The structural basis for deubiquitination by the fingerless USP-type effector TssM

  1. Thomas Hermanns
  2. Matthias Uthoff
  3. Ulrich Baumann
  4. Kay Hofmann

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Abstract

Intracellular bacteria are threatened by ubiquitin-mediated autophagy, whenever the bacterial surface or enclosing membrane structures become targets of host ubiquitin ligases. As a countermeasure, many intracellular pathogens encode deubiquitinase (DUB) effectors to keep their surfaces free of ubiquitin. Most bacterial DUBs belong to the OTU or CE-clan families. The betaproteobacteria Burkholderia pseudomallei and Burkholderia mallei , causative agents of melioidosis and glanders, respectively, encode the TssM effector, the only known bacterial DUB belonging to the USP class. TssM is much shorter than typical eukaryotic USP enzymes and lacks the canonical ubiquitin-recognition region. By solving the crystal structures of isolated TssM and its complex with ubiquitin, we found that TssM lacks the entire “Fingers” subdomain of the USP fold. Instead, the TssM family has evolved the functionally analog “Littlefinger” loop, which is located towards the end of the USP domain and recognizes different ubiquitin interfaces than those used by USPs. The structures revealed the presence of an N-terminal immunoglobulin-fold domain, which is able to form a strand-exchange dimer and might mediate TssM localization to the bacterial surface.

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  1. Version published to 10.26508/lsa.202302422
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    Reply to the reviewers

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    Hermanns et al. report a robust biochemical and structural characterization of the TSSM virulence effector of Burkholderia pseudomallei, the causative agent of melioidosis, with TSSM being the only USP-type deubiquitinase of bacterial origin. The authors demonstrate ubiquitin specificity, and the capacity of the DUB to cleave most Ubiquitin chain types in vitro. By solving crystal structures of TSSM in isolation as well as in covalent complex with Ubiquitin, the authors rationalize how the truncated fold (compared to all other eukaryotic USPs) is capable of binding ubiquitin. The most surprising finding is the conformation of the so-called little finger loop, unique to TSSM, which engages the distal Ubiquitin in a manner not seen in any other DUB. These findings are convincingly validated by point mutations in biochemical assays and bioinformatic analyses. The structures also yielded information on the fold an N-terminal Ig-like domain with unknown function.

    The authors are encouraged to address the following main points before publication can be supported:

    • On page 6, the authors state "the little finger loop contacts hydrophobic residues around Ile-44 of ubiquitin, an important interaction interface that is not observed in USP7 and is not part of the canonical USP: ubiquitin interface". The latter statement I believe is simply wrong as Ile44 is at the centre of the large canonical USP:ubiquitin interaction interface observed in all structurally studied USP proteins so far (the recognition is through varying residues, that are not always strictly hydrophobic, but it is always contacted). This is occurring at the hinge between thumb and fingers. In USP7, this is for example occurring through the hydrophobic part of Arg301. The authors are referred to O'Dea et al. Nat Chem Biol 2023, where same conformation of the equivalent residue in USP36 is discussed in the context of Ile44-Ubiquitin vs. Fubi recognition.

    We thank reviewer #1 for this detailed explanation. Initially we made this statement based on the fact that in other USPs, the interaction is not always hydrophobic. However, we now agree with reviewer #1 and have removed this statement from the manuscript.

    This should also be phrased very precisely, as Ile44 itself is not actually contacted by the little finger loop (see Fig. 3c), but other hydrophobic residues commonly included in the Ile44 patch are. However, also those are always in the Ubiquitin:USP interface.

    These section was removed in line with the previous point. Additionally, we added the information that Ile44 itself is not contacted to the main text.

    It appears as if the little finger loop takes up the exact position of what is commonly referred to as blocking loop 1 (terminology introduced by Hu et al EMBO J 2005, which the authors could also use when referring to canonical USPs). The connectivity in the domain is not equivalent to BL1 though (this could be shown with topology diagrams of a canonical USP and of TSM), but the relative position is. Importantly, BL1 in USPs typically engage the Ile36 patch with a large aromatic residue (e.g., a Trp in USP30 or a Tyr in USP7), however, in TSSM Ubiquitin is rotated such that the Ile44 patch is recognised at the exact spot where normally Ile36 is engaged. Can the authors comment on this interpretation? This reviewer would also find a comparison to the canonical USP recognition mode in the main figures beneficial (however one that is less crowded than in the SI) to make this very important finding come across well. Even if the loop is more like a BL1 (which is highly diverse in the USP family, see reference above), it can still be called "little finger", but the analogies should be carefully phrased throughout the text (e.g., page 9: "functionally but not structurally analogous" would not be correct if it does not replace the fingers which contact Phe4 as BL1 is not part of the fingers).

