-->PONE-D-26-08447-->-->Bisphenol BPAF and BPC are agonists for estrogen receptor ERα but antagonists for N-terminal domain-lacking ERα-->-->PLOS One

Dear Dr. Liu,

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.

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Sayyed Mohammad Hadi Alavi

Academic Editor

PLOS One

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“This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI grant nos. 25K15462, 22K12395, 19K12340, 15K00557, and 25740024 (to X.L.) and 22221005 and 15H01741 (to Y.S.). This work was also supported in part by a Health and Labour Sciences Research Grant for Research on the Risk of Chemical Substances from the Ministry of Health, Labor, and Welfare of Japan, under contract/grant no. H20-Chemistry-General-003 (to Y.S.).”

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“This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI grant nos. 25K15462, 22K12395, 19K12340, 15K00557, and 25740024 (to X.L.) and 22221005 and 15H01741 (to Y.S.). This work was also supported in part by a Health and Labour Sciences Research Grant for Research on the Risk of Chemical Substances from the Ministry of Health, Labor, and Welfare of Japan, under contract/grant no. H20-Chemistry-General-003 (to Y.S.).”

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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:

“This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI grant nos. 25K15462, 22K12395, 19K12340, 15K00557, and 25740024 (to X.L.) and 22221005 and 15H01741 (to Y.S.). This work was also supported in part by a Health and Labour Sciences Research Grant for Research on the Risk of Chemical Substances from the Ministry of Health, Labor, and Welfare of Japan, under contract/grant no. H20-Chemistry-General-003 (to Y.S.).”

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Additional Editor Comments:

Dear Dr. Xiaohui Liu

Thank you very much for your submission to PLOS ONE. Your MS was reviewed by two reviewers with expertise in Chemical Biology/Receptor Biology. Both provided very valuable comments that need your careful consideration upon revision. In addition to their comments, please find my comments below to include into revision. I am looking forward to receiving your revision.

Very best regards

AE COMMENTS:

- Please follow the PLOS ONE guidelines to organize the MS.

- Please clarify why P < 0.001 was considered for significant difference; usually P < 0.05 is taken into account.

- Did you check the normal distribution and homogeneity of variance for t-Test?

- Was not it possible to show the significant differences on Figures or in Tables?

- Please add number of experiments performed for each assay, both in the method, in the legend of figures, and in the title of Tables.

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- Please clarify "Competing Interests Statement"

- Please provide Ethical statement.

[Note: HTML markup is below. Please do not edit.]

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

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

Reviewer #1: Yes

Reviewer #2: 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: No

Reviewer #2: 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: No

Reviewer #2: 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: General comment :

1. All the figures, including those as supplementary ones, have to be presented with a better resolution.

Minor comment :

1. Line 110, Begin the sentence with: Indeed,

2. Line 137, Remove the last dot.

3. For clarity, add for Figure 1C, add below the structures the corresponding E2, 4-OH, ICI, BPA, BPAF, BPC

4. Line 250 to 252, simplify the sentence. Remove the semicolon in line 251 and write again the sentence

5. Line 331, “eventually reaching baseline”. Remove this part and be more precise.

6. Remove sentence from line 445 to 446.

7. For Figure 7, indicate the helices number from 1 to 12

Reviewer #2: This manuscript describes and investigation of binding and functional properties of bis-phenol A (BPA), the bis-trifluoromethylated analog (BPAF) and a gem-dichloroethylene analog (BPC) with wild-type Estrogen Receptor alpha and the modified protein that lacks the N-terminal domain [NTD(AF-1)]. The major findings are that the modified protein retains a significant amount of transcriptional activity relative to the full-length protein, however, while the simple parent compound BPA is an agonist for both proteins, the halogenated small molecules behave as antagonists in the absence of the N-terminal domain. The paper is written clearly, the experiments are described in sufficient detail, and the references are appropriate. This concluding statement is unsatisfactory and needs further elaboration: “The driving force for this H12 translocation is likely due to the halogen atoms present in BPAF and BPC, and its effect is only fully observed upon NTD(AF-1) removal.” The significance of these findings could be clarifies and strengthened for the reader by addressing several related questions. What is the broader context of these specific results of bisphenol-related environmental estrogens within the classical example of estrogen receptor alpha (ERα) as a ligand-dependent transcription factor and why should we care? What are the major structural/steric differences between these compounds evaluated and how are the halogens involved, given their ability to modify properties such as acidity through induction, and the capacity of fluorinated alkanes to act as weak hydrogen bond acceptors? Are there known differences in the biological or toxicological activity profiles of the compounds? The fact that there are numerous clinically significant mutations in the ER’s ligand-binding domain that are relevant for cancer raises the potential here to include discussion of known mutations and structural variants in or involving the N-terminal domain (NTD) that contribute to pathological conditions. One other minor comment is that in Table 1 the column titled relative “potency” should be changed to “affinity”.

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-->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

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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.]

To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures

You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation.

NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

Bisphenol BPAF and BPC are agonists for estrogen receptor ERα but antagonists for N-terminal domain-lacking ERα

PONE-D-26-08447R1

Dear Dr. Liu,

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,

Sayyed Mohammad Hadi Alavi, PhD

Academic Editor

PLOS One

Additional Editor Comments (optional):

Dear Dr Liu

Thank you for your submission to PLOS ONE. You revision has been accepted for publication.

Very best regards

Hadi Alavi

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: General comment :

1. The authors are carefully addressed my comments and improved significantly their revised manuscript.

Especially :

a) The labelling of compound name for the structures is now inserted in Fig 1C.

b) The resolution of figures is clearly better.

The authors have further stated their major findings and indicate the limit of their study, which was required.

Minor Comment:

- Page 3, line 80, Remove Cryo-EM in parenthesis as it appears one time in the manuscript

Reviewer #2: As stated in part 1. All comments have been addressed. "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." The electronic review form is indicating the the minimum character count is not met, and the form will not proceed to submission, so I am adding this additional text to meet the minimum character count.

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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

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