Crystal Structure of the N112A Mutant of the Light-Driven Sodium Pump KR2

Round 1

Reviewer 1 Report

The manuscript submitted by Nina Maliar and colleagues describes the crystal structure of the N112A mutant of the 2 light-driven sodium pump KR2. Despite the results do not allow to discover new features of the system or to find sound explanations for the known structure-function relationships, I like the manuscript, which is well written, and I think that it can be interesting not only for scientists involved in this field.

Please, not that the reference list need some attention. Reference 1 is incomplete and several other references are either incomplete or formatted in different ways.

 

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Reviewer 2 Report

In this manuscript, authors presented the crystal structure of the N112A mutant of KR2 in the ground state at the resolution of 2.4 Å. Since N112A mutant is known to have the outward pumping activity, the structural study of N112A mutant is necessary to understand the functional alteration of KR2. However, current crystal structure cannot explain the functional implication of the mutant although this study provide many valuable structural information that is necessary for structural understanding of KR2 in detail. The followings must be answered or modified to improve the quality of manuscript and provide more structural information.

(1) Table 1 must be modified to improve the data quality and credibility.

(2) It is recommend to discuss the structure-function relationship of other functionally equivalent mutants such as N112C/P/V/E/Q/L/I/M/F/W based on the crystal structure of N112A.

(3) In the abstract, it is mentioned that "Our findings also
demonstrate that for the rational design of the KR2 variants with modified ion selectivity for optogenetic applications, the structures of the intermediate states of both the protein and its functional variants are required.". However, discussion about the rational design is not well described. Therefore, it is suggested to provide more discussion on this topic.

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Reviewer 3 Report

Comments to the authors:

 

The authors determined the crystal structure of KR2, a representative, well-studied Na⁺ pumping microbial rhodopsin, to discuss the role of Asn112 (N112) for its Na⁺ pump function by referring their previous structural studies for the ground and intermediate states of the protein. I think that the problem setting, significance, and experimental technique of this study are scientifically important, original, and proper. However, I have several concerns described below.

 

Major points:

1. After reading the manuscript, I understand that the goals, which are not specified in the manuscript, of this study are to explain (i) the role of N112 for the Na⁺ pump function and (ii) the reason why the function is converted into H⁺ pump when mutated N112 into A (Line 94 - 96). (i) has been already explained in Refs. 41 and 42. By referring these studies, the authors made the conclusion described in Line 330 – 331. If the authors want to make sure or improve understanding about (i), the N112A mutant structure in the dark state is not enough. Fortunately, N112A mutant has long-lived M state (Ref. 42). The authors have chance to determine the structure of M state of the N112A mutant in the future. By using this information, (i) will be well explained. Therefore, (i) cannot be explained by any result in this work at present. On the other hand, (ii) is another significant problem that nobody answers up until now. However, the authors did not explain at all about (ii) based on the N112A mutant structure. In the end, the authors concluded as described Line 331 – 334. This does not solve any problems. To improve the manuscript, I will ask the authors to reconsider and clarify the purpose of this study to reach the goal, hopefully (ii), based on the invaluable structural information.

 

2. (Line 46 – 58) The history of microbial rhodopsins is written. I think it is redundant description because this is not a review article.

 

3. (Line 60 – 61) In Ref. 21, outward H⁺ pump activity assay at low pH was not performed. In Ref. 35, a hint for the authors’ claim (Line 60 – 61) may be written. If you claim the outward H⁺ pump activity of wild-type KR2 at low pH, please refer appropriate paper here.

 

4. (Line 153) I had the impression that the experimental results of this study are barely explained despite “3. Results” section here. Please provide full explanations for the experimental results, and then discuss by comparing to previous reports.

 

5. (Line 154) Effective use of figures that illustrate the location of surface residues can be easy to understand for the readers. I like Figure S1 to be combined with Figure 1.

 

6. (Line 155 - 157) The description is overlapped with that in the 2. Materials and Methods section. This should be simplified.

 

7. (Line 163) Please refer appropriate paper to reveal that only pentameric KR2 pumps Na⁺.

 

8. (Line 178 – 181) The authors concluded that the reason for the peak broadening in SEC chromatogram is caused by the destabilization of pentameric assembly of the N112A mutant and the change in the number of detergent molecules bound to the assembly. I can imagine that when the pentamer is destabilized the assembly becomes “loose”. If so, the number of detergents bound to the protein will be increased. As a result, the apparent molecular size will also be increased. In that situation, the chromatograph peak will be shifted toward the direction of increasing molecular size (left side). However, Figure 1a shows just tailing. One of the reasons for the chromatogram tailing is the viscosity of the sample solution. Do you have any idea that protein concentration is over the limitation of the SEC column (how much is the concentration at 2,500 a.u.?), or the sample viscosity gets higher due to the concentrated DDM ?