    The reviewer is correct. The positioning of BL1 from canonical USPs and the TssM Littlefinger loop is superficially similar. Since the Littlefinger is derived from a different part of the sequence (Fig 1a) and contacts a different patch of ubiquitin, it is still distinct from the BL1. We have incorporated this information into the main text and generated a new superposition to show the structural similarities (Supplementary Fig 3c). Additionally, we highlighted BL1 and Littlefinger in Fig1a to show the different position within the amino acid sequence. Since BL1 and Littlefinger are distinct from each other we still classify the Littlefinger as functionally (but not structurally) analogous to the fingers domains. Both serve as the main contacts to ubiquitin and (partially) interact with the Ile44 patch, while BL1 contacts the Ile36 patch.

    The assessment of the molecular weight of apo TSSM in solution by SEC is flawed in its current form (page 7, Fig. 4b). There are only two, seemingly randomly chosen, comparison proteins, and the GST-Ub is unsuitable for 36 kDa because it is an obligate dimer (due to the GST) and as a fusion protein has a much higher hydrodynamic radius than a globular protein. There are commercial reference proteins that can be used which have defined oligomerisation states and are (reasonably globular) so that the retention times can be quantitatively analysed. Such a reference, which also includes proteins of smaller sizes, must be used. Alternatively, the authors should use light-scattering (SEC-MALS or SEC-RALS) to unequivocally demonstrate the molecular weight / oligomeric state of TSSM.

    The experiment has been re-done using commercially proteins routinely used for the calibration of SEC columns. Additionally, a 75pg column was used to achieve a better resolution of the proteins. The new data confirm the original conclusion that TssM behaves like a monomer in solution.

    The abstract ends with speculation on the presence of the strand-swapping in live cells and of the role of the Ig-fold domain, but not a proper conclusion. The same applies to the ending of the results section (page 8), where it is not clear where the structural analysis of the Ig-like domains is leading. The authors then speculate about sugar binding (page 10) in the discussion. The latter could be substantiated by an analysis of the residues in the possible sugar binding site (if present in their TSSM), and the text be more rounded off at these regions.

    We admit that our manuscript does not reveal the function of the Ig-like domain and the strand swap observed in the apo structure. To really address these questions, an infection model would be required, which is outside the scope of our manuscript focussing on the TssM mechanism. As have showed that deletion of the Ig-domain does not alter the DUB properties, the remaining questions cannot be addressed in vitro. To address the concerns of the reviewer, we have changed the ending of the result section (page 8) to summarize our in vitro data on the Ig-like domain. We have also clarified the paragraph discussing the possibility of sugar binding on pages 10/11. In brief, this speculation was not based on the identification of a possible sugar binding site, but rather on the presumed positioning of the Ig-like domain relative to the bacterial surface and its evolutionary relationship to other sugar-binding domains.

    The authors show in Fig. 1b TSSM reactivity with both Ubiquitin and Nedd8 probes but then qualify the Nedd8-reactivity with the assays shown in Figs. 1cd, which currently looks like it is an issue with the comparison of probe to substrate. However, their probe assay is very long with 18 h. It should be repeated at shorted times (for Ub and Nedd8-PA only) to test if the strong Ub preference seen in the kinetic assay can also be visualized with the probes.

    We measured the requested time curve and added the data to the newly generated Supplementary Figure 1. Using very short time points of 3 – 20min, a preferential reactivity with the ubiquitin probe became visible, which supports the AMC results.

    Moreover, the authors prepare Ubl-PA probes by aminolysis of C-terminal thioesters but at very different stoichiometries as reported for Ub-PA (e.g., in Gersch et al. NSMB 2017). The precise amounts should be cross-checked. This reviewer would not be surprised if the conditions of only 2-fold excess of propargylamine over Ubl thioester and the high amount of NaOH led also to hydrolysis of the C-terminus, which by size exclusion chromatography would not be separated. For all probes, intact mass spectra of the used aliquots must be shown to demonstrate the identity of the used reagents. The presence of other species of course does not per se disqualify the assay in Fig. 1b.

    Our protocol for generating the Ub-PA probe follows exactly the one described in Gersch et al 2017. While this publication describes the amounts and concentrations of the individual reagents, our description of the protocol documents their final concentration in the reaction mixture. We double-checked the data and found them to be identical. Thus, there is no reason to assume elevated amounts of hydrolysis products. Moreover, the purity of our probes is regularly controlled by intact mass spectrometry (shown below for Ub-PA). The observed ~5% of hydrolysis product should not pose a problem for the assays, which routinely involve a 10x excess of probe over DUB. As documented in the Materials section, the Nedd-PA probe was obtained from Monique Mulder in Leiden.

    In addition, the following minor points should be addressed:

    • On page 2, the authors state that the 1-2 DUBs typically present in bacteria either target K63-chains or lack specificity (without reference), but on page 10 they state that bacteria typically only have 1 DUB with little specificity. This is contradicting and should be fixed.

