CryoEM structure and Alphafold molecular modelling of a novel molluscan hemocyanin

Gaia Pasqualetto; Andrew Mack; Emily Lewis; Ryan Cooper; Alistair Holland; Ufuk Borucu; Judith Mantell; Tom Davies; Miriam Weckener; Dan Clare; Tom Green; Pete Kille; Alex Muhlhozl; Mark T. Young

doi:10.1371/journal.pone.0287294

Peer Review History

Original SubmissionJanuary 27, 2023
11 Apr 2023 Decision Letter - Matteo De March, Editor PONE-D-23-02379CryoEM structure and Alphafold molecular modelling of a novel molluscan hemocyaninPLOS ONE Dear Dr. Young, 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. As you can see below both Reviewers positively evaluated your manuscript, which in my opinion could lie at the interface between a methodological work clearly focused on structure and a biological research paper. For the latter, one feels that some information are missed. Indeed, Reviewer #2 correctly suggested the addition of mutational and functional studies could help a better characterization of this protein improving the quality of the work. As pointed out even by this Reviewer (e.g. can explain.., can help..), I believe in this case the new experimental data are not strictly necessary but more recommended. Instead, a deeper structural characterization/discussion of the diverse interfaces responsible for the oligomerization in the result section aligned with an appropriate discussion is required to top up the gap mentioned above. Please submit your revised manuscript by May 26 2023 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. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). 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If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.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? 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: 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: Authors of this paper describe the cryoEM structure of the didecamer and tridecamer froms of a novel hemocyanin from the SLH, and use Alphafold to build large oligomeric proteins. However, I have some questions: 1. In SI page 4, line 11, Fig S4, some of them are identified by the color of the font itself, while others are identified by background highlighting，please use the same identification method to identidy functional units. 2. In paper page 18, line 395-396, "The sequence provided was split into 6 sequences which were a maximum of 800 amino acid in length with a portion overlapping". What is the basis for the truncation except for the maximum limit of Alphafold? Reviewer #2: The work of science is highly interesting which elucidates the 3D-structure of slipper limpet hemocyanin, combining cryoEM with Alphafold modelling, and demonstrated how higher-order oligomers can form in hemocyanins of the KLH class. I have some questions which I feel can help in the development of the work. 1. The authors have explained that they can see higher order oligomers in the sample produced. I would like to know which part of the protein helps in this oligomerisation. SEC-MALS studies as well as mutational studies ca explain the oligomerisation formation. 2. How important s it for hemocyanin to form oligomer for its functionality? Any in-vitro experiment elucidating the function can help in the understanding of the formation of higher order oligomers. 3. A cartoon model of the formation of oligomer from monomer can help in better understanding of the work. ****** 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: Xiaomin Ma 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.0287294.r001
Revision 1
24 May 2023 Author Response Reviewer 1 Authors of this paper describe the cryoEM structure of the didecamer and tridecamer froms of a novel hemocyanin from the SLH, and use Alphafold to build large oligomeric proteins. However, I have some questions: 1. In SI page 4, line 11, Fig S4, some of them are identified by the color of the font itself, while others are identified by background highlighting，please use the same identification method to identidy functional units. We have used the background highlighting for the functional units to keep this consistent. 2. In paper page 18, line 395-396, "The sequence provided was split into 6 sequences which were a maximum of 800 amino acid in length with a portion overlapping". What is the basis for the truncation except for the maximum limit of Alphafold? The basis for this truncation was indeed the maximum limit for Alphafold. Reviewer 2 The work of science is highly interesting which elucidates the 3D-structure of slipper limpet hemocyanin, combining cryoEM with Alphafold modelling, and demonstrated how higher-order oligomers can form in hemocyanins of the KLH class. I have some questions which I feel can help in the development of the work. 1. The authors have explained that they can see higher order oligomers in the sample produced. I would like to know which part of the protein helps in this oligomerisation. SEC-MALS studies as well as mutational studies ca explain the oligomerisation formation. This is a very interesting suggestion. We looked at using SEC-RALS to look at oligomer formation, but we were not able to resolve single peaks for any species in our SEC experiments and so we did not continue with these experiments. From visual inspection of our negative-stain and cryoEM images we always observe the core didecamer (a tail-tail interaction) in any oligomer, and then additional subunits add on in a head-tail interaction. From our fitting of the Alphafold models to the tridecamer structure, we observe a small difference in the subunit interface in the head-tail interaction compared to the tail-tail interaction. It may be that in other hemocyanins that do not show substantial higher-order oligomer formation, this interaction is prevented or disfavoured by glycosylation or simply lack of an interface. Mutational studies are an excellent suggestion to resolve this, but we are working with protein purified from a native source in an organism that is not amenable to cloning for site-directed mutagenesis, so it is not possible for us to address this point. We have included some additional information in the discussion to cover this point (page 12). 2. How important s it for hemocyanin to form oligomer for its functionality? Any in-vitro experiment elucidating the function can help in the understanding of the formation of higher order oligomers. This is a very interesting point – unfortunately we do not know about the biological relevance of the higher order oligomers, or indeed the oxygen binding or enzyme function of our SLH didecamer, but from comparison with other hemocyanins, we know that oxygen binding is cooperative (so subunit-subunit interactions are critical for this process), and that partial denaturation or proteolysis produces a functional oxidase enzyme with a ‘relaxed’ structure relative to the intact protein. Functional experiments are something that we would very much like to address in future experiments, but we think they are beyond the scope of the structural and modelling experiments that we present in this manuscript. We have included some additional information in the text (pages 12 and 13) to discuss these points. 3. A cartoon model of the formation of oligomer from monomer can help in better understanding of the work. We agree that this information would be very useful. We do not have any experimental evidence for how a decamer might form from a monomer (we assume a monomer forms a dimer ‘plate’ and then 5 ‘plates assemble to the didecamer). We have included a simple cartoon model describing how decamers may join together to assemble didecamers and higher-order structures (Fig S2C). Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0287294.r002
5 Jun 2023 Decision Letter - Matteo De March, Editor CryoEM structure and Alphafold molecular modelling of a novel molluscan hemocyanin PONE-D-23-02379R1 Dear Prof. Mark Young, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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 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, Matteo De March Academic Editor PLOS ONE Additional Editor Comments (optional): 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 ******** 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: Authors of this paper describe the cryoEM structure of the didecamer and tridecamer froms of a novel hemocyanin from the SLH, and use Alphafold to build large oligomeric proteins. And they answered my questions. I agree to publish this paper. Reviewer #2: The work of science need to accepted with no further delay. The reviews are well addressed by the authors. Further functional analysis in future will be a great addition. ****** 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: Yes: Xiaomin Ma Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0287294.r003
Formally Accepted
13 Jun 2023 Acceptance Letter - Matteo De March, Editor PONE-D-23-02379R1 CryoEM structure and Alphafold molecular modelling of a novel molluscan hemocyanin Dear Dr. Young: I'm 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 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 plosone@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. Matteo De March Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0287294.r004

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