Ub chain types not previously tested, 2) does mutation of the UIMs in USP37 selectively affect the k cat or K M of the enzyme, and 3) do the UIMs of USP37 act by engaging the proximal or distal position Ub moiety in a Ub chain. To address these questions, we have performed detailed mutational, biochemical and enzymatic characterization of the USP37 UIMs.

Results
Impact of UIMs on the Ub chain specificity of USP37. We expressed the isolated catalytic domain of USP37 from Danio rerio (residues 312-927, denoted USP37 wt ), which unlike USP37 from human, mouse and chicken, could be expressed in bacteria in stable form for biochemical and enzymatic studies. Danio rerio and Homo sapiens USP37 share identical domain architectures and strong sequence similarity (72% and 77% similarity overall and over the catalytic domain, respectively) suggesting that this ortholog would serve as a good model for understanding the mechanism of action of the human enzyme (Fig. 1a). To determine comprehensively if the UIMs of USP37 contribute to chain linkage cleavage specificity, we tested the ability of USP37 wt to cleave all 8 possible di-Ub chain types including the 6 Ub chain types not previously tested. USP37 wt displayed a preference for K6-, 11-, 33-, 48-and 63-linked di-Ub chains (Fig. 1b, top panel). Strikingly, a mutant form of USP37 (USP37 mUIM1,2,3 ) bearing mutations in all three UIMs predicted to abolish Ub binding (see methods for details) displayed no change in chain specificity. Indeed, USP37 mUIM1,2,3 still displayed a strong preference for K6-, K11-, K33-, K48-and K63-linked di-Ub chains, although with reduced activity overall relative to USP37 wt (Fig. 1b,  bottom panel).
We next performed experiments using tetra Ub chains and observed a similar overall trend ( Supplementary  Fig. S2). However, zebrafish USP37 displayed a greater preference for K48 over K63 chains, which more closely matched the reported chain specificity of the human enzyme 14,26 . Mutation of UIMs drastically decreased cleavage efficiency of USP37 against K48 and K63 tetra Ub chains without changing its preference for K48 tetra Ub chains. These findings further support the notion that the UIMs of USP37 do not confer specificity towards specific Ub chain types but are instead required for full enzymatic activity.
The effect of mutations to all three UIMs on the kinetic parameters of USP37. To determine how precisely the UIMs impact on the catalytic efficiency of USP37, we determined the effect of UIM mutations on the k cat and K M parameters of the enzyme. To this end, we performed kinetic experiments with USP37 wt and USP37 mUIM1,2,3 against the three most preferred Ub chain substrates, namely K11-, 48-, and 63-linked chains, bearing internally quenched fluorescent (IQF) di-Ub probes (Fig. 2a). In this assay format, cleavage of di-Ub liberates the fluorophore from the quencher, yielding an increase in fluorescence proportional to the amount of substrate cleaved. To estimate how well the IQF substrate mimics the unmodified substrates, we performed a gel-based cleavage assay comparing USP37wt cleavage of unmodified K48 and K63 di-Ub with IQF labeled K48 and K63 di-Ub ( Supplementary Fig. S3). We observed that IQF modifications adversely impacted the cleavage efficiency of substrate with the negative effect on K63 chains being more pronounced than K48 chains. As such, when using IQF substrates we restricted our comparisons to reactions using the same substrate type. For K48-linked chains, we observed a large reduction in k cat (decreased ~15-fold) and a smaller perturbation in K M (increased ~1.8-fold) ( Fig. 2a Fig. S4 for a zoom-in view of the USP37 mUIM1,2,3 cleavage profile) in response to all three UIM mutations. For K11-and K63-linked chains, we observed more modest reductions in both k cat (decreased ~2.6-fold for K11 and ~3.4-fold for K63) and K M (decreased ~1.7-fold for K11 and ~2.1-fold for K63) (Fig. 2a middle and right panel, Table 1). Thus, the ability of UIMs to bind Ub plays differential roles on the kinetic parameters of USP37 depending on the Ub chain type tested.

Effects of UIM mutations on the ability of USP37 to cleave K11-,K48-or K63-linked Ub chains.
