Structural analysis of the manganese transport regulator MntR from Bacillus halodurans in apo and manganese bound forms

Myeong Yeon Lee; Dong Won Lee; Hyun Kyu Joo; Kang Hwa Jeong; Jae Young Lee

doi:10.1371/journal.pone.0224689

Peer Review History

Original SubmissionSeptember 6, 2019
11 Oct 2019 Decision Letter - Eugene A. Permyakov, Editor PONE-D-19-24432 Structural analysis of the manganese transport regulator MntR from Bacillus halodurans in apo and manganese bound forms PLOS ONE Dear Dr. Jae Young Lee 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. ============================== ACADEMIC EDITOR: Please try to improve your manuscript according to the reviewers' criticism. ============================== We would appreciate receiving your revised manuscript by Nov 25 2019 11:59PM. 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. 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This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Eugene A. Permyakov, Ph.D., Dr.Sci. 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 2. We found text overlap between the current submission and your previously published works outside the Method section: - https://www.nature.com/articles/s41598-018-31676-z - http://scripts.iucr.org/cgi-bin/paper?S1399004714004118 - http://koreascience.or.kr/article/JAKO201425560113942.page In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed." 3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. 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: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: N/A ****** 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: Yes 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 paper presents crystal structure data on a further member of the MntR family of magnesium transporter regulators. As expected, the structure is largely similar to previously reported structures. But there are some new details here regarding metal binding sites. The crystallographic work appears to be mainly in order, but with one issue noted below. I have one substantive critique of the analysis and some relatively minor comments and suggestions prior to publication. Important concern: Nothing is said by the authors about anomalous scattering. They might have generated stronger anomalous signal from the Mn had they collected data at a longer x-ray wavelength, and in view of this it’s not clear why the wavelength was chosen to be at 0.979 Ang (which is a good choice for selenium studies, not applicable here). Maybe the authors could get lucky and anomalous signal in a map would be detectable even at the sub-optimal wavelength where they collected the data (f’’ for Mn looks to be about 1.3 electrons at 0.979 Ang). The authors should calculate an anomalous difference map to see if the binuclear site shows up. If it does, then they could go on to look at the other, supposedly unoccupied, site. There they inferred a Mg ion, but as noted above it doesn’t make a lot of sense that a Mg would be there when it wasn’t there in the apo form. It’s conceivable that the density in the other site is a partially occupied Mn (not Mg). And if so maybe an anomalous map would give an indication. Having a better assignment of what’s in the other site would improve the findings of the paper. Minor points: 1) There are some places where an editing for English usage will be important (e.g. recombination vs recombinant). 2) Lines 61-69 in the Introduction discuss some structural details of the MntR protein. As it is written is sounds like the authors might be describing results from the present study (which would be out of place in the Introduction), whereas they are presumably discussing what is known about the structure from prior work. This should be made clearer, e.g. “From previous structural studies it is understood that…” 3) The R-values are reported with a number of significant digits that seems one too many. 4) The legend to Fig 2 needs to explain better the distinction (in panels b AND c) between the site where the binuclear site is seen vs the manganese site. 5) The authors show 2Fo-Fc and omit (difference) maps to show the metal sites. This is ok. But they have the opportunity here to also calculate and show maps that are based on differences between the Fobs from the two data sets (i.e. Fobs(+metal) – Fobs(-metal), phased with a model without metals). This kind of map is a more direct examination of the observed differences and less model-dependent. The authors should examine this kind of map. Presumably it will show up the binuclear Mn cluster just as the Fo-Fc map did. But the authors might discern more about the unexpected magnesium site. Currently they say that no magnesium is seen in the ‘apo’ structure, despite the high concentration of Mg in the crystallization conditions. If Mg is really absent from that site in the apo form, then they should see a positive peak there in the Fo(+metal) – Fo(-metal) map. Reviewer #2: This manuscript describes MntR (from Bacillus halodurans) structures in the apo and metal bound states. The assignment of Mn2+ and Mg2+ in the binding sites was not solid. It needs to be substantialized by additional measurements such as X-ray anomalous signals. The reported structures do not provide new biological insights in addition to the many existing MntR structures. ****** 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.0224689.r001
Revision 1
18 Oct 2019 Author Response Dear Eugene A. Permyakov, Ph.D. Academic Editor, PLOS ONE Reference code PONE-D-19-24432 TITLE: “Structural analysis of the manganese transport regulator MntR from Bacillus halodurans in apo and manganese bound forms” Thank you very much for your kindly informing me of the reviewer’s comments on my manuscript submitted for a possible publication in the PLOS ONE. Please find the uploaded revised manuscript at the web address. The revised manuscript takes into account all the comments made by the reviewers and editor. A separate list is attached to this letter to describe all the changes in detail. Sincerely yours, List of changes by reviewer 1 1. << Important concern: Nothing is said by the authors about anomalous scattering. They might have generated stronger anomalous signal from the Mn had they collected data at a longer x-ray wavelength, and in view of this it’s not clear why the wavelength was chosen to be at 0.979 Ang (which is a good choice for selenium studies, not applicable here). Maybe the authors could get lucky and anomalous signal in a map would be detectable even at the sub-optimal wavelength where they collected the data (f’’ for Mn looks to be about 1.3 electrons at 0.979 Ang). The authors should calculate an anomalous difference map to see if the binuclear site shows up. If it does, then they could go on to look at the other, supposedly unoccupied, site. There they inferred a Mg ion, but as noted above it doesn’t make a lot of sense that a Mg would be there when it wasn’t there in the apo form. It’s conceivable that the density in the other site is a partially occupied Mn (not Mg). And if so maybe an anomalous map would give an indication. Having a better assignment of what’s in the other site would improve the findings of the paper. >> - We calculated an anomalous difference map with Mn2+-bound BhMntR data and found the two clear peaks showing the manganese binuclear ions in metal binding site. The additional information was added on lines 200-202, page 10, stating that “We obtained Mn2+-bound crystals by soaking with 50 mM MnCl2 in apo crystals, and confirmed using an omit map and an anomalous map showing two peaks at the counter levels even at 5σ (Fig 2A and S2 Fig).”