PONE-D-23-35512Structural bioinformatics studies of serotonin, dopamine and norepinephrine transporters and their AlphaFold2 predicted water-soluble QTY variants and uncovering the natural mutations of L->Q, I->T, F->Y and Q->L, T->I and Y->FPLOS ONE

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Comments: Minor Revision

This study applies a water solubilization design method called QTY code to monoamine transporters, computationally characterizing some properties of the designed proteins and investigating the relationship between natural mutations in these monoamine transporters and the QTY code. These findings are intriguing and hold some significance for the study on monoamine transporters and the QTY code; however, there are some issues in the manuscript should be addressed by the authors.

Specific comments:

1. Abstract：While their hydrophobic surfaces significantly reduced, this change did not cost a structural alteration.

This statement tends to be too assertive. However, after numerous sequence variations, there must be structural changes, to varying degrees.

2. Abstract：Accompanied by similarities of QTY-code pairs, our study unrevealed multiple QTY and reverse QTY variations in genomic databases.

Please provide a more explicit explanation regarding the specific aspects of similarity. Additionally, the use of the term 'QTY-code pair' in the abstract is also confusing.

3. The structure of Introduction section needs improvement. The current manuscript presents introductions to various aspects (transporter, QTY code, transporter mutation) in a somewhat confusing manner, with much interweaving. This may make it challenging for readers to quickly and clearly grasp the background knowledge on these topics.

4. The order of figures needs to be revised, as Figure S19 appears earliest and should not be the last supplementary figure.

5. Page 4: The QTY code substitutes four hydrophobic amino acids (Leucine, Isoleucine, Valine, and Phenylalanine) with hydrophilic amino acids (Glutamine = Q, Threonine = T, and Tyrosine = Y)

The content related to the QTY code has already been introduced earlier; no need to repeat. The meanings of single letters also do not need to be redundantly explained.

6. Page 4: The highly predicted Local Distance Difference Test (pLDDT) scores of the predictions are reassuring. Indicating greater confidence in predicted protein structures [33].

This sentence needs to be rewritten.

7. In Page 4, it is mentioned that the pLDDT score is used to assess the quality of AlphaFold2 predictions. It is quite perplexing that the authors have not provided the pLDDT scores for the QTY variants and their distribution in the structures.

8. In Page 4, a significant portion is dedicated to introduce hydrophilic and hydrophobic alpha-helices. It is recommended to place this information after the results and reduce its length.

9. Table 2 consists of only one row and does not need to be presented as a separate table.

Reviewer #2: This manuscript bioinformatically studies the structure and properties of monoamine transporters and their QTY designed variants based on AlphaFold2 and tools such as ClinVar and PolyPhen-2.

The author took a step forward from his previous QTY designed membrane proteins to include more genomic analysis and evolutionary conservation of specific sites related to Q, T, Y and L, I, F, so as to provide more information regarding phenotypical impacts from these substitutions, as well as providing more guidance in subsequent designs.

The study is very interesting and is suitable for publication in PLOS One following minor revision.

1. Please check the spelling and formatting of the entire manuscript. Such as, page 8, “sequance” would be “sequence”. Page 13, the format of reference 13 is inappropriate.

2. Sequence in Figure. 1 is barely visible.

3. The current research on QTY in the introduction is not comprehensive, and relevant research background needs to be supplemented. Please note that all correctly used papers should be cited.

4. There could be more discussion about whether 11 variations found to be previously reported in ClinVar archives could be discussed. This part may have referential significance for the treatment of related diseases.

Reviewer #3: The paper is well-written, and the problem this study is dedicated to is important, as justified in the introduction. The authors provide an additional GitHub repository with the structural data used in this work, making it reproducible. The evolution study is the most interesting, however, the structural part can be improved with MD simulations. Nevertheless, the authors mention all the shortcomings that can be connected to the work, making the paper of good quality. Overall, I recommend this paper for publication.

Suggestions:

"Despite this effort, structural information on the monoamine transporters is still limited and many aspects of their molecular mechanisms have remained unknown" - To understand the mechanics behind these transporters and the more realistic effects of QTY mutations on the protein's structure and function, it is worth investigating the systems in dynamics. If you cannot conduct MD, please at least add to the text a discussion of how MD simulations can influence your results and findings. However, for further studies, I do recommend including MD simulations. You can set them up in the following way: 1) native protein in the membrane, 2) native protein in solute, 3) QTY protein in membrane, 4) QTY protein in solute for at least two different proteins that perform best in the rigid models comparisons. Next, evaluate the stability and behavior of all these 8 systems (4 setups x 2 proteins). Calculate the number of hydrophobic and hydrophilic contacts for each system and conclude how hydrophobicity changes from native to QTY systems.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Submitted filename: PONE-D-23-35512.docx

Structural bioinformatics studies of serotonin, dopamine and norepinephrinetransporters and their AlphaFold2 predicted water-soluble QTY variants and uncovering the natural mutations of L->Q, I->T, F->Y and Q->L, T->I and Y->F

PONE-D-23-35512R1

Dear Dr. Zhang,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Ahmed E. Abdel Moneim

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have addressed my concerns in this round of revision. I recommend this manuscript for publication.

Reviewer #2: The authors have addressed all my comments and thus I recommend the manuscript for publication in current state.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

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