To determine if any particular UIM is strongly associated with cleavage of a specific chain linkage, we generated UIM mutations predicted to abolish Ub binding in all possible single, double and triple combinations. We then tested each protein for its ability to cleave K11-, K48-or K63-linked di-Ub chains. In characterizing the activity of the mutants against these 3 linkage types, we noted the following strong patterns of behavior (Fig. 2b).
Both UIM2 and UIM3 appeared important for the ability of USP37 to cleave all Ub chain types but to varying degrees. In the case of K11-and K63-linked substrates, individually disabling UIM2 (USP37 mUIM2 ) or UIM3 (USP37 mUIM3 ) caused partial impairment of activity, while simultaneously disabling UIM2 and UIM3 (USP37 mUIM2,3 ) resulted in an additive impairment of activity. These results are consistent with prior findings 26 , which showed that human USP37 with disabling mutations in all three UIMs had a larger decrease in activity towards K48-and K63-linked poly-Ub chains as compared to USP37 with disabling mutations in UIM2 or UIM3 alone.
In the case of the K48-linked Ub substrate, UIM3 was clearly more vital than UIM2 for the activity of USP37 (Fig. 2b). Disabling UIM3 alone (USP37 mUIM3 ) resulted in a large decrease in activity, while disabling UIM2 alone (USP37 mUIM2 ) had no observable effect on activity. However, the importance of UIM2 was revealed when UIM2 and UIM3 were disabled together, resulting in an enzyme (USP37 mUIM2,3 ) in which activity was almost completely abolished relative to WT (~4% activity remaining based on V 0 ). Such a drastic effect was not observed for the same double mutant when cleaving K11-or K63-linked chains, where USP37 mUIM2,3 retained ~20% or ~18% of WT activity (based on V 0 ), respectively. These results suggested that UIM1 does not contribute to the activity of USP37 towards K11-, K48-and K63-linked chains, whereas UIM2 and UIM3 do contribute to activity towards these chains, but to varying degrees that are dependent on the specific type of chain linkage. As the disabling mutations in the UIMs had the strongest effects on the ability of USP37 to cleave K48-linked Ub chains, we chose to focus subsequent functional characterizations on this substrate type. www.nature.com/scientificreports www.nature.com/scientificreports/

Interrogation of proximal or distal Ub binding by the UIMs of USP37.
To determine if the UIMs of USP37 specifically engage the proximal Ub (primary amine donating) or the distal Ub (carboxy terminus donating) in a K48 di-Ub chain substrate to exert their effect on cleavage activity, we first made use of the model substrate Ub-AMC in which the proximal Ub moiety is replaced with a cleavable fluorescent molecule (AMC, 7-Amino-4-Methylcoumarin) (Fig. 3a).
The proximal Ub is absent in Ub-AMC, and thus, we posited that if the UIMs act on the proximal Ub, we would observe no difference in cleavage by USP37 wt or USP37 mUIM1,2,3 (Fig. 3b). In contrast, if the UIMs act by binding to the distal Ub moiety of a di-Ub substrate, then we would expect a major difference in the cleavage of Ub-AMC by USP37 wt or USP37 mUIM1,2,3 , as the distal Ub is fully available for binding (Fig. 3b). In comparing the activity of USP37 wt and USP37 mUIM1,2,3 against Ub-AMC, we observed no difference in DUB activity (Fig. 3c). This result is consistent with the UIMs of USP37 acting by engaging the proximal Ub (Fig. 3c).
