Exploring the molecular structures that confer ligand selectivity for galanin type II and III receptors

Yoo-Na Lee; Arfaxad Reyes-Alcaraz; Seongsik Yun; Cheol Soon Lee; Jong-Ik Hwang; Jae Young Seong

doi:10.1371/journal.pone.0230872

Peer Review History

Original SubmissionDecember 9, 2019
31 Jan 2020 Decision Letter - Pere Garriga, Editor PONE-D-19-33974 Exploring the molecular structures that confer ligand selectivity for galanin type II and III receptors PLOS ONE Dear Dr Seong, 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 points raised during the review process. Based on the comments of the two reviewers and on my own reading, the manuscript is deemed scientifically sound but you should pay close attention to all the issues raised by the reviewers. In particular, it is noted that the manuscript reports on a solid molecular biology study but the variability of the conformational profile is not considered in discussing the results. This point is crucial and should be carefully discussed. Also, the possibility to conduct complementary analysis, like NMR, in order to check that the profile does not change should be taken into account. We would appreciate receiving your revised manuscript by March 2, 2020. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. 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We look forward to receiving your revised manuscript. Kind regards, Pere Garriga Academic Editor PLOS ONE Journal Requirements: 1. When submitting your revision, we need you to address these additional requirements. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf [Note: HTML markup is below. Please do not edit.] 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: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 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 ****** 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: No Reviewer #2: Yes ****** 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: This is an interesting manuscript that tries to elucidate the molecular specificities of galanin type 2 and 3 receptors for their ligands. The authors should be commended for the large body of work done and using different techniques. I have some suggestions that can improve the manuscript. 1. English needs significant improvement. For example, in the abstract they state "we identified residues in GALR2 that "attract" SG2A and residues in GALR3 that "avoid" SGA2. How can amino acids "attract" or "avoid" ligands? Suggest using scientific terms such as interact, inhibit etc., 2. Should show sequence of GAL peptide in Figure 1. 3. How similar is the Qu-SPX peptide compared to GAL? Is SPX different from GAL peptide in only the four amino acids replaced or would more complete substitution with amino acids from GAL peptide in Qu-SPX rescue GALR3 activity for the Qu-SPX peptide? This might explain why the GALR3 septuple mutant shows very subtle activity towards Qu-SPX. Reviewer #2: The authors in the present work, report the results of a study aimed at identifying residues of the GAL2R and GalR3 galanin receptors that permit ligand selectivity of a spexin analog. For this purpose, the authors carry out GAL2R and GAL3R receptor swapping and site directed mutagenesis studies. The main flaw of the manuscript regards the interpretation of the results. In the manuscript, the discussion is carried out considering that the observed differences in the binding affinity of the diverse analogs can be interpreted in terms of ligand-receptor interactions. However, peptides are flexible and their conformational profile is affected by the sequence. We may consider that one amino acid substitution may not change much the conformational profile of the analogs, but four as in Qu-SPX is risky. Accordingly, the discussion should be carried out considering a loss of an interaction and/or to a different ligand conformational behavior. These considerations are lacking in the present manuscript. Minor details: The sentence: “The strong response to WT SPX was retained by all of the GALR2/3 series a to c chimeric receptors, whereas it was drastically weakened in the GALR2/3 series d and e chimeric receptors” requires revision. I suggest referring to SPX in the same way all through the manuscript. For example, WT SPX ****** 6. 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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0230872.r001
Revision 1
29 Feb 2020 Author Response Dear Editor We thank you for allowing us to revise our manuscript entitled “Exploring the molecular structures that confer ligand selectivity for galanin type Ⅱ and Ⅲ receptors” [PONE-D_19_33974]. We carefully revised the manuscript to incorporate comments and concerns raised by the editor and reviewers. The major changes are as follows. 1. To address the comment from the editor and reviewer 2, we conducted molecular dynamics (MD) simulation study to predict the solution structures of SPX and Qu-SPX. MD-resolved structures of SPX and Qu-SPX differ each other, although both peptides may not have stable three-dimensional structures in solution. This result raises a possibility that conformations of Qu-SPX different from those of SPX may hamper full recovery of the potency toward the septuple-mutant GALR3. The result is shown in S2 Fig. As this experiment was conducted by Dr. Chul-Soon Lee we (all authors) decided to include him as a co-author of the article. 