A putative membrane-associated YdjB-like protein from Salmonella enterica exhibits a non-canonical ACT-like fold

Ju Hyeong Kim; Yong Jun Kang; Young Woo Kang; So Eun Park; Hyo Been Jin; Eunmi Hong; Hyun Ho Park

doi:10.1371/journal.pone.0336436

Peer Review History

Original SubmissionAugust 20, 2025
15 Sep 2025 Decision Letter - Georgios Pantouris, Editor Dear Dr. Park, 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. Please submit your revised manuscript by Oct 30 2025 11:59PM. 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. 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'. 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Further consideration is dependent on these concerns being addressed. 3. Thank you for stating in your Funding Statement: “. This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2025-02316334 and RS-2024-00344154).” Please provide an amended statement that declares all the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now. Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement. Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf. 4. Thank you for stating the following financial disclosure: “. This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2025-02316334 and RS-2024-00344154).” Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 5. Thank you for stating the following in the Acknowledgments Section of your manuscript: “We thank the staff at the 5C beamline at the Pohang Accelerator Laboratory (Pohang, Korea) for their assistance with data collection. This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2025-02316334 and RS-2024-00344154).” We note that you have provided funding information that is 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: “. This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2025-02316334 and RS-2024-00344154).” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 6. 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When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. 8. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 9. 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. [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? 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 Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: 1. The functional role of the protein remains confusing. No biochemical or in vivo assays were performed to validate the proposed enzymatic or regulatory function. 2. The annotation as “YdjB-like” is potentially confusing, as the protein is distinct from the known nicotinamidase YdjB (now PncA). 3. The evolutionary and physiological relevance of the protein is not explored beyond structural conservation. 4. The functional implications of the conserved groove or dimer interface are not experimentally explored. 5. The membrane localization model is speculative and not supported by cellular localization data. 6. There are minor grammatical issues and awkward phrasing (e.g., “the structure is somewhat similar to aspartokinase at first glance…”). 7. The title is overly long and could be more concise while retaining key information. Reviewer #2: This is an interesting article, I have some considerations and questions. Fist E.coli was used for transformation, however, it's know that coli has its own YdjB-like protein, there's any assay to support no background interference or Protein-Protein interactions? Please, clarify in the paper why kanamycin was used? There is a resistant sequency in the plasmid? There is any reason that only the soluble region was used and the N-terminal transmembrane region was excluded? There is some hypothesis, literature based, for the functional Implications? There is experimental evidence for membrane association or only bioinformatic predictions? Should the methodology strengths and weaknesses be cited in the article? ****** what does this mean? ). 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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 https://doi.org/10.1371/journal.pone.0336436.r001
Revision 1
20 Sep 2025 Author Response Dear Editor, We have revised our manuscript based on the reviewers’ suggestions. Thank you very much for your time and effort in editing our manuscript, and we hope that it is now suitable for publication in “Plos One”. Sincerely, Hyun Ho Park, Ph.D. Professor of Pharmacy Reviewers' Comments to Author: Reviewer 1: We thank the reviewer for the constructive comments on our work. Comments: 1. The functional role of the protein remains confusing. No biochemical or in vivo assays were performed to validate the proposed enzymatic or regulatory function. Response: We agree with the reviewer’s comment. Our current manuscript provides new structural insights into a YdjB-like protein. Although the in vivo function and enzymatic properties of this uncharacterized protein remain unknown, we believe these are important topics for future investigation. At present, due to the lack of any existing functional information, further studies must accumulate before reliable functional predictions can be made. At that point, biochemical and in vivo studies will indeed be essential. Nevertheless, we believe that our present work, as the first to report structural features of this functionally uncharacterized protein, offers meaningful and valuable information. 2. The annotation as “YdjB-like” is potentially confusing, as the protein is distinct from the known nicotinamidase YdjB (now PncA). Response: Yes, that is correct. We also agree with this comment from the reviewer. The name “YdjB-like protein” was not assigned by our research team; rather, it is the name currently listed in major gene annotation databases such as UniProt. Therefore, we specifically emphasized in our manuscript that, despite the name, this YdjB-like protein is structurally distinct from YdjB. 4. The functional implications of the conserved groove or dimer interface are not experimentally explored. Response: As mentioned in our response to Comment 1, we agree that if functional studies on this protein are to be conducted in the future, this would indeed be an essential and important research direction. We sincerely thank the reviewer for providing this valuable idea. 5. The membrane localization model is speculative and not supported by cellular localization data. Response: We fully agree that this topic ultimately needs to be validated in a cellular context. However, we would like to reiterate that the focus of our current study is the structural characterization of this protein, highlighting its novel architecture and structural differences from YdjB. In addition, we present preliminary bioinformatics data suggesting that this uncharacterized protein may localize to the membrane, despite the lack of known function. Our laboratory is currently conducting a detailed functional analysis of this protein, and we believe that the suggestions made by the reviewer will be highly valuable for that future work. Once again, we sincerely thank the reviewer for the many insightful research suggestions. 6. There are minor grammatical issues and awkward phrasing (e.g., “the structure is somewhat similar to aspartokinase at first glance…”). Response: Based on the reviewer’s suggestion, we have modified the sentence to make the meaning more precise and scientifically clear. 