To further verify that the UIMs of USP37 act by engaging the proximal Ub of K48 di-Ub, we used a mutant form of K48-linked di-Ub chains in which substitutions were incorporated into the proximal Ub moiety at position 44 (I44A or I44W). The Ile44 side chain of Ub comprises part of the canonical hydrophobic binding surface for UIMs, and substitutions at this position are likely to perturb UIM binding. If the UIMs of USP37 bind to the proximal Ub of a di-Ub substrate, then di-Ub proteins with substitutions at position 44 of the proximal Ub should be poorer substrates relative to WT di-Ub, in the case of the USP37 wt but not in the case of USP37 mUIM1,2,3 in which all UIMs are disabled (Fig. 4a). Indeed this is what we observed. Compared with cleavage of WT K48-linked di-Ub, USP37 wt displayed a reduced ability to cleave di-Ub substrates containing an I44A/W substitution (Fig. 4b top panel). In contrast, USP37 mUIM1,2,3 displayed similar activity for cleavage of all three substrates, albeit at reduced rates relative  www.nature.com/scientificreports www.nature.com/scientificreports/ to the WT enzyme and substrate forms (Fig. 4b, bottom panel). We also tested the three single site UIM mutants of USP37 for their ability to cleave K48 di-Ub substrates bearing the I44A/W substitution in the proximal Ub position. All three single site mutant proteins displayed a reduced ability to cleave the mutant substrates relative to wild www.nature.com/scientificreports www.nature.com/scientificreports/ type K48 di-Ub, similar to the behavior of USP37 wt . This expected result is consistent with a degree of functional redundancy between UIMs ( Supplementary Fig. S5). Together, these results indicate that the UIMs of USP37 act to support enzymatic activity by selectively engaging the proximal Ub of K48 di-Ub.

Interrogation of UIM function with Ub Variants. Ub variants (UbVs) are engineered forms of Ub that
bind specifically and tightly to a target protein of interest 27 . A UbV usually binds with higher affinity to preexisting Ub-binding site in a manner similar to the native interaction [27][28][29][30] . UbV reagents have been used previously to interrogate the mechanism of action and biological function of Ub-related enzymes both in vitro and in vivo 27,[29][30][31][32][33][34][35][36] .
To further verify our model that UIM2 and UIM3 act by selectively binding to the proximal Ub of K48 di-Ub, we used phage display to derive one UbV that bound to the core catalytic domain lacking the insert containing the UIMs (UbV.core), another UbV that bound to the first UIM domain of USP37 (UbV.UIM1), and a third UbV (UbV.UIM*) that bound all three the UIM domains of the enzyme (Supplementary Fig. S6). We assessed the binding specificity of each UbV using GST/MBP pulldown experiments with the UbVs as prey and each isolated UIM (His-MBP-UIM1, His-MBP-UIM2, His-MBP-UIM3), the catalytic domain (GST-USP37) or the catalytic domain lacking all three UIMs (GST-USP37 Δloop ) as bait (Supplementary Fig. S7). We also assessed the binding of the three UbVs with human USP37 and observed coincidentally that UbV.UIM* and UbV.UIM1 but not UbV. core were retained by GST-hUSP37 in pulldowns ( Supplementary Fig. S8).
We next determined the ability of each of the three UbVs to inhibit hydrolysis of Ub-AMC by zebrafish USP37 wt . Mutation of UIM1 had no effect on enzyme function, and as expected, UbV.UIM1 did not affect USP37 wt activity (Fig. 5a). In our working model, UIM2 and UIM3 engage the proximal Ub moiety of K48 di-Ub to influence catalytic activity. As Ub-AMC lacks a proximal Ub, we were not surprised to see that UbV.UIM* had little effect on hydrolysis of this substrate (Fig. 5a). In contrast UbV.core potently inhibited Ub-AMC cleavage (IC 50 ~9 nM). This result is consistent with the likelihood that UbV.core directly competed for binding to the distal Ub-binding site of USP37 (Fig. 5a).
We next determined the ability of each of the three UbVs to inhibit the activity of zebrafish USP37 for cleavage of the K48-linked di-Ub substrate bearing an internally quenched fluorophore (IQF). UbV.UIM1 had no effect on this activity (Fig. 5b), consistent with our mutational data for UIM1 (Fig. 2b). In contrast, UbV.UIM* potently www.nature.com/scientificreports www.nature.com/scientificreports/ inhibited cleavage of the K48-linked di-Ub substrate, as did UbV.core (Fig. 5b). Taken together, these results support our model in which UIM2 and UIM3 engage the proximal Ub of K48 di-Ub to enhance cleavage by USP37, whereas UIM1 is not involved in substrate recognition.