    We have rephrased the introductory sentence on page 2 and added a reference. The issue on page 10 has been corrected

    On page 3, the authors make the case for TSSM from B. pseudomallei by calling it representative. This reviewer would appreciate if the authors could expand on this towards the end of the manuscript and comment on whether they expect the identified Ubiquitin recognition mode to be present also in all (!) other DUB-TSSM proteins, at least all analysed bioinformatically. Importantly, they should include the sequence of TSSM of B. mallei (the only in vitro studied TSSM so far) into their analyses in the SI.

    We have expanded the paragraph (page 9) where we discuss the conservation of the Littlefinger-based ubiquitin recognition mode. Among the TssM-like DUBs that we identified, only those from other Burkholderia species contain the Littlefinger conservation and are predicted (by alphafold) to use this region for S1-ubiquitin recognition. The two non-Burkholderia TssM-like DUBs (one from Chromobacterium sinusclupearum, the other from an unidentified bacterial metagenome) lack the Littlefinger region and the associated ubiquitin recognition mode. This is documented in Supplementary Fig 4. We also added B. mallei to the alignment in Supplementary Fig 4a, although it is almost identical to the B. pseudomallei sequence.

    On page 4, the authors start with TSSM (193-490) but do not comment on the role of the first 192 residues. What was the rational for these boundaries?

    The N-terminal 192 amino acids are predicted to be unstructured (by alphafold, IUPRED, etc). We therefore decided to use the entire folded part of TssM for the first experiments. The information has been added to the manuscript and a domain scheme was added to the supplementary data (Supplementary Figure 1a).

    On page 4, the authors introduce the use of the fluorogenic AMC substrates with "more quantitative analysis", however, the experiments are rather minimalistic. Only one substrate concentration, and two enzyme concentrations (at least for one substrate) are used, and the data are not really analysed in a quantitative manner. Through curve fitting of the existing data (with fixed restraints) the authors may be able to determine an estimate of the Ub/Nedd8-specificity factor. Moreover, negative controls should be shown in Figure 1c to demonstrate that the Nedd8-curve is above a possible baseline drift.

    Following this reviewer’s suggestion, we estimated a 116x better cleavage of ubiquitin-AMC by determination of the initial velocities using the linear range of the data presented in Figure 1c.

    For ensuring better clarity, we have omitted the negative control data from Fig 1 panel c (comparing Ub-AMC and Nedd8-AMX) but are showing them in panel 1d (comparing Nedd8-AMC to negative control). This figure clearly shows that Nedd8-AMC is really cleaved, whereas no baseline drift is visible.

    On page 4, the authors mention a possible relationship with the Josephin family (which they later disprove through structural comparisons, page 6). It may be helpful to briefly explain the rational (i.e., why one would even consider a relationship with the Josephin family DUB given the higher homology to the USP fold).

    The rationale for considering a relationship to the Josephin family is explained at the end of the introduction section (page 3). We never considered TssM to be closer related to Josephins than to USPs. We rather speculated that the entire USP and Josephin families are distantly related to each other, and that TssM-like USPs might form a kind of missing link. This latter aspect has been disproved (page 6), since the TssM structure is no more Josephin-like than that of any other USP.

    On page 5, the "shortened C-terminus" compared to USP7 is mentioned. This is misleading, as USP7 has an elongated C-terminus compared to most other USPs, and so the TSSM C-terminus is the canonical ending of the USP fold.

    The section has been rephrased and now points out that TssM shares the canonical USP C-terminus.

    On page 6, "ubiquitin has multiple specific contacts" - why are the contacts named "specific"?

    We have removed the word ‘specific’

    On page 10, the TSSM from C. sinusclupearum appears without any context. Chromobacterium should be spelled out, and it should be discussed if this is the odd one out. It would also be appreciated if the authors could state whether their bioinformatic analysis is comprehensive (i.e., do only the known or all Burkholderia strains have TSSM with a DUB profile). And why is there a A0A1J5... sequence included in Supp Fig. 3a without any context?

    We agree that this part of the text was confusing. We have now bundled all information on other TssM-like sequences on page 9. There, we explain that TssM-like DUBs are only found in selected Burkholderia lineages, and spell out some examples. We also introduce Chromobacterium sinusclupearum with its full name and explain that the remaining non-Burkholderia TssM homolog is from an unidentified metagenomic sequence. This is also the reason why we refer to this sequence by its Uniprot accession number.

    Some minor polishing in the methods would increase consistency (cloning is only mentioned for pOPIN-K, but TSSM is also expressed from pOPIN-S; mutants are only mentioned for the 292-490 construct, but Fig. 2d shows one in the 193-490 construct; Hampton Additive Screens I-III are likely an internal name and not used by Hampton itself; "different TSSM concentrations (as indicated in the figure legends" are mentioned in the methods, but e.g. in the caption to Fig. 1 no concentration is given).

    We have added the pOPIN-S cloning method and corrected the name of the additive screen. We now provide all mutant data in the 292-490 background (Figure 2d was updated accordingly). We made sure that all figure captions mention the enzyme concentrations.

    Likewise, Table 1 needs polishing as commas and points are used interchangeably.

