PONE-D-22-26948The Q163C/Q309C Mutant of αMI-domain is an Active Variant Suitable for NMR CharacterizationPLOS ONE

Dear Dr. X. Wang,

Thank you for submitting your manuscript to PLOS ONE. As you can see below both Reviewers agree on the good impact of your manuscript that is well and clearly presented.However, they highlight some minor points that require improvement prior to acceptance.Please note that, in particular, it would be very appreciated if your experimental data will be deposited and a full account of data analysis process with representative HNCACB and HNCOCACB spectra will be included as supplementary material.

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We look forward to receiving your revised manuscript.

Kind Regards

Matteo De March

Academic Editor

PLOS ONE

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“This study was supported by funding from NIH/NIGMS (R01GM118518). We thank Drs. Brian Cherry and Samrat Amin in ASU’s Magnetic Resonance Research Center for maintenance of the NMR instruments.”

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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

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

Reviewer #1: N/A

Reviewer #2: N/A

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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: No

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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: Yes

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: This manuscript is an elegant example of rational engineering to retain function, and enable structural/physical analysis. The goal is to study an ligand-binding domain of an integrin, in the active state. Previous work proposes mutations to "stabilize" the domain, but here it is shown that such mutations do not stabilize the domain. The authors propose new mutations that would lead to an active state whereby they would stabilize with an intramolecular disulphide. All the data presented supports the conclusion of a stable active domain, although it requires glutamate/Mg2+ to maintain a state suitable for NMR analysis. The paper is well written, figures are clear. The only issue is a description of the Co+-induced pseudocontact shifts are based on the assignments. I would like these assignments to be deposited in the BMRB or if you have state the accession number. I consider this important as the BMRB will assess the quality (accuracy) of assignment. I do agree the fitting of the PCS is convincing that all is good, but you need to deposit the data.

Reviewer #2: This very clearly written and presented MS makes a strong and logically argued case for the use of an engineered domain, from a key integrin (CR3), for ligand-binding and screening studies, and in particular for studies using NMR spectroscopy. The modified domain has two non-native cysteines that form a disulfide bond, presumably stabilising an active conformation of the domain that is able to bind ligands (unlike the wild type domain) via a MIDAS motif. Because of the many reported ligands for this domain and its well-established central role in immune responses, the topic is of quite wide interest.

It would be good to show (eg in supplementary) the position of the mutated residues and perhaps a predicted structure of the disulfide-containing molecule.

Readers might be a little sceptical of how the authors were able to assign the amide nitrogens and protons for this mutant given the sub-optimal nature of the HSQC spectrum that contains many, well dispersed but very small peaks but also a region of more intense and overlapping peaks in the random-coil region. To reassure us, they should include in supplementary data a full account of this process including a method section and representative HNCACB and HNCOCACB spectra, and information regarding the extent of 15NH assignments (that should be deposited in a database) for both Mg and Co forms.

Another improvement to this MS would be to include some KD values based on SPR studies using a dilution series of their domains (binding to immobilised C3d). The authors seem to have plenty of sample for the binding partners and access to the kit so it is puzzling why they have not already included such measurements. It looks like the alpha-helix truncation mutant might bind to C3d more tightly than the double-Q (disulfide) mutant since it has a slower off-rate. That does not really weaken their conclusions but is good to know.

Small things:

Understanding.... is... (not are)

Data are... (not is)

And they seem to use the terms intracellular and intramolecular indiscriminately - I think they mean intermolecular (?)

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Reviewer #1: No

Reviewer #2: Yes: Paul N Barlow

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PONE-D-22-26948R1

The Q163C/Q309C Mutant of αMI-domain is an Active Variant Suitable for NMR Characterization

PLOS ONE

Dear Dr. Wang,

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 Feb 18 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). 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'.

We look forward to receiving your final version soon.

With Best Regards,

Matteo De March

Academic Editor

PLOS ONE

Journal Requirements:

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.

Additional Editor comments:

your manuscript can be now accepted as both Reviewers agreed with the revised version. However, Reviewer 2 stated "1 AU = 1 pg per mm2 (square mm)". Although it is a very small change, it cannot be ignored.

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: N/A

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: The authors have addressed comments and questions of the review. The deposition of data to the BMRB is important.

Reviewer #2: The authors have addressed my concerns but please correct statement regarding SPR. You say 1 AU = 1 pg per mm. This should of course be 1 pg per mm2 (square mm)

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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: Yes: Paul Nigel Barlow

The Q163C/Q309C Mutant of αMI-domain is an Active Variant Suitable for NMR Characterization

PONE-D-22-26948R2

Dear Prof. X. Wang,

we are pleased to inform you that your manuscript is now ready for publication. Within one week, you’ll receive an e-mail detailing the outstanding technical requirements that need to be addressed before scheduling your 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.

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I would like to wish you all the best for your work.

Kind regards.

Matteo De March

Academic Editor

PLOS ONE