Dear Dr. Donald,

Thank you very much for submitting your manuscript "Chiral Evasion and Stereospecific Antifolate Resistance in Staphylococcus Aureus" for consideration at PLOS Computational Biology.

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Joanna Slusky, Ph.D.

Guest Editor

PLOS Computational Biology

Nir Ben-Tal

Deputy Editor

PLOS Computational Biology

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Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: the review is uploaded as an attachment.

Reviewer #2: Chiral evasion and stereospecific antifolate resistance in Staphylococcus aureus

(PCOMBIOL-D-21-01260)

In this study, authors have used computational protein design (CPD) software suite OSPREY to explore the mechanism of this stereo-specific inhibition, namely, Staphylococcus aureus dihyrofolate reductases (SaDHFR's) chiral evasion against PLAs (propargyl-linked antifolates) enantiomers. Compared to IC50 data, K* scores (which predict Ka) produced by OSPREY successfully recapitulated the ranking of PLA enantiomers' afinity and the ranking of the impact the F98Y mutation would have in the interaction. Among all complexes, K* scores for R-27:t-NADPH:SaDHFR and S-27:β-NADPH:SaDHFR are significantly higher than for all other models, which is consistent with NADPH configuration preferences observed in crystal structure (R-27 bound with t-NADPH and S-27 bound with β-NADPH, as seen in models 6wmy and 4tu5). Ensembles of conformations of F98Y SaDHFR binding to R-27 and S-27 are predicted as well in the present study. Based on structural analysis, authors have found that different binding modes between t-NADPH (which R-27 prefers) and β-NADPH (which S-27 prefers) are likely to be a key factor of chiral evasion. Authors have shown major difference between t-NADPH and β-NADPH is how they interact with Gly93 loop on SaDHFR. Such difference may lead to clashes between Gly93 and Tyr98 in F98Y mutant, and thus may influence the thermodynamic stability of SaDHFR:NADPH binary complex, ultimately modulating the inhibition potency of R-27 and S-27.

In this whole study authors have shown how the discovery of a configuration change in NADPH can elucidate a potential mechanism of drug resistance in SaDHFR. This study suggests that the cofactor stereogenicity and chiral evasion should be taken into account when designing new drugs for F98Y SaDHFR. The use of computational drug design and protein design algorithms gained new insight, informed hypotheses in biology, and made contributions to biochemistry. The data and models authors presented in this manuscript have already been useful in medicinal chemistry campaigns for F98Y resilient inhibitors, which suggest that they will have significant value for the scientific community. I believe this is significant advancement compare to previous work from the same group and I strongly recommend for its publication.

Reviewer #3: In this manuscript, the authors combine structural analysis with simulations to propose an interesting antimicrobial resistance mechanism through “chiral evasion” from the F98Y mutant of SaDHFR. The manuscript is well written and presented.

First, I must say that I am not well versed in X-ray crystallography and so I cannot comment on or critique this aspect of the work. I do wonder though whether the reported change in the Rfree value from 0.2543 to 0.2529 when going from α-NADPH to t-NADPH should be considered a significant improvement though?

I can however say that the simulations and subsequent simulation analysis look well performed. I’m confident this article would be of interest to the readership and I would like to recommend it for publication after addressing the following minor concerns:

1. The OSPREY software is publicly available yes, but the scripts used by the authors along with the preprocessed input structures could be shared to aid reproducibility.

2. It would be instructive to provide error estimates on the bar chats for Figure 3.

3. The label sizes on Figures 4-6 should be increased.

4. Can the authors comment on the variability of the results obtained for different snapshots from their ensemble with the Probe dots analysis. It would be good to confirm the lowest energy conformer can adequately represent the ensemble obtained from OSPREY. I mean in terms of the subsequent insights taken from the Probe dots calculations.

5. Some very minor corrections I spotted along the way:

“It has been proved in Ref. 30 that K* will be equal to Ka under the condition of using exact” (add “be”).

“Under this model, the ability of DHFR to readily form binary complexes…” (replace from with form).

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Have the authors made all data and (if applicable) computational code underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data and code 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 and code 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 or code —e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: None

Reviewer #3: No: The scripts used by the authors along with the preprocessed input structures could be shared to aid reproducibility. (same as my minor comment 1).

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Rory Maurice Crean

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Attachments

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Submitted filename: PLOSCB_review.pdf

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Submitted filename: Chiral evasion and stereospecific antifolate resistance in Staphylococcus aureus.docx

Dear Dr. Donald,

We are pleased to inform you that your manuscript 'Chiral Evasion and Stereospecific Antifolate Resistance in Staphylococcus Aureus' has been provisionally accepted for publication in PLOS Computational Biology.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

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Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Computational Biology. 

Best regards,

Joanna Slusky, Ph.D.

Associate Editor

PLOS Computational Biology

Nir Ben-Tal

Deputy Editor

PLOS Computational Biology

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Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: review is attached

Reviewer #3: I thank the authors for taking the time to respond to my questions and making the extra changes. I am happy with their responses.

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Have the authors made all data and (if applicable) computational code underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data and code 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 and code 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 or code —e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

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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 #3: No

Attachments

Attachment

Submitted filename: Plosone_review_2nd_011722.pdf