    Table 1 has been corrected

    In Fig. 2c, it looks like the catalytic His is built such that there is no hydrogen bonding between Cys and Asp - is there a particular reason for this? If not, the side chain should be flipped so that the nitrogens are positioned for ideal hydrogen bonding.

    Fig 2c had been accidentally exported from an early version of the structure. It has now been replaced by a panel that uses the final and deposited version of the structure. Here, the His position is correct.

    In Fig. 3, dotted lines are introduced as "hydrophobic interactions" as per the captions, but some are clearly hydrogen bonds (e.g., from the amide backbone), and for some others one does not see as they emerge from a cartoon.

    In Fig 3, dotted lines are not generally introduced as "hydrophobic interactions". Instead, the individual panels have their own definition for the dotted lines. c) hydrophobic d) hydrogen bonds e) hydrophobic.

    In Fig. 4, context should be given to "3VYP" and why it is used here.

    We have added an explanation of 3VYP to the figure legend (The reason for showing it is also explained in the main text)

    **Referees cross-commenting**

    Reviewer comments appear to be fair, balanced and complementary.

    Re Reviewer 2's comment on the pre-print: It includes a structure of TSSM bound to ubiquitin (but not of the apo protein). I am not sure if it is appropriate to follow up on the esterase activity. Firstly, there are no tools for it commercially available or readily made in vitro, and secondly it would appear a bit "copycat"-like. Especially since the molecular determinants of what makes a DUB a good esterase are still elusive. A narrower focus of this manuscript, but done very well (also according to what reviewer 3 suggested), might be a more fitting option.

    Reviewer #1 (Significance (Required)):

    The findings are novel and of high relevance to the broad ubiquitin and bacterial pathogen communities as the study addresses an enigmatic USP-type deubiquitinase which, as the authors reveal, recognizes Ubiquitin in an entirely different way than its eukaryotic counterparts. This is basic research of pronounced conceptual and mechanistic advance, as it demonstrates that the USP domain can be much more diversified than previously assumed. The structural analysis is very thorough, the data are scholarly presented, and interactions/mechanisms carefully validated by mutations.

    The strongest point is clearly the structural analysis of Ubiquitin recognition by this extremely truncated USP fold, and the introduction of the little finger motif. The manuscript does not provide cellular validation of the findings, which is fine to this reviewer for the DUB catalysis part. For the part of the N-terminal domain, the manuscript would benefit from a cellular validation or some localisation studies, however, this is beyond what is established in the authors' lab, and therefore has not been asked. This in turn limits the study to a very thorough in-vitro analysis of this DUB for TSSMs with DUB activity, using conventional substrates like polyUb chains and fluorogenic substrates, and providing convincing conclusions.

    My expertise lies in DUBs, biochemistry, and structural biology, and I this believe to have sufficient expertise to evaluate the majority of the findings except the bioinformatic algorithms used which are however not an emphasis in this manuscript.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    In this manuscript, the authors solved crystal structures of apo-TssM and its complex with Ubiquitin. Together with the biochemical assays, the authors highlighted the differences of TssM from other USP family. TssM do not contain the classical finger domain while it has little finger domain that authors defined. The Ile44 patch on the ubiquitin is mainly used for interacting with TssM.

    For the clarity of there findings several points should be edited.

    1. In Fig 2d, the authors used different constructs for testing catalytic residues. It will be better to be consistent. Though the authors showed that the deletion of Ig-like domain does not affect the catalytic activity of TssM by showing the AMC assay, it does not guarantee that the effect of mutation on catalytic sites is same for both construct.

    The experiment has been repeated with TssM292-490 C308A and the panel in Fig 2d has been replaced

    .

    1. In fig 2d, authors need to put the label to indicate which linkage of di-Ub is used. Authors did it for figure 2f.

    Chain type was already stated in the legend, but was now also added to the respective panel.

    1. By showing AMC assay (fig 2e) and K63 Ub2 cleavage assay (fig 2f), authors concluded that the deletion of Ig-like domain does not affect the activity of TssM. However, as authors found that the Ig-like domain forms dimer at least in their crystallization condition, one cannot exclude the possibility of the role of Ig-like domain in recognising different ubiquitin chains. I would clarify the words by saying "The Ig-like domain does not affect the cleavage K63-Ub2), or authors can expand the cleavage assay with all the linkages.

    We agree with reviewer #2 and rephrased the corresponding sentence.

    1. In the apo structure, authors found a domain swapped dimer. One can expect that this dimer is crystallographic artifact and not found in the nature. Indeed, authors could not observe this dimer in the solution when they performed SEC analysis and there was no effect on the catalytic activity when the Ig-like domain is deleted. Because there is no clear evidence of functional importance of this dimer in the manuscript, authors need to clarify about this dimer.

    We agree with reviewer #2 that there is no evidence for functional importance of the dimer. We had already addressed this issue in our discussion section, where we hypothesized a potential in vivo function. We have now rephrased this section of the discussion in order to make it more precise.

    **Referees cross-commenting**

    Agree with Reviewer #1's comment on my points.