7. The title is overly long and could be more concise while retaining key information. Response: We appreciate the reviewer’s suggestion. We also agreed that the original title was too long. In response to the comment, we have revised the title to make it more concise. The new title is: A putative membrane-associated YdjB-like protein from Salmonella enterica exhibits a non-canonical ACT-like fold. Reviewer 2: We thank the reviewer for the constructive comments on our work. Comments: 1. Fist E.coli was used for transformation, however, it's know that coli has its own YdjB-like protein, there's any assay to support no background interference or Protein-Protein interactions? Response: We understand the reviewer’s concern. However, the YdjB-like protein used in our study was overexpressed and purified from E. coli, allowing us to obtain sufficient amounts of the target protein without being limited by the endogenous YdjB-like protein expressed by E. coli. 2. Please, clarify in the paper why kanamycin was used? There is a resistant sequency in the plasmid? Response: Yes, that is correct. The plasmid vector used in our study carries a kanamycin resistance gene. 3. There is any reason that only the soluble region was used and the N-terminal transmembrane region was excluded? Response: Thank you for the insightful question. In structural studies, it is often necessary to obtain large amounts of soluble protein, which can be particularly challenging for membrane proteins. To address this, it is a common strategy to remove predicted transmembrane domains and focus on the soluble regions for structural characterization. In our study, we followed this approach by removing the N-terminal region predicted to contain the transmembrane domain. This allowed us to successfully express, purify, and determine the structure of the soluble portion of the protein. 4. There is some hypothesis, literature based, for the functional Implications? Response: This protein was initially annotated as a “YdjB-like protein” based on predicted sequence and structural similarity to YdjB. However, as demonstrated in our current study, its structure is entirely different from that of YdjB. This structural divergence suggests that the protein may have a distinct function. To our knowledge, there are currently no published studies describing the biological or biochemical function of this protein. 5. There is experimental evidence for membrane association or only bioinformatic predictions? Response: Our study does not include experimental validation; it only presents bioinformatic predictions. We fully agree that this topic ultimately needs to be validated in a cellular context. However, we would like to reiterate that the focus of our current study is the structural characterization of this protein, highlighting its novel architecture and structural differences from YdjB. In addition, we present preliminary bioinformatics data suggesting that this uncharacterized protein may localize to the membrane, despite the lack of known function. Our laboratory is currently conducting a detailed functional analysis of this protein, and we believe that the suggestions made by the reviewer will be highly valuable for that future work. Once again, we sincerely thank the reviewer for the many insightful research suggestions. 6. Should the methodology strengths and weaknesses be cited in the article? Response: We believe this is an excellent suggestion. In response to the reviewer’s comment, we have added a new paragraph at the end of the “Results and Discussion” section to address both the methodological strengths and limitations of our study, as outlined below. “In this study, we analyzed the YdjB-like protein from Salmonella enterica, which appears to be a putative dimeric membrane-associated protein. Our structural analysis, combined with bioinformatic predictions, revealed that it adopts a fold that is completely distinct from the canonical YdjB structure. Despite these intriguing findings, the absence of data on cellular localization, biochemical characterization, or in vivo functional analysis highlights the need for future studies to investigate the physiological role of this uncharacterized protein” Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. 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 https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf And https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf Response: Done 2. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed: https://www.sciencedirect.com/science/article/abs/pii/S0006291X25008654?via%3Dihub 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. Response: We did. 3. Thank you for stating in your Funding Statement: “. This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2025-02316334 and RS-2024-00344154).” Please provide an amended statement that declares all the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now. Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement. Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf. Response: Done 4. Thank you for stating the following financial disclosure: “. This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2025-02316334 and RS-2024-00344154).” Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. Response: Done 5. Thank you for stating the following in the Acknowledgments Section of your manuscript: “We thank the staff at the 5C beamline at the Pohang Accelerator Laboratory (Pohang, Korea) for their assistance with data collection. This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2025-02316334 and RS-2024-00344154).” We note that you have provided funding information that is 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: “. This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2025-02316334 and RS-2024-00344154).” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. Response: Done 6. Please note that your Data Availability Statement is currently missing the DOI/accession number of each dataset OR a direct link to access each database. If your manuscript is accepted for publication, you will be asked to provide these details on a very short timeline. We therefore suggest that you provide this information now, though we will not hold up the peer review process if you are unable. Response: Included 7. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. Response: We used uncropped gel in the main figures. 8. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. Response: Ok 9. 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. Response: Confirmed Attachments Attachment Submitted filename: Response.docx https://doi.org/10.1371/journal.pone.0336436.r002
30 Sep 2025 Decision Letter - Georgios Pantouris, Editor Dear Dr. Park, Please submit your revised manuscript by Nov 14 2025 11:59PM. 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. 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 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, Georgios Pantouris, Ph.D. Academic Editor PLOS ONE Journal Requirements: 1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 2. 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. [Note: HTML markup is below. Please do not edit.] [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. https://doi.org/10.1371/journal.pone.0336436.r003
Revision 2
14 Oct 2025 Author Response Comment: For transparency, please include the wwPDB validation report generated for your structure. Response: We included the PDB validation report for our structure as requested. Attachments Attachment Submitted filename: Response_auresp_2.docx https://doi.org/10.1371/journal.pone.0336436.r004
26 Oct 2025 Decision Letter - Georgios Pantouris, Editor A putative membrane-associated YdjB-like protein from Salmonella enterica exhibits a non-canonical ACT-like fold PONE-D-25-45368R2 Dear Dr. Park, 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. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support . 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, Georgios Pantouris, Ph.D. Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0336436.r005
Formally Accepted
Acceptance Letter - Georgios Pantouris, Editor PONE-D-25-45368R2 PLOS ONE Dear Dr. Park, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, 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. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Professor Georgios Pantouris Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0336436.r006

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