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

Peer Review File - Reviewers' comments first round: 
 
Reviewer #1 (Remarks to the Author): 
 
BACKGROUND FOR REVIEW 
 
I have previously reviewed a former version of this manuscript by Kumar et al. submitted to 
another Nature journal. In my review of the present version of the manuscript, I have both 
reviewed the manuscripts in its present form and considered the changes introduced to the 
manuscript and the authors’ rebuttal in response to my review of the former version of the 
manuscript. Comments or criticism of the previous version of the manuscript that I in this review 
have assessed to have been satisfactorily addressed by the authors are not mentioned in this 
review. I have performed this review and assessment of the manuscript with its submission to 
Nature Communications in mind. 
 
REVIEW 
 
The manuscript by Kumar et al. presents the determination of structures of the alpha1 glycine 
receptor in resting (GlyR-Apo), open (GlyR-Gly/PTX) and desensitized (GlyR-Gly) conformations 
using cryo-EM. The cryo-EM structures (resolutions 3.3-3.6Å) are of the full-length GlyR 
reconstituted in lipid nanodiscs, and the functional states of the structures are assigned and 
validated by molecular dynamics simulations. Based on the structures and analysis of them, the 
authors present the overall structure of the pentameric complex, the molecular compositions of 
specific regions/domains (the binding sites of Gly and PTX, the ion pore, the ICD, the ECD/TMD 
interface and the TMD, with particular focus on intermolecular interactions formed by M4 residues) 
and the global conformational changes in the receptor complex associated with activation and 
desensitization. The authors also present GlyR-Gly/ivermectin structures (non-conducting) and 
propose that these structures are intermediate to the open and desensitized GlyR structures. While 
numerous structures of GlyRs and other pLGICs have been published over the years, of particular 
importance for this work is that the authors through the use of full-length receptor and lipid 
nanodiscs have obtained an “open” conformation of the receptor (GlyR-Gly/PTX) that is more 
believable than the “super-open” conformation previously published. 
 
The data presented in the manuscript is original and novel, and the analysis of the available 
structures of resting, open and desensitized conformations does facilitate an in-depth insight into 
the conformational transitions of the GlyR and the molecular mechanisms underlying activation 
and desensitization. However, much of the information gained from this study is more or less 
obtainable from previous GlyR structures or from previous studies delineating allosteric transitions 
between different receptor conformations for other pLGICs. This is not a criticism of the performed 
work in this study, and the obtained structures certainly will be of interest, but as a reader one is 
perhaps too often left with the impression that this is verification of previous findings rather than 
true new insights into GlyRs and pLGICs. 
 
As for the techniques and methodologies applied to address the research questions in this work, 
the structure determinations/analysis and molecular dynamics simulations performed are 
advanced and appropriate for this kind of work. The generated data is of high quality, and data 
analysis and statistical analysis appear to have been done consistently and correctly. However, 
with the exception of a few simple TEVC recordings verifying the functional properties of glycine 
and picrotoxin as an agonist and a channel blocker, respectively, at GlyR expressed in oocytes, no 
electrophysiological studies of observations made from the structures have been performed. As 
outlined specifically below, this lack of validation/verification of functional implications of observed 
intermolecular interactions represents a flaw in this nice study. 
 
The research questions, the rational for the experimental design applied, the generated data and 
the interpretation of data are largely presented in a logical and precise manner in the manuscript, 
where appropriate credit is given to previous work in the field. 
 
CONCLUSION 
 
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. 
 
When it comes to my three major concerns about the former version of this manuscript submitted 
to another Nature journal, two of these have been satisfactorily addressed by the authors in the 
present manuscript submitted to Nature Communications: 
 
1) While it is still this reviewers’ opinion that considerable parts of the information provided by this 
work are extractable from previous structural biology studies of GlyR and other pLGICs, the work 
does provide novel and interesting findings about an important neurotransmitter receptor and 
receptor class. Thus, as for this criteria I think it warrants publication in Nature Communications. 
 
2) The authors make a much stronger case for the GlyR-Gly/PTX structure being a valid surrogate 
structure for the open conformation of GlyR than in the former version of the manuscript, although 
I think it is very important that they include their arguments for this explicitly in the manuscript. 
 
3) My concern about the lack of functional studies validating/verifying observations made in the 
structures has not been addressed in the present version of the manuscript. As argued in the 
specific comment below, I do believe that the arguments by the authors against this concern are 
particularly valid. I think that this lack of validation/verification of these structural details is highly 
unfortunate since it renders these potentially interesting findings much more vague and non-
conclusive than they could – and should - be. Moreover, on this foundation the authors risk that 
their suggestion of these molecular interactions being important for GlyR function eventually could 
be contradicted by subsequent studies. 
 