    Reviewer #2 (Significance (Required)):

    The structure of TssM is recently reported in a preprint ( https://doi.org/10.21203/rs.3.rs-2986327/v1) from Pruneda (OHSU). In this preprint, they suggested the role of TssM as Ubiquitin esterase which is not explored in the this manuscript. As it is already published and freely available, authors can explore the role of TssM in that direction as well. Because the preprint do not contain the complex structure of TssM with Ubiquitin, authors can also examine the roles of Ile44 patch-interacting residues on the catalytic activities.

    Said preprint contains the TssM~Ub complex structure, but not the structure of the apo form. The role of the Ile44-patch contacting residues is also already analysed in regards of esterase activity. As explained in the ‘general points’ at the top of the rebuttal letter, the two manuscripts were meant to be submitted simultaneously. Due to a delay in our PDB deposition/validation, we submitted our version two weeks later. Thus, we share the opinion of reviewer #1 that it would be inappropriate to use our competitors’ data and analyse esterase activity in our manuscript. Instead, we added a reference to the competing preprint to our discussion section and briefly compare the key findings. The authors of the preprint have agreed to reciprocate and reference our preprint in their revised manuscript.

    Also, authors can compare their little finger structure with the preprint.

    See point above.

    In general, this manuscript is providing several interesting points to the readers working on the ubiquitin, structures of proteins, host-pathogen interactions and especially those who studying deubiquitinases.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    In this study, Hermanns et al. have examined the Burkholderia TssM deubiquitinase (DUB) for its ubiquitin chain cleavage specificity using in vitro analyses and for a structural rationalization of its specificity by solving crystal structures with and without covalently bound ubiquitin. TssM was previously shown to be a DUB of the USP family capable of cleaving several ubiquitin chain types. From bioinformatic comparisons, the authors inferred that TssM lacks the 'fingers' domain of classic USP enzymes, and this was shown in the co-crystal structure to be replaced by a 'Littlefinger' loop. The work here is well described and appears to be overall technically solid.

    My enthusiasm is reduced for several reasons. First, there is no analysis in an (infected) cell model to evaluate the significance of the TssM mutations, for example, the mutations in ubiquitin-interacting surfaces that are not seen in classic USPs. Second, there is very little quantitation of cleavage rates; while I would not demand derivation of kinetic values for all mutants, the qualitative treatment was not always convincing. For example, Y443A was said to cause "strongly reduced cleavage" (Fig. 4h) whereas E378A was said to "only mildly affect cleavage" (Fig. 4i): to my eye, these cleavage rates are only slightly different (2-3 fold?).

    In order to support the findings of our gel based assay and to get more quantitative data, we tested all mutants against Ub-AMC. The results are depicted in supplementary figures 3d/f/g and correspond to results of the chain cleavage assays.

    Finally, there is a preprint available on Research Square (doi: 10.21203/rs.3.rs-2986327/v1) that shows a potent esterase activity of TssM against ubiquitinated LPS, which is probably its key role in avoiding surveillance and elimination by the host. This paper, although still a preprint, is far more quantitative, includes similar crystallographic data as in the current paper, and describes cellular assays of function. Even without the RS preprint, the Hermanns et al. study provides a fairly modest advance in our knowledge of TssM function; with the preprint, its novelty is, unfortunately, severely reduced.

    As explained in the ‘general points’ at the top of the rebuttal letter, the two manuscripts were meant to be submitted simultaneously. Due to a delay in our PDB deposition/validation, we submitted our version two weeks later. The competing work is currently under review at a top-tier journal and far from being accepted. We therefore ask to consider the significance of our own manuscript based on the peer-reviewed state-of-the-art.

    In our revised manuscript, we have added a brief discussion of the pre-published results. The authors of the preprint have agreed to reciprocate and address our data in their revised manuscript.

    Minor comments:

    Full genus names should be spelled out when they first appear in the text (such as Burkholderia and Chromobacterium).

    Genus names are now spelled out.

    Fig. 3a is described out of sequence.

    A reference to figure 3a was missing in the beginning of the chapter. The reference was added and the figure is now described in sequence.

    In Fig 4a, there still seem to be extensive contacts between monomers but the viewing angle could be misleading.

    There are no extensive contacts between the two DUB monomers in the right panel (complex structure). We slightly changed the viewing angle in Fig 4a to make this clearer.

    **Referees cross-commenting**

    I also agree with Reviewer #1's comments

    Reviewer #3 (Significance (Required)):

    Even without the Research Square preprint (doi: 10.21203/rs.3.rs-2986327/v1), the Hermanns et al. study provides a fairly modest advance in our knowledge of TssM function; but with the preprint, its novelty is, unfortunately, severely reduced.