. In addition, the requested anomalous maps of Mn2+-bound BhMntR structures was added to supplemental figure (S2 Fig). 2. << There are some places where an editing for English usage will be important (e.g. recombination vs recombinant). >> - The English spelling was carefully checked and corrected on line 104, page 5, stating that “recombinant”. 3. << Lines 61-69 in the Introduction discuss some structural details of the MntR protein. As it is written is sounds like the authors might be describing results from the present study (which would be out of place in the Introduction), whereas they are presumably discussing what is known about the structure from prior work. This should be made clearer, e.g. “From previous structural studies it is understood that…” >> - The requested information was added as recommended on lines 62-69, page 3, stating that “From previous structural studies it is known that the metal binding site of B. subtilis MntR consists of several residues including Asp8 and Glu11 in domain 1, and His77, Glu99, Glu102 and His103 in domain 2.” 4. << The R-values are reported with a number of significant digits that seems one too many. >> - The R-values were corrected as recommended in text and table. 5. << The legend to Fig 2 needs to explain better the distinction (in panels b AND c) between the site where the binuclear site is seen vs the manganese site. >> - The original Fig2 legend sentence was changed on lines 212-217, page 10, stating that “(B) Metal binding site with binuclear manganese ions. The coordination with binuclear manganese ions and the distance between Mnc and the backbone carbonyl oxygen of Glu99 are shown in yellow and red, respectively. (C) Metal binding site with a magnesium ion. Unlike binuclear manganese ions binding, the magnesium ion (gray) forms a mononuclear cluster and the His77 made a hydrogen bond with Glu81 via a water (red).” 6. << The authors show 2Fo-Fc and omit (difference) maps to show the metal sites. This is ok. But they have the opportunity here to also calculate and show maps that are based on differences between the Fobs from the two data sets (i.e. Fobs(+metal) – Fobs(-metal), phased with a model without metals). This kind of map is a more direct examination of the observed differences and less model-dependent. The authors should examine this kind of map. Presumably it will show up the binuclear Mn cluster just as the Fo-Fc map did. But the authors might discern more about the unexpected magnesium site. Currently they say that no magnesium is seen in the ‘apo’ structure, despite the high concentration of Mg in the crystallization conditions. If Mg is really absent from that site in the apo form, then they should see a positive peak there in the Fo(+metal) – Fo(-metal) map.>> - We also calculated the Fo(+metal) – Fo(-metal) map. The two positive peaks were clearly shown around metal binding site indicating the binuclear manganese ions but no clear map was shown in magnesium binding site in the other subunit. In addition, the anomalous maps were calculated and shown in Supporting Information (S2 Fig). List of changes by reviewer 2 1. << This manuscript describes MntR (from Bacillus halodurans) structures in the apo and metal bound states. The assignment of Mn2+ and Mg2+ in the binding sites was not solid. It needs to be substantialized by additional measurements such as X-ray anomalous signals. The reported structures do not provide new biological insights in addition to the many existing MntR structures. >> - We calculated an anomalous difference map with Mn2+-bound BhMntR data and found the two clear peaks showing the binuclear manganese ions in metal binding site. The additional information was added on lines 200-202, page 10, stating that “We obtained Mn2+-bound crystals by soaking with 50 mM MnCl2 in apo crystals, and confirmed using an omit map and an anomalous map showing two peaks at the counter levels even at 5σ (Fig 2A and S2 Fig).”. In addition, the requested anomalous maps of BhMntR structures was added to Supplementary figure (S2 Fig). 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 >>. Our revised manuscript was generated with PLOS ONE style templates as requested. 2. << We found text overlap between the current submission and your previously published works outside the Method section: - https://www.nature.com/articles/s41598-018-31676-z - http://scripts.iucr.org/cgi-bin/paper?S1399004714004118 - http://koreascience.or.kr/article/JAKO201425560113942.page In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed." >> - We carefully checked text overlaps between the current submission and your previously published works outside the Method section. Main overlaps were found in Acknowledgement Section and the other minor overlap was rephrased on lines 291-293, page 13, stating that “Although the functional assignment of metal binding site for BhMntR is tentative, this structural model is applicable to other MntR homologous structures.” 3. << We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. >> - We already provided the accession numbers (6KTA and 6KTB) for PDB coordinates and structure factors and those data were included in Supporting Information. Attachments Attachment Submitted filename: rebuttal_letter.docx https://doi.org/10.1371/journal.pone.0224689.r002
21 Oct 2019 Decision Letter - Eugene A. Permyakov, Editor Structural analysis of the manganese transport regulator MntR from Bacillus halodurans in apo and manganese bound forms PONE-D-19-24432R1 Dear Dr. Jae Young Lee, We are pleased to inform you that your manuscript 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, Eugene A. Permyakov, Ph.D., Dr.Sci. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0224689.r003
Formally Accepted
24 Oct 2019 Acceptance Letter - Eugene A. Permyakov, Editor PONE-D-19-24432R1 Structural analysis of the manganese transport regulator MntR from Bacillus halodurans in apo and manganese bound forms Dear Dr. Lee: 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 Prof. Eugene A. Permyakov Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0224689.r004

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