Dear DR Sulkowska,

Thank you very much for submitting your manuscript "Amino acid variants of SARS-CoV-2 papain-like protease have impact on drug binding" for consideration at PLOS Computational Biology.

As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

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Sincerely,

Alexander MacKerell

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: The manuscript 'Amino acid variants of SARS-CoV-2 papain-like protease have impact on drug binding' by Perlinska et al.

presents a study on the effect of mutations on the binding mode of potential inhibitors to the Papain-like Protease (PLP)

of SARS-CoV2. The study combines Molecular Dynamics (MD) simulations with molecular docking, sequence analysis, MMPBSA

calculations and experimental enzyme activity assays to characterize the impact of five mutations : P247S, E263D-Y264H and

T265A-Y268C on the affinity of potential non-covalent inhibitors of PLP.

The manuscript is well-structured and all methods that have been used in this study are described in detail.

The authors' conclusions are supported by the results, although this referee has some remarks on the methodology

used in their study (see below). This referee recommends this manuscript to be published after a minor revision.

In the following, I will point out my remarks that appeared to me during the study of this manuscript :

1. Sequence redundancy in the selected dataset :

The selected data of PLP-sequences seems to be highly redundant, which means that the sequence-similarity

among a large fraction of the selected CoV-2 datasets seems to be redundant as well. To remove artefacts that

arise from sequence-redundancies, only sequences with a similarity that lies below a certain threshold should

be considered < 80 %. (see the mutation rates in Table 1).

The mutation-rates can be reflected using a position-dependent heat-mapped color-coding on Figure 1.

2. Median of MMPBSA-interaction energies :

The median values and the variances of the measured energies (see Figure 3 D) indicate that there is no

evident difference in the energies between the mutational variants and the wild-type, because the error-bars

are crossing each of the individual median values.

A running averaging that also considers the statistical error might be more indicative for the energy

differences : 100 ps, -> av_2 1 ns -> av_3 10 ns -> av_4 100 ns -> av_5 500 ns (final result) + the statistical error.

As MMPBSA is a quite inaccurate method for the calculation of interaction energies, the study could be improved

using thermodynamic integration (TI) or Free energy perturbation (FEP) calculations.

3. The RMSDs of the residues in the drug-binding pocket over time can be added for each mutational variant, as this metric might

be an indicator for the stability of the drug-binding site compared to the wild-type. Further, it might explain the

differences in the enzymatic activity that has been observed experimentally.

Reviewer #2: In this manuscript, the authors tried to clarify different sequences of PLpro and then studied the influence of these mutations on the binding process of ligands to PLpro via docking and MD simulations. In vitro works were then performed to validate the observation. It is of great interest to read the manuscript. A large work was completed, however, there are some comments to improve the manuscripts.

1. The structural change of the PLpro under the effect of mutation would significantly impact the binding free energy and binding pose of ligands to PLpro. So, the MM/GBSA calculation should be carried out over the equilibrium snapshots of the complex, which were obtained from MD simulations instead of molecular docking only.

2. According to the previous comment, the MM/GBSA calculation over docking simulation probably does not make sense since the obtained results are not significantly different eg. the binding energy range from -27.4 +/- 6.1 to -32.28 +/- 5.7 (line 286), or -28.1 +/- 3.6 to -29.0 +/- 3.5 kcal/mol (line 314 - 315), etc. The obtained results are not different within the error bar, authors may wish to discuss about this.

3. The MM/GB(PB)SA calculation (Per-residue free energy decomposition) should be carried out since the obtained results will clarify the contribution of each residue of PLpro. The interaction picture would thus be a clarifier.

4. The MMGBSA terms should be reported in the manuscript.

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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: Yes

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Reviewer #1: No

Reviewer #2: No

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Dear DR Sulkowska,

We are pleased to inform you that your manuscript 'Amino acid variants of SARS-CoV-2 papain-like protease have impact on drug binding' 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.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

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,

Alexander MacKerell

Academic Editor

PLOS Computational Biology

Nir Ben-Tal

Section 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: This referee thinks that the article is suitable for publication.

Reviewer #2: Authors answer all of my question. The manuscript should be published at the current 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: No: 

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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 #2: No