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    Referee #3

    Evidence, reproducibility and clarity

    In this study, Hermanns et al. have examined the Burkholderia TssM deubiquitinase (DUB) for its ubiquitin chain cleavage specificity using in vitro analyses and for a structural rationalization of its specificity by solving crystal structures with and without covalently bound ubiquitin. TssM was previously shown to be a DUB of the USP family capable of cleaving several ubiquitin chain types. From bioinformatic comparisons, the authors inferred that TssM lacks the 'fingers' domain of classic USP enzymes, and this was shown in the co-crystal structure to be replaced by a 'Littlefinger' loop. The work here is well described and appears to be overall technically solid.

    My enthusiasm is reduced for several reasons. First, there is no analysis in an (infected) cell model to evaluate the significance of the TssM mutations, for example, the mutations in ubiquitin-interacting surfaces that are not seen in classic USPs. Second, there is very little quantitation of cleavage rates; while I would not demand derivation of kinetic values for all mutants, the qualitative treatment was not always convincing. For example, Y443A was said to cause "strongly reduced cleavage" (Fig. 4h) whereas E378A was said to "only mildly affect cleavage" (Fig. 4i): to my eye, these cleavage rates are only slightly different (2-3 fold?). Finally, there is a preprint available on Research Square (doi: 10.21203/rs.3.rs-2986327/v1) that shows a potent esterase activity of TssM against ubiquitinated LPS, which is probably its key role in avoiding surveillance and elimination by the host. This paper, although still a preprint, is far more quantitative, includes similar crystallographic data as in the current paper, and describes cellular assays of function. Even without the RS preprint, the Hermanns et al. study provides a fairly modest advance in our knowledge of TssM function; with the preprint, its novelty is, unfortunately, severely reduced.

    Minor comments:

    Full genus names should be spelled out when they first appear in the text (such as Burkholderia and Chromobacterium).

    Fig. 3a is described out of sequence.

    In Fig 4a, there still seem to be extensive contacts between monomers but the viewing angle could be misleading.

    Referees cross-commenting

    I also agree with Reviewer #1's comments

    Significance

    Even without the Research Square preprint (doi: 10.21203/rs.3.rs-2986327/v1), the Hermanns et al. study provides a fairly modest advance in our knowledge of TssM function; but with the preprint, its novelty is, unfortunately, severely reduced.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, the authors solved crystal structures of apo-TssM and its complex with Ubiquitin. Together with the biochemical assays, the authors highlighted the differences of TssM from other USP family. TssM do not contain the classical finger domain while it has little finger domain that authors defined. The Ile44 patch on the ubiquitin is mainly used for interacting with TssM.

    For the clarity of there findings several points should be edited.

    1. In Fig 2d, the authors used different constructs for testing catalytic residues. It will be better to be consistent. Though the authors showed that the deletion of Ig-like domain does not affect the catalytic activity of TssM by showing the AMC assay, it does not guarantee that the effect of mutation on catalytic sites is same for both construct.
    2. In fig 2d, authors need to put the label to indicate which linkage of di-Ub is used. Authors did it for figure 2f.
    3. By showing AMC assay (fig 2e) and K63 Ub2 cleavage assay (fig 2f), authors concluded that the deletion of Ig-like domain does not affect the activity of TssM. However, as authors found that the Ig-like domain forms dimer at least in their crystallization condition, one cannot exclude the possibility of the role of Ig-like domain in recognising different ubiquitin chains. I would clarify the words by saying "The Ig-like domain does not affect the cleavage K63-Ub2), or authors can expand the cleavage assay with all the linkages.
    4. In the apo structure, authors found a domain swapped dimer. One can expect that this dimer is crystallographic artifact and not found in the nature. Indeed, authors could not observe this dimer in the solution when they performed SEC analysis and there was no effect on the catalytic activity when the Ig-like domain is deleted. Because there is no clear evidence of functional importance of this dimer in the manuscript, authors need to clarify about this dimer.

    Referees cross-commenting

    Agree with Reviewer #1's comment on my points.

    Significance

    The structure of TssM is recently reported in a preprint ( https://doi.org/10.21203/rs.3.rs-2986327/v1) from Pruneda (OHSU). In this preprint, they suggested the role of TssM as Ubiquitin esterase which is not explored in the this manuscript. As it is already published and freely available, authors can explore the role of TssM in that direction as well.

    Because the preprint do not contain the complex structure of TssM with Ubiquitin, authors can also examine the roles of Ile44 patch-interacting residues on the catalytic activities.

    Also, authors can compare their little finger structure with the preprint.

    In general, this manuscript is providing several interesting points to the readers working on the ubiquitin, structures of proteins, host-pathogen interactions and especially those who studying deubiquitinases.

  5. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Hermanns et al. report a robust biochemical and structural characterization of the TSSM virulence effector of Burkholderia pseudomallei, the causative agent of melioidosis, with TSSM being the only USP-type deubiquitinase of bacterial origin. The authors demonstrate ubiquitin specificity, and the capacity of the DUB to cleave most Ubiquitin chain types in vitro. By solving crystal structures of TSSM in isolation as well as in covalent complex with Ubiquitin, the authors rationalize how the truncated fold (compared to all other eukaryotic USPs) is capable of binding ubiquitin. The most surprising finding is the conformation of the so-called little finger loop, unique to TSSM, which engages the distal Ubiquitin in a manner not seen in any other DUB. These findings are convincingly validated by point mutations in biochemical assays and bioinformatic analyses. The structures also yielded information on the fold an N-terminal Ig-like domain with unknown function.

