Characterization of the dynamic resting state of a pentameric ligand-gated ion channel by cryo-electron microscopy and simulations

  1. U Rovšnik
  2. Y Zhuang
  3. L Axelsson
  4. BO Forsberg
  5. V Lim
  6. M Carroni
  7. C Blau
  8. RJ Howard
  9. E Lindahl

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

Ligand-gated ion channels are critical mediators of electrochemical signal transduction across evolution. Biophysical and pharmacological development in this family relies on high-quality structural data in multiple, subtly distinct functional states. However, structural data remain limited, particularly for the unliganded or resting state. Here we report cryo-electron microscopy structures of the Gloeobacter violaceus ligand-gated ion channel (GLIC) under resting and activating conditions (neutral and low pH). Parallel models were built either manually or using recently developed density-guided molecular simulations. The moderate resolution of resting-state reconstructions, particularly in the extracellular domain, was improved under activating conditions, enabling the visualization of residues at key subunit interfaces including loops B, C, F, and M2–M3. Combined with molecular dynamics simulations, the cryo-electron microscopy structures at different pH describe a heterogeneous population of closed channels, with activating conditions condensing the closed-channel energy landscape on a pathway towards gating.

Article activity feed

  1. eLife

    ###Reviewer #3:

    In their manuscript "Characterization of the dynamic resting state of a ligand-gated ion channel by cryo-electron microscopy and simulations", Rovšnik et al. describe a structural study of the GLIC ion channel under 3 pH conditions combining cryo-electron microscopy and molecular dynamics simulations. Their aim is to shed light on the resting state (neutral pH) structure of this ion channel, that has previously been described by a crystallographic study with intriguing observations. Although the authors do not really say so explicitly, it seems their interpretations of the new data largely confirm the conclusions of that previous work. This is a major point that needs to be made explicit: does their study confirm (and to what extent) the one by Delarue (ref [27]) and how similar are the structures. Here a comparison of the pH7 cryo-EM and x-ray density maps could be a welcome analysis. The important related question is: what new information (in terms of the ion channel function etc., not in terms of structure determination methodology) do we learn from this study compared to ref [27]? This should also be made more explicit and be implemented by taking into account intrinsic uncertainties in the study (see next paragraph).

    One concern - quite honestly raised by the authors themselves - is to what extent the cryo-EM maps obtained at ph3 and ph5 may represent the expected functional state, or incorporate some artefactual conformational substates, as they seem to lack a few key features of an open/active state that would be expected under these conditions. For the ph7 state as well, it cannot be excluded that the observed conformation bears some traits of desensitized or intermediate states, as is mentioned in the present manuscript. These overall uncertainties are somehow convoluted with the interpretation and analysis of the data, and in the present version of the manuscript it needs to be made clear much earlier that most of the interpretations only hold/make sense if one assumes certain hypotheses (eg that the pH7 structure is a resting one and not any of the other possibilities for instance, etc.), which otherwise is perfectly fine.

    The last major concern about the manuscript concerns the computer simulations. The protonation states adopted to represent activating or resting simulations are not explicitly given in the paper, nor the choices discussed and justified in any way, whereas this seems to be a rather controversial issue for the simulation of this particular pH-gated channel as literature attests, and obviously a central one with respect to the questions studied in the present work. Also, are there indications in the cryo-EM derived structures on specific protonation states (eg two acidic side chains very closeby may indicate at least one is deprotonated, etc.)? The next issue that has not been mentioned, but seems quite critical to assess whether activating simulations actually go the right way, is about the wetting/dewetting of the channel pore. Are they stably water-filled in any of the simulations? This is one of the metrics actually used in ref. [21] and a few of which have been adopted for the analysis in Fig. 5 of this paper. A more detailed comparison with that computational work seems rather commendable, as well as probing more of the metrics that are employed there. Also, the discussion of Fig. 5 results should be extended, as it is not clear how to interpret this important figure. Why were the simulations ordered as they are? And how consistent are the observed trends for ECD radius, twist and upper spread?

  2. eLife

    ###Reviewer #2:

    This article reports 3 new structures by cryo-EM of a bacterial pentameric ligand-gated ion channel (pLGIC) known as GLIC, in its resting form, at 3 different pH: pH 7, pH 5 and pH 3. The resolution extends from 4.1 Å for the first one and to 3.4-3.6 Å for the last two. Since GLIC is gated by protons, one should see at least two different forms, resting and active, at the various pHs. The main results are the following:

    1. The structure at pH 7 is in a resting state and is highly flexible

    2. It becomes much less flexible at pH 5 or pH 3, but the pore remains closed

    3. All three structures were obtained in detergent (not in nano-discs)

    In itself, this is a valuable article with a lot of new interesting information. However, I suggest to consider the 4 following points to improve the manuscript. In a nutshell, I see 3 main points in the analysis of the structures that should be addressed, plus a methodological issue.

