Peer review of: Legionella effector MavC targets the Ube2N~Ub conjugate for noncanonical ubiquitination (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

In this paper, Puvar K. et al. reported an interesting discovery that the Legionella effector MavC 
targets the activated, thioester-linked Ube2N~Ub conjugate as the real substrate, not free Ub or 
Ube2N as previous studies reported, to catalyze an intramolecular transglutamination reaction and 
covalently crosslink the Ube2N and Ub subunits for noncanonical ubiquitination. They presented a 
series of biochemical data and four complex structures of MavC with Ube2N and Ub. The structures 
clearly uncovered the molecular basis for recognition of the Ube2N~Ub conjugate and uncovered 
the catalytic mechanism with protein dynamics in the transglutamination reaction. The whole 
manuscript is well-written and should be published in Nature Communication after revision. 

I have the following concerns: 
1, In the Table S1 for structure determination, the I/sigma I value of the last shell in MavC1-
384_Ub-SS-UbE2N were much less than 2. The authors should cut it to be more than 1.5 or 2 and 
re-refine the structure. 
2, In Figure 4C, the authors should analyze the effects of the mutants of MavC on the 
transglutamination reaction, not on the deamindation reaction. 
3, In Figure 5D, the authors did not show the effect of Tyr47 of MavC. This mutant should be 
included. In addition, the role of Tyr47 in substrate recognition or structural stability of MavC 
should be presented in a structural figure. 
4, The data in Figure 5E is not consistent fully with those in Figure 5F. The YYE mutant still inhibits 
NF-kB signaling. The author should test the effects on NF-kB activation by TRAF6 overexpression. 
5, All mutants of the Ub- and Ube2-binding residues of MavC (in Figures 4 and 5) should be test 
for their effects on the transglutamination reaction in vivo. The completely inactive mutants of the 
binding residues of MavC also need be tested during infection. 
6, In Figure 7A, the author tested the effect of adding Uev1 on the transglutamination reaction. 
Uev1A did not significantly affectted the modification. But the authors need to test the effect of the 
crosslinking of Ube2N-Ub on the interactions between Ube2N and Uev1A for further confirming 
their conclusion. 

Reviewer #2 (Remarks to the Author): 

In their manuscript, Puvar et al. explore the structural basis of MavC catalyzed ubiquitination of 
the Ub conjugating enzyme Ube2N. In two previous studies, MavC has been reported to act as a 
transglutaminase that catalyzes isopeptide bond formation between the Ub Q40 and Ube2N K92 
side chains without requiring Ub activation by a canonical E1 enzyme or ATP. Instead, MavC 
deamidates Ub Q40 to form a thioester intermediate with the MavC catalytic Cys prior to reacting 
with the Ube2N K92 amine. 

In this study, the authors identified the Ube2N~Ub thioester as primary MavC substrate. The 
authors determined crystal structures of a Ube2N~Ub G76C disulfide as a thioester mimic and the 
isopeptide linked Ube2N-Ub in complex with a catalytically incompetent MavC C74A mutant. The 
structures of the substrate and product bound states of MavC offer valuable snapshots of 
conformational changes in MavC and Ube2N along the reaction trajectory and are validated with 
biochemical assays. 

The manuscript is an insightful contribution to our understanding of how bacterial effectors target 
Ub signaling pathways and elucidates how MavC favors Ub transglutamination over deamidation 
activity. Overall, the paper is based on solid experimental data that support the authors’ 
conclusions. However, the findings reported in the last subsection of the manuscript seem 
somewhat preliminary. Moreover, the interpretation of the structures could be more detailed and 

many figure legends should be considerably more explicit in order for this study to be suitable for 
publication in Nature Communications. 

Major issues: 
- The affinities of MavC to Ube2N alone and the Ube2N-Ub thioester mimic are virtually identical 
(Fig. 2a). However, the Ube2N-thioester linked Ub interacts with MavC in the crystal structures 
and in the NMR binding studies. The Ub binding surface should contribute to the Kd of the Ube2N-
SS-Ub:MavC complex. How do the authors explain this apparent discrepancy? 

