PONE-D-22-33788Amino acid substitutions in human growth hormone affect secondary structure and receptor bindingPLOS ONE

Dear Dr. Samuel Coulbourn Flores,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses ALL the points raised by the two reviewers during the review process.

Please submit your revised manuscript by February 16, 2023. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Sabato D'Auria

Academic Editor

PLOS ONE

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When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript:

“SF gratefully acknowledges a grant from the Swedish Foundation for International

Cooperation in Research and Higher Education (STINT). The Lars Hierta Memorial

Foundation provided on year of funding to AR. SF receives partial salary support from

the Swedish Research Council grant VR-M 2016-06301, the National Research School in

Medical Bioinformatics.

The SciLifeLab Drug Discovery and Development Platform provided differential

scanning fluorimetry equipment. Faruck Morcos helped us look for a coevolutionary

signal in some rather limited sequence alignments. The authors have no conflict of

interest.”

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“We gratefully acknowledge support from the Swedish Foundation for International Cooperation in Research and Higher Education (STINT) to SF. The Lars Hierta Memorial Foundation provided salary support for AR. SF is funded by the Swedish Research Council grant VR-M 2016-06301, the National Research School in Medical Bioinformatics.”

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Reviewers' comments:

Reviewer's Responses to Questions

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: No

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

Reviewer #1: I Don't Know

Reviewer #2: No

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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: No

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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: No

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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: The manuscript by Andrei Rajkovic et al. entitled “Amino acid substitutions in human growth hormone affect secondary structure and receptor binding” describes the interaction between human Growth Hormone (hGH) and hGH Receptor (hGHR) to select substitutions at single hGH positions to increase affinity of hGH against hGHR.

The authors conclude that the results point to a link between coiled-coilingstructure, zinc binding, and hGHR-binding affinity in hGH, and also suggest rules for choosing affinity- increasing substitutions.

I believe, I believe that reading the work is tiring. In my opinion the work should be much improved.

I admit this manuscript with major revision.

In particular:

- I would like to suggest to the authors to modify the introduction. It should contain an introductory part and then go into details.

- I would suggest trying to introduce mutations.

- The resolution of the figures is poor

- the discussion has no logical thread

- even if there are results, they are presented badly

- the work must be rewritten

The data is not well organized

Reviewer #2: The manuscript by Rajkovic et al. entitled “Amino acid substitutions in human growth hormone affect secondary structure and receptor binding” presents a study of the interaction between the human growth hormone and its receptor. Using a computational and experimental approach to find an action mechanism of 21 discovered mutations of hGH and their impact on the complex with hGHR. The mutations were modeled by FoldX. The mutations expressed and purified were experimentally studied with SPR and CD. About a previously known E174A mutation, the authors propose a mechanistic explanation for the increase in affinity, that involves part of a zinc-binding triad and packed in a coiled-coil interface. The aim of this work is interesting (and this reviewer appreciates it), although there are many inconsistencies between the theoretical and the experimental data that should be addressed in more detail. The paper is not well done in almost all parts, and the manuscript in its present form demands a deep revision before it can be published, so this reviewer suggests to reject the paper.

The paper should be modified in some parts.

Major issues:

1) The abstract needs to be revised according to the following suggestions.

2) The introduction section is too poor and unclear; nothing is reported on the aim of the paper.

3) The section Materials and Methods need to be reorganized and reordered. In this way is difficult to read and understand the methodologies used in the paper. About the CD, there are some inconsistencies regards the sample preparation (for example: by 2 cycles of dialysis is not possible to remove all the salt in the sample) and nothing is reported about the analysis of the collected data. Where is table 1? The SPR results are not well described.

4) As a consequence of the point before also the discussion section needs to be revised, this paragraph is really poor, no correlations were reported between the methodologies used, just hypothesis and open questions are reported. Where is table 1?

9) The conclusions section is expected by the reader, but in this paper is not present, mainly because there is nothing to conclude.

Minor issue:

1) Please renumbered the references, is difficult to consult them.

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Reviewer #1: No

Reviewer #2: No

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-22-33788R1Amino acid substitutions in human growth hormone affect secondary structure and receptor bindingPLOS ONE

Dear Dr. Samuel Coulbourn Flores,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the minor points raised during the review process.

Please submit your revised manuscript by March 9, 2023. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Sabato D'Auria

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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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: I Don't Know

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: Dear Authors, the data has been better organized, even if the images of the graphs have low resolution.

Reviewer #2: (No Response)

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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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Amino acid substitutions in human growth hormone affect secondary structure and receptor binding

PONE-D-22-33788R2

Dear Dr. Samuel Coulbourn Flores,

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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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Sabato D'Auria

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: