The crystal structure of bromide-bound GtACR1 reveals a pre-activated state in the transmembrane anion tunnel

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1. 

   Version published to 10.7554/elife.65903 on eLife

   May 17, 2021
2. 

   eLife

   May 16, 2021

   Author Response:

   Reviewer #1 (Public Review):

   The dark structure of GtACR1 has been almost simultaneously published at the end of 2018 and beginning of 2019 by the Deisseroth and Spudich groups, respectively. Both groups did not manage to solve a structure with an ion bound and there is very limited information on the open conformation of the channel. Both groups identified a central constriction site as being central for the gating mechanism but the Spudich group proposes two additional constrictions (C1 and C3). In this work Li et al are able to solve the structure of a GtACR1 with a bromide bound near C3, which clearly represents a significant step towards understanding the mechanism of light gated anion channels. The structure reveals that Br binds to the intracellular constriction site (C3) resulting in a small opening of C3. …

   Author Response:

   Reviewer #1 (Public Review):

   The dark structure of GtACR1 has been almost simultaneously published at the end of 2018 and beginning of 2019 by the Deisseroth and Spudich groups, respectively. Both groups did not manage to solve a structure with an ion bound and there is very limited information on the open conformation of the channel. Both groups identified a central constriction site as being central for the gating mechanism but the Spudich group proposes two additional constrictions (C1 and C3). In this work Li et al are able to solve the structure of a GtACR1 with a bromide bound near C3, which clearly represents a significant step towards understanding the mechanism of light gated anion channels. The structure reveals that Br binds to the intracellular constriction site (C3) resulting in a small opening of C3. The data support the notion that the partial electropositivity of Pro58 together with two tryptophans play a critical role in anion interaction at C3, which was also confirmed by mutagenesis studies. In addition, there was a noteworthy conformational change in the Bromide bound protein in the extracellular constriction (C1), a 180 degree flip of Arg 94 resulting in a salt bridge to Asp 234 and a slight opening of the C1 constriction.

   While the data and conclusions are sound, the lack of discussion of their data in the context of the work of others is a bit surprising.

   We thank the reviewer for thorough reading of our submission and constructive criticism, which helped us to improve the quality of our manuscript. As requested, we added the following paragraph at the end of the Results section (lines 219-233):

   “Studies in 3 different laboratories have concluded that Asp234 is neutral in the dark state from measurements of the D234N mutant of GtACR1 by UV-vis absorption spectroscopy (Kim et al., 2018; Sineshchekov et al., 2016), Resonance Raman spectroscopy (Yi et al., 2016), and FTIR (Kim et al., 2018). Both studies of independently determined crystal structures of GtACR1 attribute the major component of its neutralization to hydrogen-bonding to Tyr207 and Tyr72 (Kim et al., 2018, Li et al., 2019), leaving open partial electronegativity of Asp234 participating in hydrogen-bonding to the protonated Schiff base (PSB). The Asp234 residue is expected to be functionally important given its proximity to the PSB and its nearly universal conservation in microbial rhodopsins. Kim et al (Kim et al., 2018) conducted an extensive analysis of Asp234 and report that the D234N mutation nearly abolished photocurrents. Reduced photocurrents to 20% of wild-type from the D234N mutation were also observed by Sineshchekov et al. (Sineshchekov et al., 2015). Differences in extent of photocurrent reduction are likely attributable to different assay conditions used in these studies. The electrostatic interaction of Arg94 with Asp234 in the pre-activated state may be correlated with the change in the electron conjugation of the retinylidene polyene chain in the dark that we observed by FTIR.”

   Reviewer #2 (Public Review):

   In the manuscript entitled "The Crystal Structure of Bromide-bound GtACR1 Reveals a Pre-activated State in the Transmembrane Anion Tunnel", Li et al. analyzed the effect of bromide binding to GtACR1 by X-ray crystallography and electrophysiology. The authors propose that a bromide ion is bound to the intracellular pocket in the dark, inactivated state and induces a structural transition from an inactivated to a pre-activated state.

