Peer review of: Mechanisms of activation and desensitization of full-length glycine receptor in lipid nanodiscs (2020)


RR2
REVIEWERS' COMMENTS second round: Reviewer #1 (Remarks to the Author):
  In their revised manuscript Kumar et al. have addressed most of my concerns from the reviews of
the two previous versions of the manuscript that I have reviewed.
As for my bigger concerns and changes implemented in response to them:
  • The authors have now included mentioning and justification of the approach of using the
 Gly/PTX-GlyR structure as a surrogate for the open GlyR concentration, so that the reader now is
 made aware of this issue and can explore and use this particular structure while keeping this in
 mind.
 • The inclusion of the ivermectin-structures in the first version of the manuscript submitted to Nat.
 Comm. represents a substantial contribution to the study, as they add more information about the
 conformational transitions of the GlyR and potentially also other pLGICs.
 • Although some of the conclusions drawn by the authors based on this study mainly confirm and
 substantiate findings already extractable from previous GlyR and pLGIC structures, the study does
 introduce novel information and add to the knowledge about the closed, open and desensitized
 conformations and the transitions between them.
 • When it comes to addressing my concern about the lack of functional verification of the putative
 interactions between specific residues depicted in the former Figs. 4A and 4B that potentially could
 be of functional importance, the authors have been less responsive:
 As also mentioned in their rebuttal to my comments to a former version of this manuscript, the
 authors point out that they include references to previous studies showing the functional
 importance of residues forming network of interactions in the ECD/TMD interface. However, this
 network is shown in Fig. 3, and not in Fig. 4A and 4B which was the figures my comment was
 aimed at. In their rebuttal to this version of the manuscript the authors acknowledge (at least I
 interpret their comment to mean that that) that further functional characterization is needed in
 order to assess the functional importance of the interactions previously depicted in Fig. 4A and 4B,
 and they have also revised the two figures so that these putative interactions are now longer
 shown in them. I acknowledge that this means that the authors no longer claim or hint to these
 interactions being of functional importance without having investigated (and substantiated) this.
 While this certainly is a good thing and this version of the manuscript thus is improved on this
 account compared to the former version, I can not help feeling that the authors are missing a
 golden opportunity here: to dive further into and use this information provided by their structures
 and through functional studies translate a potentially interesting observation into solid conclusions
 about the specific molecular basis underlying the conformational transitions of the GlyR. In their
 rebuttal to a previous version of this manuscript, the authors argued that such an analysis would
 be very elaborate, but I would argue that a fairly focused functional characterization of a small
 series of selected GlyR mutants could provide solid experimental data to either substantiate or
 disprove a significant functional importance of these interactions.
 In conclusion, when it comes to this issue, the authors are no longer vaguely hinting to a possible
 functional importance of these interactions in the manuscript, but they are not “breathing life” into
 their structures by investigating the putative importance of these either.
 As I also stated as my overall conclusion based on review of the former version of this manuscript,
 this is a very nice study that provides high-quality structures of the GlyR in its three major
 conformations as well as additional proposed intermediate conformations using advanced cryo-EM
 and molecular dynamics techniques. Thus, my overall assessment of the work is that this is a
 study that will be of considerable interest to the GlyR and pLGIC fields and to the neuroscience and
 structural biology fields in general.
 Anders A. Jensen

 Reviewer #2 (Remarks to the Author):
Reviewer #3 (Remarks to the Author):
 I think authors did good job in addressing all of the critiques and the manuscript is now acceptable
 for publication in Nature Communications.
  This is my second review on the work. In the previous review, I pointed out two major concerns
 and three minor ones. In their revised manuscript, they answered my questions and suggestions
 clearly so that I don't have any more. I basically agree the publication of this work.
 Only one concern is that in their revised manuscript, they mentioned that "We have removed the
 sections for unrestrained simulations from the current version". I don't understand the reason why
 they have removed them although in the previous manuscript they mentioned that the pore
 features of the three conformations were stably maintained in the MD simulations without
 positional restraints. I guess that the resolution of this model might not be enough for
 unrestrained MD simulations. If so, I would like to suggest the authors to add one sentence for the
 reliability of unrestrained MD simulation based on the experimental data, if they could in future
 revision or proof.