Discussion
Our results are consistent with a model whereby UIM2 and UIM3 of USP37 bind the proximal Ub of K48 di-Ub to increase catalytic efficiency. In the case of K48-linked substrates, binding of the UIMs to the proximal Ub increased catalytic efficiency of USP37 largely through k cat and to a lesser degree through K M . This result was somewhat counter-intuitive since we expected that binding of the UIMs of USP37 to Ub to have a more pronounced effect on K M (a proxy for binding affinity) since others have shown that UIM mutations perturbed the ability to pull down Ub conjugates 26 . Furthermore, we observed that in the case of K48-linked di-Ub substrate, UIM3 was more vital than UIM2 for the proteolytic activity of USP37. This behavior was not noted in the same previous study 26 . A possible explanation for our findings relates to the main type of Ub conjugates investigated, which involved chains of variable length greater than 2 moieties 26 versus Ub chains of fixed length equal to 2 Ub moieties used here. Characterizing the kinetic parameters of cleavage of longer Ub chains by USP37 could help to address this point. The issue of how the binding of the UIMs of USP37 to the proximal Ub of K48 di-Ub enhances activity remains an open question. We envision a model whereby the UIMs optimally position a di-Ub chain across the active site of the USP domain for more efficient catalysis of the inter-Ub isopeptide bond by selectively binding the proximal Ub position. We expect the UIMs to act upon the same substrate engaged by the USP domain active site (i.e. in cis) as zebrafish USP37 displays no tendency to multimerize in solution as assessed by size exclusion chromatography (Supplementary Fig. S9). Whether this model is generalizable to other Ub chain types, requires further verification. Furthermore, as our experiments were largely focused on di-Ub substrates, it is possible that the UIMs could function in additional capacities for longer chains types. A co-structure of USP37 with a Ub chain substrate would be highly informative for resolving the remaining mechanistic questions.
We developed UbVs that bound the core catalytic domain or the UIMs of USP37 and functioned as potent inhibitors of catalytic activity in vitro. The UbVs targeting the UIMs represent the first report of UbV DUB inhibitors that act by targeting domains distinct from the catalytic domain. Notably, UbVs have been shown to inhibit DUBs in cells 27 , and we posit that the UbVs developed here may prove useful as reagents for future studies interrogating the in vitro biochemical functions and in vivo biological functions of USP37.
While inhibitors of USP enzymes have been developed, specificity has been a recurring problem 37 . This may be due to the lack of differentiating features between the catalytic sites of USP family members. Only recently have groups demonstrated effective specific inhibitors against a USP enzyme, namely USP7 [38][39][40][41] . Interestingly, one of these inhibitors did not target the conserved active site directly, but rather, acted by targeting a USP7 loop distinct from other USP family members 39 . In an analogous manner, we exploited the unique UIMs of USP37 to derive a specific inhibitor of USP37.
As noted, USP37 stabilizes the proto-oncogene c-MYC and the oncogenic fusion PLZF/RARA in cells 17,18 . The demonstration that knockdown of USP37 decreases the stability of these oncogenic proteins, raises the possibility that specific and potent inhibitors of USP37 may provide a viable therapeutic strategy to treat human cancers. To this end, targeting UIM2 and UIM3 may provide the basis for potent and specific inhibition of USP37 DUB activity. www.nature.com/scientificreports www.nature.com/scientificreports/

Materials and Methods
Protein Purification. For biochemical experiments, a codon optimized gene (GeneArt) encoding for Danio rerio USP37 wt (312-927) containing a N-terminal GST tag was expressed in Escherichia coli BL21(DE3)-RIL cells. Cells were lysed by homogenization in lysis buffer (50 mM HEPES pH 8, 500 mM NaCl, 5% glycerol, 5 mM DTT) with 0.5 mM phenylmethane sulfonyl fluoride (PMSF). After clarification via centrifugation, supernatant was incubated via gravity column with glutathione resin and washed with lysis buffer. Protein bound to glutathione resin was incubated overnight in presence of TEV protease at 4 °C. Protein was eluted with lysis buffer and concentrated to ~4 mg/mL and subsequently flash diluted to ~0.2 mg/mL in low salt buffer (25 mM HEPES pH 8, 50 mM NaCl, 5 mM DTT) and loaded onto an HiTrap Q HP anion exchange column (GE Healthcare). Protein was eluted via a linear gradient with low and high salt buffer (25 mM HEPES pH 8, 500 mM NaCl, 5 mM DTT). Fractions containing USP37 wt were pooled, concentrated and injected onto a Superdex 200 column, pre-equilibrated with sizing buffer (25 mM HEPES pH 8, 150 mM NaCl, 5 mM DTT). Fractions containing USP37 wt were pooled, concentrated and stored at −80 °C. USP37 mutants (V680G and S684A for UIM1, A774G and S778A for UIM2, and A796G and S800A for UIM3) were cloned from the WT construct using the QuikChange method (Stratagene) and expressed and purified in similar fashion.