    1. The fact that GLIC at pH 7 in its resting form is highly flexible was already known before this study and has been extensively documented in the article that describes the x-ray structure at 4.4 Å (Sauguet et al., 2014, Ref. 27) because the asymmetric unit of the crystal contains in fact 4 different pentamers in different conformations. This should be better discussed in the article, in particular in relation with Figure 4 of Ref 27, where the dynamical nature of the resting state is clearly mentioned.

    2. While the analysis of differences between GLIC structures at 3 different pH is well conducted, there is no detailed comparison with the other crystal structures of the same ion channel GLIC, which are listed in the manuscript (p. 2, line 27 to p. 3 line 6): the crystal structures of the resting state, the activated state, a locally-closed state and a possible desensitized state. One should expect at least a panel in a principal Figure of a detailed comparison between these structures. To understand the differences between the 3 structures presented here (pH 7, pH 5 and pH 3) and other known structures of GLIC, a projection of these 3 structures on various 2D maps should be presented using relevant variables (RMSD are rather useless here), along with representative structures of all other known forms of GLIC: the open form (4HFI), the 4 structures in 4NPQ and the locally closed form in 3TLT. See B. Lev et al, PNAS 2017 for such variables, in Figure 4 and 5 (ECD radius, beta expansion, M2-M1(-) distance, ECD twist).

    3. While it is surprising to observe that the pH 3 structure is still in a resting form, it is possible to interpret this as the left side of the minimalist reaction path of the allosteric transition that looks like this:

    pH 7 closed <-> open

    ^ ^

    | |

    v v

    pH 4 closed <-> open

    However, the reaction path of the gating transition is unlikely to be this simple. The dynamics of the gating transition in GLIC has been extensively studied in B. Lev et al., PNAS 2017 by long MD simulations and the string method. Unfortunately, this article is not cited in the present work, nor any detailed comparison of its conclusions with the proposed pathway presented in Figure 6A. In particular, Lev et al. insist on the role of the salt-bridge D32-R192, that gets broken to form another salt bridge D32-K248 in the open form. Do the 3 new GLIC structures solved in this new work confirm the importance of this salt bridge in driving the transition or not? In p. 6 the authors analyze specifically the conformations of the side-chain K248 but do not mention this possibility.

    1. Methodology (p. 10) The paper reports both a new and interesting method to refine models in cryo-EM maps using MD simulations with adaptive constraints and the resulting refined models. But the validation of the method itself on well documented test cases is missing (unless I missed something). In other words, there is some sort of a circular argument here: a new method is presented that allows good sampling and flexibility in the refinement under experimental constrains, but the justification is simply the output of the method, namely fitted -and flexible- models. While it is possible that the new method is superior to other extant and validated methods in speed, is it as accurate - or more?

    Specific comment on the Figures:

    Figure 1: The structure at pH 3 has (overall) a slightly higher local resolution than at pH 5. Any comment?

    Figure 2: Does K248 makes a salt bridge with D122 (Panel B)?

    Figure 4: Rmsd do not bring a lot of information. Could the authors map their structures, along with all other known GLIC structures, on 2D maps with essential parameters such as ECD twist angle, M2-M1(-) distances as in Figure 4 and Figure 5 in Lev et al., PNAS, 2017?

    Figure 5: Again Rmsd -and their distribution- plots do not bring a lot of information. Also,

    1. Which pentamer has been used for the pH 7 X-ray form? (there are 4 of them in the asymmetric unit). Would the result be different with a different pentamer?

    2. I strongly oppose the names of the so-called pH5 and pH3 cryo Activating forms: they are not Activating, but merely the same structures with different sets of electrostatic charges. This is misleading, the reader might think it is an experimental structure (cryo). Best if the words Resting and Activating are changed to Deprotonated and Protonated, respectively.

    Figure 6: Panel A should be compared and discussed with Figures 3 & 4 in Sauguet et al., PNAS 2014, as well as with the Discussion in Lev et al., PNAS 2017.

  3. eLife

    ###Reviewer #1:

    This manuscript reports cryo EM structures of the GLIC channel under resting (high pH), partially (pH 5) and fully (pH 3) activating conditions. The structures reveal some features that were not so well resolved in previous X-ray structures and use simulations to suggest a dynamic structure at high pH, indicative of an ensemble of resting state conformations, compared to a more compact and well-defined structure under activating conditions. This idea is not entirely new, however, as it was a conclusion of the resting state X-ray structure paper of Delarue and co-workers (ref.27). The study also sees changing structural elements that might imply roles in gating, such as with loop F and interactions of E243, though also suggested in past X-ray structures. It is surprising that all structures, including under maximally activating conditions, are completely closed, and the explanation for this is not compelling. Another surprising outcome is that the distributions from simulations of the resting state at high pH based on the new cryo EM structure are so different to those obtained using the past X-ray structure, and there are indications of lack of convergence of these simulations.