   I agree that some of the amino acid residues in the current crystal structure change their conformations compared to the previous one reported in 2019 (Li et al., 2019), and it is very impressive that the authors determined the structure using state-of-the-art crystallography technique, ISIMX. However, unfortunately, most of the conclusions and claims described in the manuscript are not well supported by the authors' data.

   1. The most serious problem is that the evidence of bromide binding is too weak. The authors showed the composite omit map in Supplementary Figure 1A, but they should present an anomalous difference Fourier map to validate the bromide binding. The authors also claim that they replaced the bromide ion to the water, run the PHENIX refinement, and observed a strong positive electron density at the bromide position in the Fo-Fc difference map (Supplementary Figure 1B). However, when I do the same thing using the provided coordinate and map (I really appreciate the honesty and transparency of the authors), I could not reproduce their result; a weak positive electron density is observed between the bromide position and Pro58 in chain A and there is no positive peak at the position in chain B (Fo-Fc, contoured at 3σ). I am wondering the occupancy and B-factor of the water molecule they show in Supplementary Figure 1B.

   We appreciate the reviewer’s effort in analysis of our structure. As described in the Discussion section (lines 238-248), the identification of bromide is supported by multiple lines of evidence: (1) the composite omit map indicates the presence of bromide at the cytoplasmic port (Suppl. Fig. 1A-1B); (2) we exclude the possibility of a water at the bromide position as demonstrated in the Fo-Fc difference map (Suppl. Fig. 1C-1D); (3) the bromide binding site exhibits a similar chemical conformation seen in chloride-binding structures (Auffinger et al., 2004); (4) functional analysis of W250F and W246F are consistent with the H-bond interaction in the bromide binding site (Fig. 2B); (5) Specific interaction of GtACR1 with bromide in the dark state was further demonstrated by FTIR analysis (Fig. 3). Differences in major bands that reflect the ethylenic (C=C) stretch mode of the retinylidene chromophore show a large bromide-induced alteration in the electron conjugation of the retinylidene polyene chain in the dark, confirming that bromide causes a significant structural change. In sum, these data confirm the bromide binding conformation in the structure.

   We agree with the reviewer that the signal of bromide in chain A is stronger than in chain B. We now address the difference throughout the main text and Suppl. Fig. 1. The datasets were collected at 0.91882 Å wavelength, but we did not detect any strong bromide signals in the anomalous difference Fourier map. This may be due to preferential orientation of the thin-plate GtACR1 crystals in the IMISX plate. The weak Br signals may also be attributed to the weak bromide binding conformation, its partial occupancy, and poor intrinsic order. It is not unusual that anomalous signals are influenced by the location of the scatter. For example, in our previous structural determination of YfkE (Wu, PNAS 2013), Seleno-methionine was used to label 12 native Met residues. However, we could identify only 10 Se positions and the other 2 Se were undetectable in the anomalous difference map, despite the dataset collection at the Se absorption peak wavelength. Therefore, the lack of strong anomalous signals does not exclude the presence of bromide in the structure.

   Regarding the reviewer’s question, the occupancy of the water is 1 and its B-factor is 71.

   In addition to the insufficient evidence, the current models of bromide ions have significant steric clashes. The PDB validation report shows that the top 5 serious steric clashes observed in the coordinate are the contacts between the bromide ions and surrounding residues (PDB validation report, Page 10). I analyzed them and found that the distance between the bromide ion and CG and CD atoms of Pro58 in chain A are only 2.43Å and 2.36Å, respectively. The authors claim that such a close proline-halide interaction has also been observed in the structure of the chloride-pump rhodopsin CIR, but in the structure (PDB ID: 5G28), the distances between the chloride ion and CD and CG atoms of Pro45 are much larger (3.43 and 3.91Å, respectively) and there is no steric clash. Moreover, the authors claim that Pro58 changes its conformation by bromide binding, but it is very possible that the PHENIX program just displaces Pro58 to alleviate the steric clash between the proline and the bromide ion, so the authors should carefully check the possibility.