    The authors are encouraged to address the following main points before publication can be supported:

    • On page 6, the authors state "the little finger loop contacts hydrophobic residues around Ile-44 of ubiquitin, an important interaction interface that is not observed in USP7 and is not part of the canonical USP: ubiquitin interface". The latter statement I believe is simply wrong as Ile44 is at the centre of the large canonical USP:ubiquitin interaction interface observed in all structurally studied USP proteins so far (the recognition is through varying residues, that are not always strictly hydrophobic, but it is always contacted). This is occurring at the hinge between thumb and fingers. In USP7, this is for example occurring through the hydrophobic part of Arg301. The authors are referred to O'Dea et al. Nat Chem Biol 2023, where same conformation of the equivalent residue in USP36 is discussed in the context of Ile44-Ubiquitin vs. Fubi recognition.
    • This should also be phrased very precisely, as Ile44 itself is not actually contacted by the little finger loop (see Fig. 3c), but other hydrophobic residues commonly included in the Ile44 patch are. However, also those are always in the Ubiquitin:USP interface.
    • It appears as if the little finger loop takes up the exact position of what is commonly referred to as blocking loop 1 (terminology introduced by Hu et al EMBO J 2005, which the authors could also use when referring to canonical USPs). The connectivity in the domain is not equivalent to BL1 though (this could be shown with topology diagrams of a canonical USP and of TSM), but the relative position is. Importantly, BL1 in USPs typically engage the Ile36 patch with a large aromatic residue (e.g., a Trp in USP30 or a Tyr in USP7), however, in TSSM Ubiquitin is rotated such that the Ile44 patch is recognised at the exact spot where normally Ile36 is engaged. Can the authors comment on this interpretation? This reviewer would also find a comparison to the canonical USP recognition mode in the main figures beneficial (however one that is less crowded than in the SI) to make this very important finding come across well. Even if the loop is more like a BL1 (which is highly diverse in the USP family, see reference above), it can still be called "little finger", but the analogies should be carefully phrased throughout the text (e.g., page 9: "functionally but not structurally analogous" would not be correct if it does not replace the fingers which contact Phe4 as BL1 is not part of the fingers).
    • The assessment of the molecular weight of apo TSSM in solution by SEC is flawed in its current form (page 7, Fig. 4b). There are only two, seemingly randomly chosen, comparison proteins, and the GST-Ub is unsuitable for 36 kDa because it is an obligate dimer (due to the GST) and as a fusion protein has a much higher hydrodynamic radius than a globular protein. There are commercial reference proteins that can be used which have defined oligomerisation states and are (reasonably globular) so that the retention times can be quantitatively analysed. Such a reference, which also includes proteins of smaller sizes, must be used. Alternatively, the authors should use light-scattering (SEC-MALS or SEC-RALS) to unequivocally demonstrate the molecular weight / oligomeric state of TSSM.
    • The abstract ends with speculation on the presence of the strand-swapping in live cells and of the role of the Ig-fold domain, but not a proper conclusion. The same applies to the ending of the results section (page 8), where it is not clear where the structural analysis of the Ig-like domains is leading. The authors then speculate about sugar binding (page 10) in the discussion. The latter could be substantiated by an analysis of the residues in the possible sugar binding site (if present in their TSSM), and the text be more rounded off at these regions.
    • The authors show in Fig. 1b TSSM reactivity with both Ubiquitin and Nedd8 probes but then qualify the Nedd8-reactivity with the assays shown in Figs. 1cd, which currently looks like it is an issue with the comparison of probe to substrate. However, their probe assay is very long with 18 h. It should be repeated at shorted times (for Ub and Nedd8-PA only) to test if the strong Ub preference seen in the kinetic assay can also be visualized with the probes.
    • Moreover, the authors prepare Ubl-PA probes by aminolysis of C-terminal thioesters but at very different stoichiometries as reported for Ub-PA (e.g., in Gersch et al. NSMB 2017). The precise amounts should be cross-checked. This reviewer would not be surprised if the conditions of only 2-fold excess of propargylamine over Ubl thioester and the high amount of NaOH led also to hydrolysis of the C-terminus, which by size exclusion chromatography would not be separated. For all probes, intact mass spectra of the used aliquots must be shown to demonstrate the identity of the used reagents. The presence of other species of course does not per se disqualify the assay in Fig. 1b.

