Peer review of: A deubiquitylase with an unusually high-affinity ubiquitin-binding domain from the scrub (2020)

Reviewers' comments: 

 

Reviewer #1 (Remarks to the Author): 

 

NCOMMS-19-37565 

Review comment: 

Berk et al., “A Deubiquitylase with an Unusually High-affinity Ubiquitin-binding Domain from the 
Scrub Typhus Pathogen Orientia tsutsugamushi” 

 

Effector proteins of pathogenic bacteria can disturb and/or hijack immune signaling pathways of 
host cells. Such signaling pathways are controlled by ubiquitylation, and therefore, some effector 
proteins possess E3 ubiquitin (Ub) ligase activity or deubiquitylating (DUB) activity. The CE clan/Ulp1-
like proteases are a major DUB family of bacterial effectors. Functional and structural mechanisms 
underlying their DUB activities have been extensively studied. In this study, Berk et al. present the 
apo- and Ub-bound structures of the CE clan/Ulp1-like protease domain in a likely effector protein of 
the obligate intracellular bacterium Orientia tsutsugamushi (OtDUB). OtDUB binds to three Ub 
molecules, which occupy the S1 and S2 sites in the catalytic domain and the high-affinity binding site 
in the additional Ub-binding domain (UBD). The S2 site or the additional UBD is not included in the 
CE clan/Ulp1-like protease domains of known structures. However, unfortunately, physiological 
significance of these two structural properties is unclear. The structure suggests the preference to 
K63 linkage, but OtDUB seems to cleave K48-linked chains as well as K63-linked chains. The 
functional role of the additional UBD is also unclear. I would like to suggest the following points to 
be improved if this study would be published. 

 

Major points: 

1. Orders of figures are inconsistent (e.g., Fig. 1c is cited before Fig. 1b). 

2. In Fig. 1a, VR-2, CR, and VR-3 should be indicated. Also, the color of VR-3 is indistinguishable. 

3. Fig. 1c is difficult to interpret. Please align the standard cartoon models with the conserved CE 
clan fold in similar orientations. Do not superpose the structures. 

4. It would be better if tetra M1- and K11-linked Ub chains could be tested as substrates for OtDUB. 

5. The difference between the apo- and Ub-bound OtDUB structures should be mentioned. 

6. In Fig. 2b, please compare the OtDUB-Ub(S1) and other bacterial DUB-Ub/Ubl(S1) structures with 
Ub(S1) in similar orientations. 

7. In Fig. 3b, Ile44 of Ub(S2) should be indicated. 

8. In Fig. 3c, K48-linked diubiquitin seems to be cleaved. Please explain this. Other data show that 
OtDUB cleaves triubiquitin or longer chains. 

9. In Fig. 6a, please indicate the positions of the S1 and S2 sites and the UBD. 

10. In Fig.6b and Supplementary Fig. 1c, the cleavage of K48-linked Ub chains by O. chuto DUB (1-
299) and O. tsutsu DUB (1-177) should be tested. 

11. Phe59 of OtDUB is replaced by Thr in O. chuto DUB (Fig. 6a). In Fig. 3c, the F59T mutation of 
OtDUB decreases the cleavage activity for K63-linked chains. However, in Fig. 6b, O. chuto DUB 
shows substantial activity for K63-linked chains. Does O. chuto DUB really have the S2 site? Please 
explain this. 

12. Is pre-incubation performed in Fig. S1b also effective for UBD mutants (V203D, F207D and 
L221D)? 

13. In Fig. S2, please compare OtDUB-Ub with OTUB1-Ub, where two Ub molecules are aligned so as 
to form a K48 linkage. 

14. In Methods, twin refinement is mentioned. Twinning parameters should be shown. 

 

Minor points: 

1. The terms “OTT_1962” and “OtDUB” are used in the figures. Please use either one. 

2. In Fig. 2, the position of Lys48 should be indicated. 

3. “Supplemental Figure” may be “Supplementary Figures” in Nature style. 