    The findings and discussion of Delarue and co-workers in Ref27 could be more prominent, including in the introductory statement, which could be cited along with refs 11,14,15 as a solved resting state, and not just described as being of low resolution. I refer to Fig.3c of ref.27 which conveys the idea of the diverse resting state distribution in that paper.

    In regards to the "relative novelty" of the methods used for MD fitting to cryo EM data, it is not obvious how different the approach is to standard MDFF flexible fitting strategies. Although there is brief mention in the discussion section, it is not clear from the introduction and methods how novel the approach is. I do suggest, however, that it does not make sense to refine the structure with simulations of GLIC in a POPC lipid bilayer, when the cryo EM involved detergent solubilised particles. Fitting MD should have been done in micelles as it is not appropriate to refine in a different environment to which it was solved.

    The authors claim higher RMSD for pH 7, but fig.4A suggests divergence of simulations in 1us. It seems the simulations would need to run longer to reach an equilibrium distribution. It is curious that such divergence is not evident in high pH X-ray structure simulations in the same figure. Does this suggest the cryo EM structure at high pH is unstable? Is this increasing RMSD spread uniformly or due to changes in particular parts of the protein during MD? I note that subsequent analysis, such as fig.5, revealing no maximum in the distribution for ECD bloom compared to X-ray simulations at high pH, may be due to not yet converging on an equilibrium for the resting state (and pre-equilibration period not being excluded).

    Despite the pH 7 cryo EM simulations likely being not yet equilibrated, leading to some uncertainty about the meaning of the distributions in Fig.5, it is clear that low pH leads to a more tightly bound ECD bloom range than pH 7 in that figure. Although the effects of pH are similar between cryo EM and X-ray starting structures, why is the peak in Fig5b ECD twist also so different for pH 7? This also could be an artefact of lack of equilibration. Differences are also noted at low pH.

    Fig5c is striking. It suggests cryo EM at low pH has failed to capture an open pore, whereas X-ray was able to capture an enlarged pore radius. The authors write that this was initially surprising, having all low pH structures closed, but consistent with past X-ray with one structure partially closed. But here all structures look completely closed, whereas a fairly even mix of open and closed TMDs may have been anticipated at low pH, at worst. The possible artefact due to interaction with the glow-discharged cryo EM grid could be better explained for the reader. On page 16, the authors say the closed pores do not look like they would expect for a desensitised state. This also needs a better explanation with more specifics. They then suggest it may be because at low pH the pore can flicker and the open pore has a high free energy. Why is the open state expected to be high free energy at low pH? Doesn't the pH50 of 5 suggest the equilibrium is shifted to the open channel (lower free energy) by pH 3, as also suggested by previous free energy analysis in ref.21? While fig.6 is used to illustrate a reduction of number of closed states to the left with lowered pH, "priming" the protein for gating, again it does not make sense to me that at low pH the free energy of the open state on the right is higher than the closed state on the left.

  4. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.

    ###Summary:

    This manuscript reports cryo-EM structures of the pentameric ligand-gated ion channel GLIC at pH 7, 5 and 3. The reviewers have appreciated several aspects of the manuscript, which combines experiment with simulation to describe the GLIC channel's resting state. However, concerns have been raised. The reviewers have questioned what has been gained in addition to previous work on the structure and mobility of the resting state (Sauguet et al. PNAS 2014; ref.27), not described in this manuscript. How do the new structures compare to past X-ray structures/density maps? The reviewers raise questions about the functional states found. In particular, while rigidification at pH 5 or 3 is interesting, normally it should switch to the open state, especially at pH 3, and why this has not occurred is not explained well. Several concerns have been raised about the simulations and what is learned. This includes protonation state choices (not discussed or justified), why flexible fitting was conducted in a bilayer instead of a micelle (which may impact regions of the map less well defined), and have the simulations converged? The reviewers note lack of informative analysis, leaving us in the dark as to the functional states visited. It has been suggested that analysis in collective variable space would be needed, such as defined in Ref.21 (not discussed in this manuscript), so that the reader can observe if structural features change, despite maintaining an apparent resting conformation (e.g. does the D32-R192 salt bridge break; does the pore wet/dewet)?

  5. Version published to 10.1101/2020.06.19.161356v1 on bioRxiv