Peer review of: Tetrameric architecture of an active phenol-bound form of the AAA+ transcriptional (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

In this manuscript, Park et al. study the oligomerization of DmpR, a transcriptional enhancer that 
regulates response to aromatic compounds in Pseudomonas sp. The authors use a battery of 
biochemical, structural and biophysical approaches to demonstrate that DmpR protein forms 
tetramers. The authors show that the tetrameric assembly is required for ATPase activity and 
binding to the effector protein, sigma54. The manuscript is well written and the data are 
convincing. I only have a few minor comments that may strengthen the manuscript. 

1. Although the blue native gels provide evidence for protein oligomerization, the single-molecule 
data presented may benefit from presenting a positive control. What would be the expected 
distribution for photobleaching steps from a pure tetrameric protein? Can the authors deconvolve 
the photobleaching data and recapitulate the distribution between dimer and tetrameric species 
observed in the native page assays? The authors should comment on how the data are classified 
as precipitates and what fraction of spots are in this category. Is it possible to remove precipitates 
by say, ultracentrifugation? 

2. Does treatment with DNase affect the oligomerization of full-length protein? Does the addition 
of the cognate DNA sequence affect oligomerization status? 

3. Data in Fig. 4b suggests that there is an approx. 400kDa species on blue native gels. The 
authors should comment on this data in the text. Is a similar species also observed with wt DmpR? 

4. Why is the photobleaching data presented in Fig. 6g different from Fig. 1? Specifically, why does 
immobilization with anti-GFP result in a substantially high fraction of 3-step photobleaching events 
as compared to immobilization with anti-His antibody? 

Ankur Jain 

Reviewer #2 (Remarks to the Author): 

In this manuscript the authors report the interesting observation that phenol-bound DmpR forms a 
tetramer and DmpR interacted with the N-terminal peptide of σ54 only in the presence of phenol 
and ATPγS. This indicates that DmpR-like bEBPs tetramers activate σ54-dependent transcription 
by using a mechanism distinct from that of hexameric AAA+ ATPases. 
Overall, this manuscript is interesting. However there are many important problems remaining to 
be addressed: 
1. The lack of phenol-free DmpR structure makes structural comparison of DmpR with and without 
phenol binding impossible. If the crystallization is not successful the authors should perform some 
more experiments to provide more information about the conformational change induced by 
phenol binding. 
2. P4, L20. Is there an equilibrium existing between DmpR dimer and tetramer? Moreover, the 
supplementary figure 1A and 1B are not consistent. There 1B shows much more tetramer than 1A 
does even both have phenol. In figure 1A, although more tetramer form in the presence of phenol 
the effect is not very significant. 
3. P5, L4. The presence of a tetrameric subpopulation before the addition of phenol presumably 
resulted from the binding of aromatic metabolites derived from E. coli. Is there any solid evidence 
that support this statement? Did the authors try to purify phenol-free DmpR to confirm this 
argument? 
4. P5, L6-22. Monomeric DmpR is observed. Does usage of the antibiody affects the 
oligomerization of protein? 
5. P6, L6. What is the stoichiometry between DmpR and phenol since this protein shift from dimer 

to tetramer during the titration? 
6. P7, L11. The binding specificity of phenol should be analyzed. Is phenol the best ligand for 
DmpR? 
7. P8, L12. “ANP-PNP”, “AMP-PNP”? How about the KD value？
8. P9, L6. Does the interaction between DmpR and σ54 has an effect on the ATPase activity? 
9. P11, L9-11. The stoichiometry of the ITC indicates a 1:1 ratio. However Supplementary Figure 
7b indicates too weak a band for σ54. 
10. The authors did not provide data showing how phenol affects DNA binding ability of DmpR. 
This could be important since transcription depend on DNA binding. 
11. The resolution of DmpR is low (3.422Å). The quality of the structure could be questionable.