- In previous studies, free Ub and even a Ub G75A/G76A mutant that cannot form a thioester 
intermediate with Ube2N were shown to be linked to Ube2N by MavC. The authors should discuss 
these results. 

- The quality of the Ube2N-thioester linked Ub 15N spectra in Fig. 2d is quite poor and contains 
many signals with low intensities. Are these due to chemical exchange or due to impurities or 
degradation of the sample? It would be helpful if the authors showed an overlay of the 15N spectra 
of free Ub with the Ube2N-SS-linked Ub in the Supplement and labeled the Ub resonances. Do the 
CSPs in Fig. 2d agree with the Ub binding surfaces of the crystal structures? 

- Fig. 2e: The high concentration of DTT (1 mM) in the reaction buffer compared to the Ube2N~Ub 
disulfide (25 uM) should reduce the disulfide to free Ube2N and Ub. The authors should show 
loading controls in the absence of MavC for each time point to demonstrate the integrity of the 
Ube2N~Ub disulfide during the reaction. 

- Fig. 5e,f: Although the YYE mutant is virtually incapable of performing transglutamination, it still 
effectively down-regulates NF-kB compared to the C74A mutant. The authors should discuss this 
result. Fig. 5f: The MavC gel (six lanes) does not match with the Tubulin gel (seven lanes). What 
does the bar below the bar chart refer to? Significance should be tested in the bar chart and the 
gels should be labeled. The methods for measuring NF-κB activity should be described in more 
detail. The error bars and number of experiments are not described. 

- The paper falls short of analyzing the conformation of the Ube2N~Ub thioester and Ube2N-Ub 
complex. The authors should compare / superimpose their structures with known structures of 
Ube2N (or Ubc13)~Ub conjugates. Would E3 binding clash with the MavC interaction? A more 
thorough analysis of the Ube2N-Ub isopeptide would be helpful to understand the mechanism 
underlying E2 inhibition and NF-κB inactivation. Does the structure of the product complex provide 
any cues in this direction? A CSP analysis of <sup>15</sup>N Ub isopeptide linked to Ube2N 
might be useful in this respect. 

- The subsection describing the role of Uev1a is somewhat preliminary. The authors should 
therefore move Fig. 7a-b to the Supplement and substantiate their speculations by adding a 
structural model of the Ube2N-Ub:MavC complex including Uev1a. The units in Fig. 7a for the time 
points are missing. 

- Fig. S5 nicely summarizes the structural data. I would therefore recommend replacing Fig. 7a,b 
with Fig. S5. The Ube2N C87 and K92, Ub Q40 and MavC C74 side chains should be labeled. The 
isopeptide linked Ube2N-Ub / product complex should be shown as well. It would also be helpful to 
include these cartoons on top of the structures shown in Fig. 6a-c. 

Minor issues: 
- The formatting of Greek letters and superscripts is missing / has been lost at several positions. A 
single font type should be used for all figures. 

- Throughout the text, the authors should make clear which catalytic Cys / thioester is referred to, 
Ube2N or MavC. 

- Ube2N is also referred to as Ube2N and UBE2N. The spelling should be consistent throughout the 
paper. Similarly, a space should always be included between values and their units. 

- Fig. S2: Fig. S2d: The 15N axis should be labeled, since it should be different from Fig. S2c 
(HSQC vs. TROSY spectrum). Fig. S2e and f: The corresponding NMR spectra should be shown. 
What is the CSP cut-off used in Fig. 2f? Fig. S2h: The units for the time points are missing. 

- Fig. S3: The numbering of the panels is not consistent with the text, e.g. Fig. S3e is mentioned 
in the text before Fig. S3a. Fig. S3a: The YYE mutant should be spelled out in the same way as the 
other mutants. Fig. S3c: The units for the time points are missing. Fig. S3e: The gel is not 
described in the legend. 

- Fig. 4c: The figure legend should be more explicit. How are the Ub species visualized? What does 
the “Ctrl” lane refer to? 

- Fig. S4d: The figure legend should be more explicit. “corresponds to maximum rmsd” of what? 

- Constructs and purification of E2 enzymes other than Ube2N and of Cif are not described. 

- Software used for multiple sequence alignments is not described.