 

9. (Line 197) In this section, the authors do not explain Figure 2a. For many readers, it is easy to understand that the Figures should be explained in the exact order. If Figure 2a is not important, the authors should reconsider the compositions of the figures and perhaps the whole structure of the manuscript.

 

10. (Line 206 – 207) The authors discussed here the orientation of helix C in the wild-type KR2 (6YC3, yellow) and N112A mutant (6YT4, violet). As long as I compare these two structures, no distortion is found in the region of 109 – 115 residues. The distortion will be emphasized when you compare the structures with “compact” conformation (4XTN, orange) and monomeric form (4XTL, cyan). I think the discussion here is unreasonable without explanation about the latter two structures.

 

11. (Line 209 – 210) Crystal structure clearly showed the Na⁺ binding site in the dark at around BC-loop containing D102, which is one of the essential residues for Na⁺ binding (Kato et al. Nature 2015). In Ref. 21, the BC-loop deleted mutant was analyzed and the FTIR analysis for this mutant revealed that Na⁺ binding ability was lost. A part of the Na⁺ binding site has already been identified by FTIR. I mean that these facts are not “in contrast” or “inconsistent” with each other (crystal vs FTIR) as you described in the manuscript.

 

12. (Line 255 – 256) The proton pump activity of KR2 at low pH was not demonstrated in Refs. 35 and 37. Even in the supporting materials for Ref. 37, pH change was not abolished in the presence of CCCP at pH lower than 7, meaning the secondary H⁺movement caused by probably Na⁺ transport by KR2. Therefore, the following sentence (Line 256 - 258) should be reconsidered. I think the “compact” and “expanded” conformations in the dark state are not the determinant for the H⁺ and Na⁺ pumping modes, respectively. Moreover, these conformation difference does not explain the H⁺/Na⁺

 

13. (Line 268 – 271) Figure 2a does not explain this sentence.

 

14. (Line 285) This section is not described based on the experimental results in this study. I mean this section is just discussion. In addition, the number is not 3.3, but 3.4.

 

15. (Figure S3) Figure S3b is a model structure of O state for KR2-N112A mutant. I think that the result of modeling will help to interpret the role of N112 for the ion selectivity (Section 3.4). However, there is no explanation. The result, and also the method, should be written in the main text when necessary. In addition, I do not understand the necessity for the modeling result of N112G mutant here. Please explain the importance of N112G mutant for your discussion.

 

16. (Figure 4) I do not know whether Figure 4 presented in Conclusion section is proper or not. I think this figure shows the overview of the N112A structure and thus should be explained earlier in the text.

 

 

Minor points:

1. (Line 23) eikastus
2. (Line 74 - 76) Check the position of parentheses again.
3. (Line 91) Fourier-transformed infrared (FTIR) spectroscopy
4. (Line 153) This section contains discussion. Rename “3. Results” into “3. Results and Discussion” if it is allowed by the Journal guideline.
5. (Line 205) RMSD is a new word. The explanation for this abbreviation should be described.
6. The authors use “variant” and “mutant” in the manuscript. It is confusing. Please reconsider the use of this term.
7. A lot of subjective descriptions are made. For example, “surprisingly”, “perfectly”, “easily”, etc.

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Round 2

Reviewer 2 Report

The questions are well addressed.

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Reviewer 3 Report

The great efforts by the authors improved the manuscript. For my comment, I was satisfied with all but one, about the proton pump activity of KR2 at low pH. This is the minor point.

 

As you say, the ref. 35 (Kovalev et al. 2019) performed the pump activity measurements at pH 4.3 (Fig. S9) and discussed the proton pump activity by the wild-type KR2. However, it is very hard for me to accept the fact because;

• The signals of pH change were too small to judge whether the KR2 has the activity or not. You claimed that the signal was decreased when the CCCP was added, which is unique for the proton pump rhodopsins. But, I think it is dangerous to argue on this basis. When the pH electrode itself is illuminated, you will see the pH increase as same as you observed at pH 4.3. Can you deny the possibility of the artifact from the pH electrode ?
• At pH 4.3, the Schiff base counterion Asp116 will be protonated because its pKa is around 7 (Here, the pKa of Asp116 and Asp251 are shown; see supplementary figure S8 of the ref. 21). The neutralized mutant of this residue (D116N, moreover D251N) does not show any pump activity at all (both Na⁺ and H⁺ pump activities; see the ref. 21). It is generally understood that when Asp116 is protonated nothing happens in KR2. Do you have any idea that will solve such inconsistence ? In addition, “At the same time, the KR2 predominantly pumps protons under pH lower than 6” (Line 273 – 274) is therefore the extended interpretation.

I know the ref. 35 is a great work for understanding the molecular mechanism of KR2 and will be helpful for your manuscript. However, as long as you discuss the mechanism for Na⁺-to-H⁺ pump conversion based on the N112A mutant structure, I hope you have to understand, interpret, and refer the results carefully and correctly.

Author Response

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