   Overall, the authors should analyze the density again, provide more solid evidence for the bromide binding such as anomalous difference Fourier map, and if they could, they should correct the current significant steric clashes in their models.

   We thank the reviewer for pointing out the steric clashes. We have corrected them in the revised structure as demonstrated in the latest validation report. As described in the Results section (line 107-109), the distance between the bromide ion and CG and CD atoms of Pro58 in chain A are now 3.6 Å and 3.1 Å (see the updated structure pdb), respectively, and the distance between the bromide ion and CG and CD atoms of Pro58 in chain B are 4.0 Å and 3.2 Å, respectively, similar to those distances between the chloride ion and CD and CG atoms of Pro45 in ClR (3.43 and 3.91Å, respectively). These modifications do not alter the structure beyond the local binding site of the bromide, and do not change our conclusions.
   We do not agree that the Br--induced conformational changes are due to the refinement program. To further confirm the Pro58 position, we have performed a refinement by removing Pro58 and adjacent residues using PHENIX. The resulted electron density map shows a positive electron density at the Pro58 position, confirming the conformational changes induced by bromide binding.

   1. To analyze the functional importance of putative bromide binding, the authors prepared W246E and W250E mutants and analyzed their electrophysiological properties. Because tryptophan and glutamate are so different in terms of volume and charge, they should analyze other mutants as well. The authors claim that bromide is stabilized by a hydrogen bond interaction formed by the indole NH group of W246, so they should at least test the W246F mutant.

   We thank the reviewer for this important suggestion, which helps confirm the bromide binding conformation. The glutamate substitutions were chosen to assess the specific anion selectivity and conductivity of GtACR1 due to the negative charge of its side chain. We now include the data of W246F and W250F in Fig 2B. W250F shows reduction of the current amplitude by 50%, whereas W246F behaves like WT. These results are consistent with the structural observations in which W250, but not W246, stabilizes bromide via H-bond interaction. These results are provided in the Results section (lines 136-142) and in the revised Fig. 2B.

   1. The authors claim that the bromide binding in the intracellular pocket induces the conformational change of R94, but the causal relationship is doubtful. As mentioned in the manuscript, R94 forms a salt-bridge with D234 in chain A. However, the arginine has a completely different conformation and does not have any interaction with D234 in chain B. If the bromide binds both in chain A and B and induces the conformational change of R94, why only R94 in chain A interacts with D234? The authors change the pH in the crystallization condition compared to their 2019 study (Li et al., 2019), so the pH may affect the protonation state of D223 and/or other titratable residues and induces the conformational change of R94. The authors should provide more solid evidence for the causal relationship between the bromide binding and the conformational change of R94.

   We did not change the pH in the crystallization condition compared to our previous crystallization of GtACR1. Both structures were obtained at pH 5.5 as noted in the manuscript. In our structure, the only bromide binding site was identified near C3 and no bromide was found at C1. We address this result in Discussion (lines 276-286) as follows:

   “The conformational change of Arg94 near C1 is not likely to be directly induced allosterically by bromide binding at distant C3 since it is only observed in chain A, not in chain B. Instead, this conformational change may reflect the intrinsic flexibility property of Arg94 in the tunnel in the bromide-bound state. Although both Arg94 of GtACR1 (in chain A) and Arg95 of CIR adopt a similar conformation (Fig. 4B), these two counterpart residues appear to be stabilized by distinct H-bond networks. In GtACR1, inward Arg94 only forms a salt-bridge with Asp234 and an H-bond with a water molecule (Suppl. Fig. 2A). However, in the CIR structure, in addition to the salt bridge, R95 is further stabilized by three polar residues, Asn92, Gln224, and Thr228, via two water molecules from the extracellular side of the protein (Suppl. Fig. 2B). The absence of these polar residues and waters in the vicinity may liberalize Arg94 and facilitate its flip-flopping in the tunnel of GtACR1.”

   1. The authors assume that the conformational change of R94 creates a functional anion binding site with the Schiff base in GtACR1, but it is too speculative. If the anomalous difference Fourier map does not support the idea, they should delete it.