All in all, 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. 
 
 
SPECIFIC COMMENTS 
 
“Abstract” 
 
• line 6: maybe “GlyR protein complex” instead of “GlyR protein”? 
• line 12: maybe “single-channel conductance properties”? 
 
“Introduction” 
 
• p.2, line 1 and 5-6: The abbreviations of receptor should be uniform. The authors have three 
different abbreviation styles: “glycine receptors (GlyRs)”, “GABAA receptors (GABAA)”, “nicotinic 
acetylcholine receptors (nAChR)” and “5HT3 receptors (5HT3R)”. Be consistant about including an 
“R” and a plural “s” for “receptors” in the abbreviations. 
• P.2, line 18: it should be “channel” instead of “channels”. 
 
“Results and Discussion” 
 
• P.5, first paragraph, P.10-11, p.15-17: One of my principle concerns regarding the manuscript in 
its former version was the use of a Gly- and PTX-bound structure to reflect the open conformation 
of GlyR. While I understood the rational for this approach and the need to obtain the channel 
blocker-bound structure to access the putative open conformation, the authors was asked to argue 
for the validity of the structure for this purpose. In their rebuttal, the authors convincingly argue 
for this point, including outlining the essentially identical 9’, 6’ and 2’ regions in the GlyR-Gly and 
GlyR-Gly/PTX structure, and they have included new GlyR-ivermectin structures that are proposed 
to represent intermediate states between the open and the desensitized GlyR. PTX is observed not 
to induce changes in its binding region and the authors conclude that it acts allosterically on the 
intracellular P-2’ gate and prevents it from closing. In this reviewers’ opinion, this still raises the 
question whether this intracellular gate in its open conformation in the GlyR-Gly/PTX structure has 
to be identical to that of the “true” open GlyR conformation. In the “true” open GlyR, the channel 
is open because the collapse of the channel in the course of the desensitization process has not 
happened yet, in GlyR-Gly/PTX structure the channel is open because the bound ligand prevents 
this collapse. While I fully agree with the authors that structural arrangement of also this part in 
the GlyR-Gly/PTX structure very likely is a good representation of it in the “true” open GlyR, I do 
not think one can be absolutely sure. 
 
In conclusion, I find the authors’ explanations/arguments and the added experimentation to be 
very solid, and thus the authors have been very responsive to this comment. The only thing I 
would ask for here is that the good arguments provided for the Gly/PTX-GlyR structure as the 
“open” conformation in the rebuttal also are presented in their discussion of the structure in the 
manuscript text. I think it would be important to convey the use of this structure as a “surrogate” 
for the open conformation and to substantiate the validity of this use as such to the reader, and 
when the authors have so strong arguments as they have, it only strengthens their case for this 
claim. 
 
• p.5, lines 6-12: It would be good with a reference to Supplemental Figure 1 in this segment with 
the effects of glycine and picrotoxin are outlines. It comes later in the paragraph but then it is a 
reference for the strategy for the structures. 
 
• General comment: Picrotoxin is an equimolar mixture of picrotonin and picrotoxinin. The authors 
appear to have used the latter compound in for example Fig. 2D-E, which is fine since it is the 
more potent GlyR inhibitor. It is also fine to refer to the structures at picrotoxin, but somewhere in 
the manuscript there should be a mentioning of this, since there strictly speaking is no such thing 
as a picrotoxin molecule. 
 
• p.6, 3rd line from bottom: It should be “pLGICs” 
 
• p. 12, top segment & p. 14, bottom segment: The Å2 values should be given without the space 
between the first two and the last three digits. 
 
• p.12 bottom half – p. 13, lines 1-6 & Fig. 4: Another principle concerns regarding the manuscript 
in its former version was the lack of investigations of the functional importance of any of the many 
interactions between residues identified in specific regions of the GlyR structures by 
electrophysiological recordings (interactions shown in Fig. 4A-B in this manuscript, and in Fig. 5 in 
the former version). The authors have not included any data on this in the present version of the 
manuscript. 
 
The authors argue in their rebuttal that they have identified an extensive network of interactions 
at interdomain interfaces that are altered between different conformational states, and that they in 
the present version of the manuscript have highlighted residues previously investigated in 
functional studies of the hyperekplexia-causing mutations and included references to these 
studies. I certainly acknowledge that, but these additions are all given with regard to the ECD-ICD 
cross-interactions shown in Fig. 3, and not for those interactions that my comment was regarding, 
those formed residues in post-M4 to beta8/9- and pre-M1-residues (presented in Fig. 4A in the 
present manuscript) and those formed between M4-preM4 residues and lipid molecules (presented 
in Fig. 4B in the present manuscript). The authors do not themselves validate or present previous 
studies validating the functional implications of these interactions. In the case of the latter 
interactions, they state that these investigations are ongoing but argue that the results from these 
can not be included in the manuscript due to its length. 
 