4. In Methods, “DNAse” should be “DNase”. 

 

 

 

Reviewer #2 (Remarks to the Author): 

 

In their submitted manuscript, Berk & Lim et al. present a thorough structural and biochemical 
analysis of a novel deubiquitinase domain from the scrub typhus pathogen Orientia tsutsugamushi. 
This work is a fascinating addition to our understanding of a growing family of bacterial 
deubiquitinases from the CE clan of proteases and describes several discoveries that are thus far 
unique to OtDUB and related Orientia homologs. In particular, the preference for longer 
polyubiquitin chains and surprising high-affinity interaction with monomeric ubiquitin are very 
striking and new to this family of deubiquitinases. The work is of high quality and will be of broad 
interest to researchers studying the ubiquitin system or host-pathogen interactions. Barring several 

comments outlined below, I find this manuscript to be well-suited for publication in Nature 
Communications. 

 

1) The authors seem to explain the lack of activity against di-ubiquitin chains as resulting from a 
requirement for longer (K63-linked) chains binding into the S2 site. If this is true, then why is OtDUB 
also more active against longer K48-linked chains, when based on the structure the S1-S2 
connectivity appears to be K63-specific? 

2) On a related note, why is it that K48 chains can be cleaved to produce monoUb, while K63 chains 
cannot (see Fig. 1e-f)? And why is the enzyme active against the simple Ub-AMC substrate? 

3) Considering the above comments, I am curious if the active site (S1’-S1 connection) is 
incompatible with K63-linked chains, while other linkages (e.g. K48 or Ub-AMC) can be cleaved. Have 
the authors considered the possibility that OtDUB targets hybrid chains? 

4) In Figure 4, the authors present mutations along one face of VR-1 that target the S1 site. These 
mutations appear to affect cleavage of K48 chains but not K63 chains, presumably because the 
effect on K63-linked chains is masked by the additional S2 interaction. Can the authors support this 
with a Ub-AMC assay? And possibly with S1-site mutations in the VR-2 region as well? 

5) Given the disorder-order transition observed in VR-1 upon Ub binding (Fig. 5f), I am surprised that 
blocking this interaction with the V203D mutation does not maintain the disordered state enough to 
affect Ub binding to the S1 site and thus DUB activity (Fig. S4a). By eye this mutant might be slightly 
slower than the WT enzyme. Quantitation of this assay and/or kinetic analysis with the Ub-AMC 
substrate could be more telling and possibly indicate a role for the OtDUB VR-1 UBD in activating 
DUB function (akin to the interesting regulation of OTUB1 in humans). 

 

Minor comments: 

1) The OcDUB appears to be more active against di-ubiquitin substrates. Have the authors 
considered performing the full di-ubiquitin cleavage panel with this enzyme to profile specificity 
across the active site? 

2) Without experimentation, claims that the UBD is specific for mono-Ub over chains are perhaps 
overstated. 

3) In future assays, the “time zero” samples would be more representative if the individual reaction 
components were mixed directly into SDS sample buffer. 

4) The authors might consider discussing the tUI ubiquitin sensor published by the Cohen lab as a 
higher affinity (though artificial) ubiquitin binder. 

5) Please include CC1/2 values in Table 1, or otherwise justify why such an aggressive I/sigI cutoff 
was acceptable. 

6) Why did the authors choose to not use the higher resolution native dataset for apo OtDUB? 

 

Sincerely, 

Jonathan Pruneda 

 

 

Reviewer #3 (Remarks to the Author): 

 

