Tracing the function expansion for a primordial protein fold in the era of fold-based function prediction: β-trefoil

Moushmi Goswami; Subhashini Srinivasan

doi:10.1371/journal.pone.0320177

Peer Review History

Original SubmissionFebruary 21, 2025
Decision Letter - Sushil Mishra, Editor Dear Dr. Srinivasan, 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 May 31 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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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: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: N/A Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: No Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: No Reviewer #2: Yes ****** Reviewer #1: The authors present an interesting investigation into the application of AlphaFold and fold-recognition software for the efficient discovery and functional annotation of novel protein folds. They argue that this approach, leveraging structural data in conjunction with sequence homology, enhances functional annotation. While the methodology is generally well-presented, several concerns require attention. Major Concerns: 1. Code Availability: The absence of publicly available code limits the reproducibility and broader applicability of the presented methodology. Providing the code as a tool for general use would significantly enhance the study's impact. 2. Trefoil Fold Representation and Analysis: A detailed graphical representation of the trefoil fold's architectural characteristics is essential. Furthermore, a comprehensive explanation of the eighteen variant structures used in the second iteration is required. This explanation should clearly articulate their distinct features compared to the canonical trefoil domain and provide a robust justification for their selection as representative instances. 3. Mutation Tolerance Analysis: The claim that "trefoil folds appear to be highly tolerant of amino acid changing mutations" requires substantiation through a mutation survey analysis. 4. CATH Database Perspective: A thorough discussion of the CATH database's classification and perspective on the trefoil fold is necessary. Minor Concerns: • Language and Presentation: o The language requires refinement to achieve a more formal and precise style. o Sentence structure should be improved to eliminate ambiguity. o Data presentation, including table conciseness, figure clarity, and image resolution, needs enhancement. • "Thirty-two Functions": The mention of "thirty-two functions" lacks context and reference. Clarification is needed regarding their relevance and potential categorization. • Wording Improvements: o Replace the informal phrases "decades where nothing happened" and "slowly but surely" with more precise and academic language when describing the progression of homology-based structure prediction. o The list of novel domain architectures requires clearer and more concise presentation. • Approach Clarity: The description of the "bottom-up approach" is confusing and lacks clarity. A more precise explanation of the methodology is needed. • Sentence Refinement: The sentence describing the improvement of structure prediction methods through multiple templates, secondary structure prediction, ab initio loop modeling, and backbone-dependent rotamer libraries requires a more streamlined and coherent structure. In summary, while the study presents a valuable methodology, addressing these concerns, particularly regarding code availability, trefoil fold analysis, mutation tolerance, and language refinement, is essential for improving the rigor and impact of the research. Reviewer #2: This is an interesting analysis using the beta-trefoil fold. It highlights several interesting aspects of of evolution of this fold, notably that it is likely on a trajectory of convergent evolution. The overall writing style is also fine and it is clear why they did what they did. ****** 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: Yes: Neshatul Haque 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. https://doi.org/10.1371/journal.pone.0320177.r001
Revision 1
30 May 2025 Author Response Dear Reviewers, Below find point-by-point response to the reviewers. 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: N/A 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: No Most of the data presented are from public domain such as PDB and AFdb. We do provide accession IDs for all the proteins we identified, which are available under the respective databases. We have now provided the command-lines used in our research. 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 We have changed the flow and readability significantly.. 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: The authors present an interesting investigation into the application of AlphaFold and fold-recognition software for the efficient discovery and functional annotation of novel protein folds. They argue that this approach, leveraging structural data in conjunction with sequence homology, enhances functional annotation. While the methodology is generally well-presented, several concerns require attention. THANK YOU. Major Concerns: 1. Code Availability: The absence of publicly available code limits the reproducibility and broader applicability of the presented methodology. Providing the code as a tool for general use would significantly enhance the study's impact. We have used publicly available tools and we have now provided command lines along with options used in this work. (lines 1153-1159 and 1169-1194) 2. Trefoil Fold Representation and Analysis: A detailed graphical representation of the trefoil fold's architectural characteristics is essential. Furthermore, a comprehensive explanation of the eighteen variant structures used in the second iteration is required. This explanation should clearly articulate their distinct features compared to the canonical trefoil domain and provide a robust justification for their selection as representative instances. ● We have shown the structural arrangement of beta-hairpins, which constitutes the trefoil in the nineteen functional spoke in Figure 1. ● We have now explained it in the text (lines 333-337). It says that we selected to represent diverse domain architecture as shown in the outmost layer in Figure 1. ● We have now compared the representative structures with 5bow, which has a canonical trefoil fold, and report r.m.s.d and sequence identity within each functional spoke in Figure 1. The diversity in r.m.s.d provides the justification for selection as representative for a given function. 