In my opinion, this segment and the findings conveyed in Figs. 4A-B constitutes the weakest part 
of this nice study, and I would argue that electrophysiological verification of selected observations 
made in ones structures are to be expected. In their rebuttal the authors claim that detailed 
functional analysis is needed to investigate these functional implications, and while that may be 
true the authors could come a long way by studying the functional properties of mutants 
comprising mutation of the 6 or more residues (such as Asp218, Arg220, Glu 244, Arg437, 
Gln444, Arg406) identified to form these interactions. As perhaps best evidenced by the sentence 
immediately before this segment (p.12, lines 8-11), where the authors reference previous studies 
to substantiate that the ECD-ICD cross-interaction have substantial functional implications, such as 
electrophysiological validation/exploration of the actual functional implications of these identified 
interactions would bring a whole other level of impact to these findings. The lack of such 
investigation makes the present findings purely observational and in effect renders the author 
unable to do anything beyond hand-waving when it comes the functional importance of these 
interactions. 
 
• P.13, bottom third – P. 15, line 4, P. 17, Fig. 5 and Supp Fig 8-10: In this version of their 
manuscript the authors have included structures of ivermectin/Gly-bound GlyR structures, which in 
MD experiments are found to be non-conducting. In addition to the comparison of the basic 
features of these structures to previously published IVM-GlyR structures (the authors need to 
reference these structures and studies in this section) and to the Gly-GlyR and Gly/PTX-GlyR 
structures, the authors interestingly find a difference in the Pro-2’ side-chains in these structures 
compared to Gly-GlyR and Gly/PTX-GlyR and propose that these structures could represent 
intermediate states to the desensitized and “open” conformations. These added structures and the 
analysis of them add to the impact of the study – and, specifically, also helps building the case for 
the validity of the Gly/PTX-GlyR structure for the “open” GlyR conformation. 
 
 
Anders A. Jensen 
 
 
Reviewer #2 (Remarks to the Author): 
 
Kumar et al. present new structures of GlyR in lipid nanodiscs obtained in the presence of different 
ligands. Comparing the structures, the authors observe conformational changes in the protein and 
interpret them as representing functional gating dynamics, also complementary analyzed using MD 
simulations. While the study is important and interesting, there are some issues for authors to 
consider. 
 
Starting from the title, the manuscript intends to oversale the significance of the protein 
preparation in nanodiscs. Nanodiscs are not membranes and there is no proof that they are any 
better for capturing membrane proteins in conformations more physiological than using 
detergents, amphipols or SMAs. Referencing one paper, even if it is published in Nature (Laverty et 
al., 2019), does not make the opinion about lipid nanodiscs as a mimic of natural lipid 
environment “generally accepted”. The title therefore should be changed to refer to “glycine 
receptor in lipid nanodiscs”. Similarly, “in a physiological environment” should be removed from 
the last sentence of the Abstract. 
 
The quality of models should be significantly improved. Not only the side chains do not often fit the 
density (very apparent for the bulky side chains of Arg, Lys and aromatic residues) but even the 
backbone is often traced incorrectly. For many regions of the models, maps are extremely weak or 
missing, especially for the extracellular or intracellular ends of the peripheral transmembrane 
helices. Precision of models in these regions must be critical for estimating intra-subunit and inter-
subunit cavities illustrated in Fig. 4C. Similarly, many of the modelled sugars are not supported by 
the densities or show extremely poor fit. It is understandable that there were probably more 
complete structures available from before but for each of these new presented structures, the 
models need to be trimmed so that they only contain what is visible in density. 
 
There are lipid molecules fitted into non-protein densities surrounding the TM region. The quality 
of these densities is way too poor to unambiguously fit any lipid or detergent molecules and surely 
not good enough to specify the type of lipid/detergent. The corresponding models of lipids have to 
be removed from the corresponding PDBs. If authors want to illustrate the non-protein densities 
surrounding the TM region, they can show them in cryo-EM maps (alongside the protein density at 
the same contour level) and call “lipid-like” densities. 
 