    In addition, the following minor points should be addressed:

    • On page 2, the authors state that the 1-2 DUBs typically present in bacteria either target K63-chains or lack specificity (without reference), but on page 10 they state that bacteria typically only have 1 DUB with little specificity. This is contradicting and should be fixed.
    • On page 3, the authors make the case for TSSM from B. pseudomallei by calling it representative. This reviewer would appreciate if the authors could expand on this towards the end of the manuscript and comment on whether they expect the identified Ubiquitin recognition mode to be present also in all (!) other DUB-TSSM proteins, at least all analysed bioinformatically. Importantly, they should include the sequence of TSSM of B. mallei (the only in vitro studied TSSM so far) into their analyses in the SI.
    • On page 4, the authors start with TSSM (193-490) but do not comment on the role of the first 192 residues. What was the rational for these boundaries?
    • On page 4, the authors introduce the use of the fluorogenic AMC substrates with "more quantitative analysis", however, the experiments are rather minimalistic. Only one substrate concentration, and two enzyme concentrations (at least for one substrate) are used, and the data are not really analysed in a quantitative manner. Through curve fitting of the existing data (with fixed restraints) the authors may be able to determine an estimate of the Ub/Nedd8-specificity factor. Moreover, negative controls should be shown in Figure 1c to demonstrate that the Nedd8-curve is above a possible baseline drift.
    • On page 4, the authors mention a possible relationship with the Josephin family (which they later disprove through structural comparisons, page 6). It may be helpful to briefly explain the rational (i.e., why one would even consider a relationship with the Josephin family DUB given the higher homology to the USP fold).
    • On page 5, the "shortened C-terminus" compared to USP7 is mentioned. This is misleading, as USP7 has an elongated C-terminus compared to most other USPs, and so the TSSM C-terminus is the canonical ending of the USP fold.
    • On page 6, "ubiquitin has multiple specific contacts" - why are the contacts named "specific"?
    • On page 10, the TSSM from C. sinusclupearum appears without any context. Chromobacterium should be spelled out, and it should be discussed if this is the odd one out. It would also be appreciated if the authors could state whether their bioinformatic analysis is comprehensive (i.e., do only the known or all Burkholderia strains have TSSM with a DUB profile). And why is there a A0A1J5... sequence included in Supp Fig. 3a without any context?
    • Some minor polishing in the methods would increase consistency (cloning is only mentioned for pOPIN-K, but TSSM is also expressed from pOPIN-S; mutants are only mentioned for the 292-490 construct, but Fig. 2d shows one in the 193-490 construct; Hampton Additive Screens I-III are likely an internal name and not used by Hampton itself; "different TSSM concentrations (as indicated in the figure legends" are mentioned in the methods, but e.g. in the caption to Fig. 1 no concentration is given).
    • Likewise, Table 1 needs polishing as commas and points are used interchangeably.
    • In Fig. 2c, it looks like the catalytic His is built such that there is no hydrogen bonding between Cys and Asp - is there a particular reason for this? If not, the side chain should be flipped so that the nitrogens are positioned for ideal hydrogen bonding.
    • In Fig. 3, dotted lines are introduced as "hydrophobic interactions" as per the captions, but some are clearly hydrogen bonds (e.g., from the amide backbone), and for some others one does not see as they emerge from a cartoon.
    • In Fig. 4, context should be given to "3VYP" and why it is used here.

    Referees cross-commenting

    Reviewer comments appear to be fair, balanced and complementary.

    Re Reviewer 2's comment on the pre-print: It includes a structure of TSSM bound to ubiquitin (but not of the apo protein). I am not sure if it is appropriate to follow up on the esterase activity. Firstly, there are no tools for it commercially available or readily made in vitro, and secondly it would appear a bit "copycat"-like. Especially since the molecular determinants of what makes a DUB a good esterase are still elusive. A narrower focus of this manuscript, but done very well (also according to what reviewer 3 suggested), might be a more fitting option.

    Significance

    The findings are novel and of high relevance to the broad ubiquitin and bacterial pathogen communities as the study addresses an enigmatic USP-type deubiquitinase which, as the authors reveal, recognizes Ubiquitin in an entirely different way than its eukaryotic counterparts. This is basic research of pronounced conceptual and mechanistic advance, as it demonstrates that the USP domain can be much more diversified than previously assumed. The structural analysis is very thorough, the data are scholarly presented, and interactions/mechanisms carefully validated by mutations.

    The strongest point is clearly the structural analysis of Ubiquitin recognition by this extremely truncated USP fold, and the introduction of the little finger motif. The manuscript does not provide cellular validation of the findings, which is fine to this reviewer for the DUB catalysis part. For the part of the N-terminal domain, the manuscript would benefit from a cellular validation or some localisation studies, however, this is beyond what is established in the authors' lab, and therefore has not been asked. This in turn limits the study to a very thorough in-vitro analysis of this DUB for TSSMs with DUB activity, using conventional substrates like polyUb chains and fluorogenic substrates, and providing convincing conclusions.

    My expertise lies in DUBs, biochemistry, and structural biology, and I this believe to have sufficient expertise to evaluate the majority of the findings except the bioinformatic algorithms used which are however not an emphasis in this manuscript.

  6. Version published to 10.1101/2023.08.01.551441v1 on bioRxiv