Size-exclusion chromatography. USP37 wt was purified by glutathione resin and anion exchange chromatography as detailed in the protein purification methods section. USP37wt was concentrated and injected (1 mL of ~2 mg/mL) onto a Superdex S200 (GE Healthcare) previously equilibrated with 25 mM HEPES pH 8, 150 mM NaCl, 5 mM DTT. Absorbance at 280 nm was used to monitor the elution of the protein and determination of effective molecular weight was performed using a formula derived from protein standards.
Gel-based deubiqutination assays. Gel-based deubiquitination assays were performed in 25 mM Hepes pH 8, 150 mM NaCl, 5 mM DTT, 0.1 mg/ml BSA. For data in Fig. 1, 1 nM USP37 enzyme was incubated with 9 µM di-Ub substrate at 20 °C. Di-Ub chains were enzymatically produced by our group (K11, K48, and K63) or Boston Biochem (K6, K27, K29, and K33), with the exception of Met1 di-Ub chains, which were produced as a tandem fusion directly in E. coli. Met1-linked Ub chains were produced according to published methods 42 . K48-and K63-linked Ub chains were produced as described 43 , with the use of a K48R and K63R Ub mutant (for distal Ub) and with the variation of using a Ub with the last two Gly residues deleted (Ub∆GG, for the proximal Ub). K11-linked Ub chains were produced similarly (K11R Ub for distal Ub and Ub∆GG for proximal Ub), with the exception of using 30 µM Ube2S as the E2 conjugating enzyme according to published methods 44 . K6-, K27-, K29-and K33-linked Ub chains were obtained from a commercial source (Boston Biochem, catalog #: UC-11B, UC-61B, UC-81B, and UC-101B, respectively). Time points were analyzed by SDS-PAGE and staining with gel-code blue (Thermofisher, catalog # 24590). Images were taken on a Bio-Rad ChemiDoc MP Imaging System. Images were cropped using Image Lab (Bio-Rad).
For data in Fig. 4b and Supplementary Fig. S5, 5 nM USP37 enzyme was incubated with 18 µM di-Ub substrate at 20 °C. Mutant K48-linked di-Ubs were produced with the same method as WT K48-linked di-Ub using K48R Ub (for distal Ub) and I44A/W Ub∆GG (for proximal Ub). Time points were analyzed by SDS-PAGE and staining with Coomassie blue. Images were taken on a Bio-Rad ChemiDoc MP Imaging System. Images were cropped using Image Lab (Bio-Rad).
For data in Supplementary Fig. S2, 10 nM USP37 enzyme was incubated with 6 µM tetra-Ub at 20 °C. K48 and K63 tetra-Ub chains were obtained from a commercial source (Boston Biochem, catalog #: UC-210B and UC-310B, respectively). Time points were analyzed by SDS-PAGE and staining with Coomassie blue. Images were taken on a Bio-Rad ChemiDoc MP Imaging System. Images were cropped using Image Lab (Bio-Rad).
For data in Supplementary Fig. S3, 1 nM USP37 enzyme was incubated with 9 µM of the indicated di-Ub substrate at 20 °C. Time points were analyzed by SDS-PAGE. Gels were fluorescently scanned using a Bio-Rad