Berk et al. reported the structural characterization and ubiquitin chain linkage specificity of a 
deubiquitylase from Orientia tsutsugamushi. The crystal structures of the DUB domain of OtDUB 
alone and bound to three molecules of ubiquitin were presented. The structure of OtDUB reveals a 
similar core fold of CE clan proteases with distinct variable regions that are involved in ubiquitin 
binding. Surprisingly when co-crystallizing OtDUB with free ubiquitin, there were three ubiquitin 
bound in the structure. One bound at the S1 site, one at the S2 site and the other bound to a unique 
backside surface of the C-terminal region of OtDUB which the authors referred to as the ubiquitin-
binding domain (UBD). Interestingly the C-terminus of ubiquitin at S2 is in proximity of K63 of 
ubiquitin at S1 leading to the hypothesis of K63-ubiquitin chain (n>3) recognition. The authors 
showed that OtDUB has preference for cleaving longer K63 ubiquitin chain (n>2) and does not cleave 
di-ubiquitin. Likewise it also cleaves K48-ubiquitin chain. The structure explains a model for how 
OtDUB could bind a tri-K63-ubiquitin. Mutation in the S2 binding site reduced cleavage of K63-tetra-
ubiquitin but had little effect on K48-tetra-ubiquitin. While it is unclear how OtDUB recognizes K48-
ubiquitin chain, the structure showed that K48 of S1 ubiquitin is accessible for conjugation with 
ubiquitin. Lastly the authors showed that the backside of UBD has a strong binding affinity for 
ubiquitin (Kd of 5 nM), which is two-orders of magnitude tighter than previously reported UBD-
ubiquitin interaction. 

 

This work reports a novel structure of OtDUB in complex with ubiquitin at S1 and S2 sites and 
explains how it recognizes K63-ubiquitin chain. In addition, it identifies a high-affinity UBD. These 
findings will be of interests to researchers in the ubiquitin field in understanding ubiquitin 
recognition and chain specificity. There are few concerns see below. 

 

Comments 

1. It is a bit confusing that in some assays 1-259 (Figure 3c, 4b, 6b, S4) was used and in others 1-311 
was used (Figure 1d-f, S1B). Do both constructs have similar activity? It seems that 1-259 was used 
when comparing with mutants. Please explain. 

 

2. This reviewer assumes that the same amount of OtDUB was used in the assay for WT and mutant. 
Please show a SDS-PAGE of OtDUB WT and mutants used in this study to illustrate their purity. 

 

3. In the supplementary text and supplementary Figure 1B, the authors showed that in the absence 
of added free ubiquitin, OtDUB failed to cleave K63-tri-ubiquitin completely due to product 
inhibition. Is free ubiquitin added in all assays in other figures? It seems that K63-tetra-ubiquitin (Fig. 
3b, 4b versus supplementary Figure 1B) is less susceptible to inhibition (by 10 min nearly all tetra-
ubiquitin is cleaved). Is this due to tri vs tetra-ubiquitin? Is this specific to K63 or the same for K48-
ubiquitin chain? 

 

The authors stated in the supplementary text that 1-177 (Fig. S1B) is more efficient in cleaving K63-
tetra-ubiquitin compared to 1-311 (Fig. 1E) suggesting that UBD could compete for ubiquitin binding 
thereby inhibiting the cleavage. First, these reactions were performed on separate western blots and 
it should be performed on the same gel to make such comparison. Second, Fig S4A showed that 
V203D abrogated UBD-ubiquitin interaction and exhibited similar activity as WT. If UBD has an 
inhibitory role one would expect V203D to be more active. Lastly, Fig.4A right panel showed that VR-
1 makes extensive contact with S1 ubiquitin and 1-177 construct lacks this whole surface. One would 
predict to have a more severe defect than the point mutations in Fig. 4B and could impact on the 
cleavage of K63 chain. Please explain. 

 

It would be useful to speculate the role of UBD-ubiquitin interaction in the discussion. 

 

4. In Figure 3C, it seems that K63-tetra-ubiquitin is cleaved to di-ubiquitin whereas K48-tetra-
ubiquitin is cleaved to mono-ubiquitin. Please discuss this difference. Also in Figure S1B, K48-di-
ubiquitin was not cleaved at all, but when tetra-ubiquitin was used even di-ubiquitin was cleaved. 
Please explain. 

 

5. Figure 1D legend missing. Please specify the concentration of Ubl-AMC or 1X 

 

6. p.16 top “Instead the backside of the UBD domain (VR-1) is necessary for optimal K48-chain 
cleavage (Figure 4B and 4C)”. Throughout the manuscript backside was referred to UBD-ubiquitin 
interaction. Please revise to S1 binding site.