3. Mutation Tolerance Analysis: The claim that "trefoil folds appear to be highly tolerant of amino acid changing mutations" requires substantiation through a mutation survey analysis. Since, a structure-based sequence alignment of diverse trefoil domains identified here represent mutations accepted during natural selection, we have computed Shannon’s entropy at each alignment position with less than 10 % gaps. The high entropy at 94% at the positions suggest a fold highly tolerant to amino acid changing mutations. Even at the positions with low entropy, multiple hydrophobic amino acids are tolerated. We have now added and whole new section titled “Interrogation of mutation tolerance in β-trefoil” this to the manuscript along with Figure 6 in lines 747-765. 4. CATH Database Perspective: A thorough discussion of the CATH database's classification and perspective on the trefoil fold is necessary. We have now added CATH classification both in the last paragraph of the Introduction and Discussion sections in lines 219-223 and 783 respectively. We believe that the CATH database relies on annotation based on the functions of the major domains, which in majority of the cases, are more than 50% of the trefoil domain and are also more conserved at the sequence-level. Since Trefoil domain is very divergent at the sequence-level, often misses annotation based on sequence comparison. Here, we have identified many proteins from PDB and AFdb that contain at least one trefoil domain. See lines 223-228. Minor Concerns: • Language and Presentation: o The language requires refinement to achieve a more formal and precise style. o Sentence structure should be improved to eliminate ambiguity. We have read and changed the style to make the manuscript more readable as may be clear from tracked changes throughout the manuscript. o Data presentation, including table conciseness, figure clarity, and image resolution, needs enhancement. We have removed the pie charts from the Table 1 and all figures are produced at high-resolutions and submitted separately for publication. • "Thirty-two Functions": The mention of "thirty-two functions" lacks context and reference. Clarification is needed regarding their relevance and potential categorization. Thanks for pointing this out. In line 32-36 under the Abstract we have elaborated this with context and reference. • Wording Improvements: We have done this throughout the document. o Replace the informal phrases "decades where nothing happened" and "slowly but surely" with more precise and academic language when describing the progression of homology-based structure prediction. We have replaced this in lines 155-163. o The list of novel domain architectures requires clearer and more concise presentation. • Approach Clarity: The description of the "bottom-up approach" is confusing and lacks clarity. A more precise explanation of the methodology is needed. Thanks for pointing this out. Now we have added what we consider top-down to clarify why we call our approach bottom-up in lines 201-213. In the top-down approach one clusters all structures into fold/functional classes using structure-comparison similar to CATHdb clustering using sequence-based comparison. In one such applications (top-down) they report 63 million clusters based on structure comparison from AFdb. Again for multidomain proteins containing trefoil fold along with addition fold with higher sequence identify, may misplace trefoil folds into different clusters. Here we take a single fold to explore the entire database from functional classification point of view giving preference to trefoil domain and drag other domains with it for annoation • Sentence Refinement: The sentence describing the improvement of structure prediction methods through multiple templates, secondary structure prediction, ab initio loop modeling, and backbone-dependent rotamer libraries requires a more streamlined and coherent structure. We have modified the sentence to read better. Line 151-154. In summary, while the study presents a valuable methodology, addressing these concerns, particularly regarding code availability, trefoil fold analysis, mutation tolerance, and language refinement, is essential for improving the rigor and impact of the research. Thank you for your valuable comments. To our knowledge we have addressed all these concerns. Reviewer #2: This is an interesting analysis using the beta-trefoil fold. It highlights several interesting aspects of of evolution of this fold, notably that it is likely on a trajectory of convergent evolution. The overall writing style is also fine and it is clear why they did what they did. THANK YOU. 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: Yes: Neshatul Haque Reviewer #2: No Attachments Attachment Submitted filename: TrefoilRebuttal.docx https://doi.org/10.1371/journal.pone.0320177.r002
Decision Letter - Sushil Mishra, Editor Tracing the function expansion for a primordial protein fold in the era of fold-based function prediction: beta-trefoil PONE-D-25-08365R1 Dear Dr. Srinivasan, 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. If you have any questions relating to publication charges, 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 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, Sushil Mishra, Ph.D Academic Editor PLOS ONE Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #1: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #1: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes ****** Reviewer #1: The authors have addressed all my concerns, and the manuscript is now ready for publication. However, I couldn't locate lines 1153-1159 and 1169-1194 as referenced. While some commands are present, the code sharing could be improved by using standard formats like R, Python, or shell scripts. Although I couldn't always match the suggested changes to specific line numbers, I did confirm their implementation in the manuscript. Finally, the images in the PDF I received were unclear; hopefully, the journal has higher-resolution versions. ****** 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: Yes: Neshatul Haque ******** https://doi.org/10.1371/journal.pone.0320177.r003
Formally Accepted
Acceptance Letter - Sushil Mishra, Editor PONE-D-25-08365R1 PLOS ONE Dear Dr. Srinivasan, 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. 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 Dr. Sushil Mishra Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0320177.r004

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