Proton transfer pathway in anion channelrhodopsin-1
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Evaluation Summary:
The authors report continuum electrostatics, molecular dynamics and QM/MM simulations to probe the protonation pattern of key residues in anion channelrhodopsin (GtACR1). The findings provide new mechanistic insights into the gating mechanism of GtACR1, and the study will be of potential interest for the community focused on biophysical chemistry, protein simulations and optogenetics. More generally, the study helps highlight that due to potential compensatory effects, care needs to be exercised when interpreting absorption spectra of mutant proteins.
(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. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Anion channelrhodopsin from Guillardia theta ( Gt ACR1) has Asp234 (3.2 Å) and Glu68 (5.3 Å) near the protonated Schiff base. Here, we investigate mutant Gt ACR1s (e.g., E68Q/D234N) expressed in HEK293 cells. The influence of the acidic residues on the absorption wavelengths was also analyzed using a quantum mechanical/molecular mechanical approach. The calculated protonation pattern indicates that Asp234 is deprotonated and Glu68 is protonated in the original crystal structures. The D234E mutation and the E68Q/D234N mutation shorten and lengthen the measured and calculated absorption wavelengths, respectively, which suggests that Asp234 is deprotonated in the wild-type Gt ACR1. Molecular dynamics simulations show that upon mutation of deprotonated Asp234 to asparagine, deprotonated Glu68 reorients toward the Schiff base and the calculated absorption wavelength remains unchanged. The formation of the proton transfer pathway via Asp234 toward Glu68 and the disconnection of the anion conducting channel are likely a basis of the gating mechanism.
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Evaluation Summary:
The authors report continuum electrostatics, molecular dynamics and QM/MM simulations to probe the protonation pattern of key residues in anion channelrhodopsin (GtACR1). The findings provide new mechanistic insights into the gating mechanism of GtACR1, and the study will be of potential interest for the community focused on biophysical chemistry, protein simulations and optogenetics. More generally, the study helps highlight that due to potential compensatory effects, care needs to be exercised when interpreting absorption spectra of mutant proteins.
(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. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The authors conducted continuum electrostatics, molecular dynamics and QM/MM calculations to probe the protonation states of two key amino acids, Asp234 and Glu68, in the anion channelrhodopsin (GtACR1). Previous spectroscopic experiments using different mutants of GtACR1 suggested that both residues are likely protonated, leaving open to the question what residues stabilize the protonated Schiff base. The current calculations strongly suggest that Asp234 is, in fact, deprotonated while Glu68 is protonated in the wild type. The protonation pattern changes in some mutants; for example, Glu68 is deprotonated in the D234N mutant, which helps explain why limited spectroscopic perturbation was observed in the mutant. The observed protonation patterns in the WT and mutant proteins also help explain how the Schiff …
Reviewer #1 (Public Review):
The authors conducted continuum electrostatics, molecular dynamics and QM/MM calculations to probe the protonation states of two key amino acids, Asp234 and Glu68, in the anion channelrhodopsin (GtACR1). Previous spectroscopic experiments using different mutants of GtACR1 suggested that both residues are likely protonated, leaving open to the question what residues stabilize the protonated Schiff base. The current calculations strongly suggest that Asp234 is, in fact, deprotonated while Glu68 is protonated in the wild type. The protonation pattern changes in some mutants; for example, Glu68 is deprotonated in the D234N mutant, which helps explain why limited spectroscopic perturbation was observed in the mutant. The observed protonation patterns in the WT and mutant proteins also help explain how the Schiff base is stabilized in the relevant kinetic states, and how the photocurrents are affected by various mutations. Overall, this careful study helps highlight the value of detailed atomistic calculations in mechanistic analysis as well as providing explanations for spectroscopic measurements.
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Reviewer #2 (Public Review):
This manuscript addresses the protonation state of two carboxyl acids, D234 and E68, in the active site of a channelrhodopsin. The active site is composed of a protonated retinal Schiff base which is positively charged. The authors find that D234, which is next to the Schiff base is deprotonated and E68 is protonated.
The strengths of this work is the broad range of computational methods ranging from membrane embedded molecular dynamics to QM/MM calculations. The simulations are supported by expressing a mutant that was not reported before and measuring its spectrum.
The weaknesses of this manuscript are that some methods are not state of the art and some conclusions are not justified. The method for the calculation of the absorption maxima is not accurate or might not even be physical. These computed …
Reviewer #2 (Public Review):
This manuscript addresses the protonation state of two carboxyl acids, D234 and E68, in the active site of a channelrhodopsin. The active site is composed of a protonated retinal Schiff base which is positively charged. The authors find that D234, which is next to the Schiff base is deprotonated and E68 is protonated.
The strengths of this work is the broad range of computational methods ranging from membrane embedded molecular dynamics to QM/MM calculations. The simulations are supported by expressing a mutant that was not reported before and measuring its spectrum.
The weaknesses of this manuscript are that some methods are not state of the art and some conclusions are not justified. The method for the calculation of the absorption maxima is not accurate or might not even be physical. These computed absorption maxima are crucial for the conclusion of the paper. Also the gating mechanism is discussed on the basis of the structure of resting state. While the change of some amino acids is mentioned, the retinal is still in all trans and conformational changes of the protein are neglected in the discussion.
This anion channelrhodopsin is considered to be an important to tool in the field of optogenetics, where living cells can be controlled by light. Hence, a molecular level understanding of how this protein works could be highly valuable and could assist further tailoring of this channelrhodopsin for specific applications.
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Reviewer #3 (Public Review):
This work probes the role of the acidic residues, E68 and D234, proximal to the Schiff base in the anion channel rhodopson from Guillardia theta (GtACR1). The authors present experimentally measured and QM/MM calculated absorption spectroscopic shifts for a new mutant, E68Q/D234N, as well as pKa estimates for several mutants. Their results further support the notion that Asp234 is deprotonate and Glu68 is protonated in the crystal structure and in the wild type channel, as previously proposed. They also suggest that the absorption spectra are not shifted in the D234N mutant because Glu68 deprotonates and rotates to stabilize the protonated Schiff base. This is potentially valuable both to the rhodopsin field and to the general domain of understanding the sometimes-nuanced influence of Asp to Asn or Glu to …
Reviewer #3 (Public Review):
This work probes the role of the acidic residues, E68 and D234, proximal to the Schiff base in the anion channel rhodopson from Guillardia theta (GtACR1). The authors present experimentally measured and QM/MM calculated absorption spectroscopic shifts for a new mutant, E68Q/D234N, as well as pKa estimates for several mutants. Their results further support the notion that Asp234 is deprotonate and Glu68 is protonated in the crystal structure and in the wild type channel, as previously proposed. They also suggest that the absorption spectra are not shifted in the D234N mutant because Glu68 deprotonates and rotates to stabilize the protonated Schiff base. This is potentially valuable both to the rhodopsin field and to the general domain of understanding the sometimes-nuanced influence of Asp to Asn or Glu to Gln mutations. The rotation of deprotonated Glu68 in the D234N mutant puts it in direct path with the ion channel, potentially explaining why photocurrent is abolished in this mutant.
This work would be improved by including E68(H)/D243(-) in their analyses to delineate the potential role of this intermediate in the wild type system. Many of the conclusions drawn could be similarly explained by this protonation state, thus direct comparisons are warranted. Additionally, error analysis is lacking for both measured and calculated quantities and must be added in order to verify several conclusions, including the proposed protonation states. The chosen level of QM should also be justified with higher level benchmarks for this system. Finally, delineating which findings/conclusions are new and which are supporting previous work should be clarified.
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