Glycine molecule in the ligand binding pocket fits a shapeless blob of density. The fit of glycine 
molecule is therefore based on the chemical environment of the binding site. While some of glycine 
molecules are fitted with the carboxyl group of glycine facing R89 as anyone would expect, others 
are flipped 180 degrees with the carboxyl group facing away. Is there experimental evidence for 
such drastically different placement? 
 
What is considered the density for PTX is at the level of noise and cannot be seriously considered a 
reason for placing the molecule of PTX at the corresponding location. The authors might think of 
repeating the corresponding experiment, may be at a high PTX concentration, in order to convince 
the reader that the corresponding binding site is real. After PTX is modeled correctly, it will be 
worth making side-by-side comparisons with GluCl, compared to which the current binding site is 
much higher, and GABAA, where the location is also a bit different. 
 
The Aricescu GABAA receptor pore in the presence of PTX adopts a conformation essentially 
identical to the resting state, which is quite different from that observed here with the GlyR. Many 
studies have been performed with PTX on glycine receptors; it would be helpful to cite some of 
them. In Suppl. Fig. 1, if PTX blocks desensitization as stated in the text, why is the channel more 
desensitized after PTX application than before? 
 
The issue of the previously reported widely open state has become quite contentious with many 
groups working to resolve this question. There are conflicts even between the computational 
groups. It is important to not overstate the strength of the data in either direction to help get to 
the right answer eventually. In the Introduction, the authors suggest that truncation of the ICD 
may explain why the Gouaux open state from 2015 was so wide and they cite two papers they 
suggest support this idea: “The ICD, besides its role in trafficking, has also been implicated to alter 
single-channel conductance and gating behavior (refs. 3, 25).” However, these references have 
nothing to do with the ICD. Is there actually evidence that the truncation causes any sort of 
functional behavior change in anionic pLGICs? 
 
In single particle processing for the open state, did the authors find and exclude 3D classes with 
widely open pores? 
 
The more than 4-fold difference in conductance predicted by MD simulations (20 pS) versus the 
experiment (80-88 pS, ref. 60) is explained by missing residues in the ECD (page 16). However, 
the most significant effect of mutating the basic cluster of residues in the ECD was only a change 
from 92 to 60 pS (ref. 61). Please expand a bit the discussion on computational conductance 
measurements vs. those from single-channel experiments. How good now is computational 
electrophysiology at predicting/nailing down real physiological values? Is it possible for MD to 
make any realistic predictions, when positional restraints are placed on the protein backbone 
during the simulations (page 15), while the transmembrane potential is +500 mV and the sodium 
concentration is 500 mM (page 16), which are so much different from physiological values? 
 
Minor: 
 
Page 3, third line from the bottom: “preformed” should be removed. 
 
Page 5, first sentence. The sentence should be rewritten: authors would like to believe that the 
conditions of their experiment favor physiological conditions but they cannot be so sure of this. 
 
Page 10, last line. There is no panel D in Figure 3. 
 
Why are the initial particle numbers approximate in Suppl. Table 1? 
 
Are the map sharpening B factors correct? They seem very small (in absolute value). 
 
 

Reviewer #3 (Remarks to the Author): 
 
In this work, structural basis for gating, ion selectivity, and single-channel conductance of GlyR in 
membrane was investigated based on the cryo-EM structures and molecular dynamics simulations. 
I particularly evaluate molecular dynamics simulation parts of this work. 
The molecular dynamics simulations were performed with standard computational technique. The 
comparison between the simulations and the cryo-EM structures is scientifically meaningful. 
However, there are a number of uncertain points for this reviewer, which should be clarified in a 
revised manuscript. 
 
Main Comments: 
1) In Page 12, the authors suggested that the interactions between 
subunits were changed, and the overall buried surface area was decreased 
in the GlyR-Gly/PTX and GlyR-Gly. However, relationship between 
interaction change and surface area change is not so clearly described. 
Why did the authors quantify the overall surface area change? And also, 
what is the "buried surface" area? 
 
2) In Page 17, the authors described that the pore features of the three 
conformations were stably maintained in the MD simulations without 
positional restraints. Is the pore still dewetted at Leu9’ in Gly-Apo or 
wetted in GlyR-Gly/PTX and GlyR-Gly, as shown in Figure 6C? 
 
Minor comments: 
1) Typo: In Pages 10 and 11, Fig3D and Fig 3E should be Fig 2D and 
Fig2E. There is no explanation about Fig2F in the main text. 
 
2) Panels in all figures are very busy. For example, there is a clash 
between Panels A and B in Figure 1. Position of all panels should be 
rearranged well. 
 
3) It is difficult to recognize some labels in the figures (e.g., Figure 
2C, Panel A (right) in Figure 3, and Figure 4)