   Our hypothesis (not an assumption) is based on the following facts: (1) both rhodopsin proteins GtACR1 and ClR transport the same halide substrates; (2) the chain A of GtACR1 adopts a nearly identical chemical conformation to that in the chloride-binding site (site 1) of CIR, in which the counterpart residue R95 forms a chloride binding site with the Schiff base (Fig. 4B); and (3) Arg94 is important to anion conductivity of GtACR1 (Li et al. eLife 2019). It is reasonable to hypothesize that Arg94 forms a putative anion binding site with the Schiff base in GtACR1. To make this hypothesis clear, we listed these facts in the text and rephrased our hypothesis as follows (lines 217-219): “Based on the similar chemical conformations (Fig. 4B), it is possible that Arg94 rotates its side chain to form an anion binding site with the Schiff base in GtACR1.”

   Read the original source
3. 

   eLife

   May 16, 2021

   Reviewer #2 (Public Review):

   In the manuscript entitled "The Crystal Structure of Bromide-bound GtACR1 Reveals a Pre-activated State in the Transmembrane Anion Tunnel", Li et al. analyzed the effect of bromide binding to GtACR1 by X-ray crystallography and electrophysiology. The authors propose that a bromide ion is bound to the intracellular pocket in the dark, inactivated state and induces a structural transition from an inactivated to a pre-activated state.

   I agree that some of the amino acid residues in the current crystal structure change their conformations compared to the previous one reported in 2019 (Li et al., 2019), and it is very impressive that the authors determined the structure using state-of-the-art crystallography technique, ISIMX. However, unfortunately, most of the conclusions and claims described in the manuscript …

   Reviewer #2 (Public Review):

   In the manuscript entitled "The Crystal Structure of Bromide-bound GtACR1 Reveals a Pre-activated State in the Transmembrane Anion Tunnel", Li et al. analyzed the effect of bromide binding to GtACR1 by X-ray crystallography and electrophysiology. The authors propose that a bromide ion is bound to the intracellular pocket in the dark, inactivated state and induces a structural transition from an inactivated to a pre-activated state.

   I agree that some of the amino acid residues in the current crystal structure change their conformations compared to the previous one reported in 2019 (Li et al., 2019), and it is very impressive that the authors determined the structure using state-of-the-art crystallography technique, ISIMX. However, unfortunately, most of the conclusions and claims described in the manuscript are not well supported by the authors' data.

   1. The most serious problem is that the evidence of bromide binding is too weak. The authors showed the composite omit map in Supplementary Figure 1A, but they should present an anomalous difference Fourier map to validate the bromide binding. The authors also claim that they replaced the bromide ion to the water, run the PHENIX refinement, and observed a strong positive electron density at the bromide position in the Fo-Fc difference map (Supplementary Figure 1B). However, when I do the same thing using the provided coordinate and map (I really appreciate the honesty and transparency of the authors), I could not reproduce their result; a weak positive electron density is observed between the bromide position and Pro58 in chain A and there is no positive peak at the position in chain B (Fo-Fc, contoured at 3σ). I am wondering the occupancy and B-factor of the water molecule they show in Supplementary Figure 1B.

      In addition to the insufficient evidence, the current models of bromide ions have significant steric clashes. The PDB validation report shows that the top 5 serious steric clashes observed in the coordinate are the contacts between the bromide ions and surrounding residues (PDB validation report, Page 10). I analyzed them and found that the distance between the bromide ion and CG and CD atoms of Pro58 in chain A are only 2.43Å and 2.36Å, respectively. The authors claim that such a close proline-halide interaction has also been observed in the structure of the chloride-pump rhodopsin CIR, but in the structure (PDB ID: 5G28), the distances between the chloride ion and CD and CG atoms of Pro45 are much larger (3.43 and 3.91Å, respectively) and there is no steric clash. Moreover, the authors claim that Pro58 changes its conformation by bromide binding, but it is very possible that the PHENIX program just displaces Pro58 to alleviate the steric clash between the proline and the bromide ion, so the authors should carefully check the possibility.