2. The GAL sequence is included in Fig 1 as reviewer 1 suggested. 3. The manuscript is revised to meet PLOS ONE's style requirements. ________________________________________ Response to reviewer #1 We appreciate your valuable and helpful comments on our manuscript. Point 1: English needs significant improvement. For example, in the abstract they state "we identified residues in GALR2 that "attract" SG2A and residues in GALR3 that "avoid" SGA2. How can amino acids "attract" or "avoid" ligands? Suggest using scientific terms such as interact, inhibit etc. →We used scientific terms in the revised manuscript. Please see page 2 line26, and line 31. Please see page 4 line 86 line. Please see page 17 line 465, and 466. Point 2 : Should show sequence of GAL peptide in Figure 1. →We added the GAL peptide sequence in the revised Figure 1 (please see Page 5 line 102 and line 104). Point 3 : How similar is the Qu-SPX peptide compared to GAL? Is SPX different from GAL peptide in only the four amino acids replaced or would more complete substitution with amino acids from GAL peptide in Qu-SPX rescue GALR3 activity for the Qu-SPX peptide? This might explain why the GALR3 septuple mutant shows very subtle activity towards Qu-SPX. → We agree on reviewer’s opinion that additional substitutions with amino acids from GAL peptide in Qu-SPX rescue GALR3 activity more. In our previous report (Reyes-Alcaraz et al., 2016, Ref# 26), we explained in detail that four amino acids replacement (Asn5, Ala7, Leu11/Phe11, and Pro13 substitution) in SPX completely abolished potency to GALR3 while mutations in other residues of SPX did not influence GALR2/GALR3 selectivity. Therefore, we focused on these 4 residue substitutions. We include these points in the revised manuscript. (Please see page 4 line 80). In addition, we conducted molecular dynamics (MD) simulation analysis to predict the solution structures of SPX and Qu-SPX. The result shows that structures of SPX and Qu-SPX differ each other, although both peptides may not have stable three-dimensional structures in solution. This result raises a possibility that conformations of Qu-SPX different from those of SPX may hamper full recovery of the potency toward the septuple-mutant GALR3. This notion is included in the revised text. Please see S1 text for Molecular Dynamics and S2 Fig. Response to reviewer #2 We appreciate your valuable and helpful comments on our manuscript. Point 1: The main flaw of the manuscript regards the interpretation of the results. In the manuscript, the discussion is carried out considering that the observed differences in the binding affinity of the diverse analogs can be interpreted in terms of ligand-receptor interactions. However, peptides are flexible and their conformational profile is affected by the sequence. We may consider that one amino acid substitution may not change much the conformational profile of the analogs, but four as in Qu-SPX is risky. Accordingly, the discussion should be carried out considering a loss of an interaction and/or to a different ligand conformational behavior. These considerations are lacking in the present manuscript → To address this point, we tried to predict the solution structures of SPX and Qu-SPX using a molecular dynamics (MD) simulation method. MD trajectory analysis was used for the clustering of peptides. Indeed, MD-resolved structures of SPX and Qu-SPX differ each other, although both peptides may not have stable three-dimensional structures in solution. This result raises a possibility that conformations of Qu-SPX different from those of SPX may hamper full recovery of the potency toward the septuple-mutant GALR3. This notion is included in the revised text. Please see S1 text for Molecular Dynamics and S2 Fig. Minor details: “The strong response to WT SPX was retained by all of the GALR2/3 series a to c chimeric receptors, whereas it was drastically weakened in the GALR2/3 series d and e chimeric receptors” requires revision. I suggest referring to SPX in the same way all through the manuscript. For example, WT SPX → WT SPX is changed to SPX throughout the revised manuscript. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0230872.r002
11 Mar 2020 Decision Letter - Pere Garriga, Editor Exploring the molecular structures that confer ligand selectivity for galanin type II and III receptors PONE-D-19-33974R1 Dear Dr. Seong We are pleased to inform you that your revised manuscript, after taking into account all the reviewers comments, has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Pere Garriga Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0230872.r003
Formally Accepted
16 Mar 2020 Acceptance Letter - Pere Garriga, Editor PONE-D-19-33974R1 Exploring the molecular structures that confer ligand selectivity for galanin type II and III receptors Dear Dr. Seong: I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Pere Garriga Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0230872.r004

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