      Overall, the authors should analyze the density again, provide more solid evidence for the bromide binding such as anomalous difference Fourier map, and if they could, they should correct the current significant steric clashes in their models.

   2. To analyze the functional importance of putative bromide binding, the authors prepared W246E and W250E mutants and analyzed their electrophysiological properties. Because tryptophan and glutamate are so different in terms of volume and charge, they should analyze other mutants as well. The authors claim that bromide is stabilized by a hydrogen bond interaction formed by the indole NH group of W246, so they should at least test the W246F mutant.

   3. The authors claim that the bromide binding in the intracellular pocket induces the conformational change of R94, but the causal relationship is doubtful. As mentioned in the manuscript, R94 forms a salt-bridge with D234 in chain A. However, the arginine has a completely different conformation and does not have any interaction with D234 in chain B. If the bromide binds both in chain A and B and induces the conformational change of R94, why only R94 in chain A interacts with D234? The authors change the pH in the crystallization condition compared to their 2019 study (Li et al., 2019), so the pH may affect the protonation state of D223 and/or other titratable residues and induces the conformational change of R94. The authors should provide more solid evidence for the causal relationship between the bromide binding and the conformational change of R94.

   4. The authors assume that the conformational change of R94 creates a functional anion binding site with the Schiff base in GtACR1, but it is too speculative. If the anomalous difference Fourier map does not support the idea, they should delete it.

   Read the original source
4. 

   eLife

   May 16, 2021

   Reviewer #1 (Public Review):

   The dark structure of GtACR1 has been almost simultaneously published at the end of 2018 and beginning of 2019 by the Deisseroth and Spudich groups, respectively. Both groups did not manage to solve a structure with an ion bound and there is very limited information on the open conformation of the channel. Both groups identified a central constriction site as being central for the gating mechanism but the Spudich group proposes two additional constrictions (C1 and C3). In this work Li et al are able to solve the structure of a GtACR1 with a bromide bound near C3, which clearly represents a significant step towards understanding the mechanism of light gated anion channels. The structure reveals that Br binds to the intracellular constriction site (C3) resulting in a small opening of C3. The data support the …

   Reviewer #1 (Public Review):

   The dark structure of GtACR1 has been almost simultaneously published at the end of 2018 and beginning of 2019 by the Deisseroth and Spudich groups, respectively. Both groups did not manage to solve a structure with an ion bound and there is very limited information on the open conformation of the channel. Both groups identified a central constriction site as being central for the gating mechanism but the Spudich group proposes two additional constrictions (C1 and C3). In this work Li et al are able to solve the structure of a GtACR1 with a bromide bound near C3, which clearly represents a significant step towards understanding the mechanism of light gated anion channels. The structure reveals that Br binds to the intracellular constriction site (C3) resulting in a small opening of C3. The data support the notion that the partial electropositivity of Pro58 together with two tryptophans play a critical role in anion interaction at C3, which was also confirmed by mutagenesis studies. In addition, there was a noteworthy conformational change in the Bromide bound protein in the extracellular constriction (C1), a 180 degree flip of Arg 94 resulting in a salt bridge to Asp 234 and a slight opening of the C1 constriction.

   While the data and conclusions are sound, the lack of discussion of their data in the context of the work of others is a bit surprising.

   Read the original source
5. 

   eLife

   May 16, 2021

   Evaluation Summary:

   This manuscript reports a significant contribution towards an improved mechanistic understanding of light gated anion channels. The studies, which use the recently established method of in meso in situ serial data collection (IMISX), provide a basis for optimizing the anion channelrhodopsin GtACR1 from the alga Guillardia theta as a neuron-inhibiting optogenetics tool. The work will be of interest to anyone using optogenetics for functional studies. The reviewers had a few comments regarding technical aspects of the work.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors)

   Read the original source
6. 

   Version published to 10.1101/2020.12.31.424927v1 on bioRxiv

   Jan 2, 2021