Dear Prof. Reuter,

Thank you very much for submitting your manuscript "Specificity of Loxosceles α clade phospholipase D enzymes for choline-containing lipids: role of a conserved aromatic cage" 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.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Guanghong Wei

Associate Editor

PLOS Computational Biology

Nir Ben-Tal

Deputy Editor

PLOS Computational Biology

***********************

Reviewer's Responses to Questions

Comments to the Authors:

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

Reviewer #1: This manuscript presents mainly a set of molecular dynamics (MD) simulations of spider venom PLD structures focusing on the aromatic cage and interactions with the PC headgroup. Comparisons are made with a protein in the b clade (St_bIB1) and how it interacts with the membrane when lacking this aromatic cage. Overall, this work is interesting but there lacks proof of equilibration of membrane-protein interaction and statistics associated with the experimental work. Details on this and other aspects are provided below.

General Issues:

1. Membrane-protein equilibration: The authors focus on a simple metric of RMSD of protein structure but do not focus on presenting a key metric associated with the proteins binding to the membrane. There needs to be some quantitative proof that the proposed bound states are near equilibrium. Key plots of distance of the protein relative to a metric in the membrane is a start. Moreover, plots of block averages of protein density profiles in comparison with the membrane is important. The concern is that the binding of peripheral membrane proteins can be more on the microsecond timescale and either long simulations or enhanced sampling is typically needed to probe stable binding.

2. St_bIB1 with POPC: The authors base low affinity on how this protein doesn’t bind to the POPC membranes but experimental with SM/Chol membrane suggests some binding. It appears that the timescale might not be enough (300ns) to probe binding for these PC headgroups. Have you tested this binding with longer simulations or with enhanced sampling?

3. Liposome Binding Assay: The binding assay is relatively crude and the figure lacks statistical information and the text on page 8 suggests some variance but unclear how this was obtain or if this is standard error or standard deviation. Quantifying intensity of bands with numerical values is qualitative at best with this method. Can one statistically state that the mutated St_bIB1 shows more binding? The band intensity suggest maybe but is this reproducible and do you have the same amount of mutated protein in comparison with the wildtype?

4. St_bIB1 other factors in binding: The liposomal assay suggests other factors are contributing to weak binding beyond the lack of an aromatic cage. The discussion lacks potential details on what the other factors might be. Can simulations provide some insight?

Specific Comments

Abstract: Please define PLD.

Ionizable residues: Do you have ionizable residue with this protein? Was a pKa estimate made on the protein to verify the proper states of these residues?

Initial protein setup: The authors provide a center of mass distance describing the placement of the protein relative to the center of the bilayer. This is not that informative and should be supplemented with a measure of minimal distance with the protein or some other distance related to key membrane binding domain/s on the protein.

Figure 3A: The density profiles are not described in the figure or figure captions. I can figure this out but the protein vs. membrane density should be clearly stated.

Cutoff LJ: The authors forgot to mention the cutoff method and length used in these simulations.

Reviewer #2: Review uploaded as an attachment

Reviewer #3: Moutoussamy et al. set out to study a hypothesis that a key reason why a certain set of phospholipases (α-clade) has a higher affinity towards phosphatidylcholine (PC) lipid headgroups than a related set (β-clade), is that α-clade typically contains a structural element dubbed 'aromatic cage' (comprising 2 to 4 tryptophans or tyrosines), whereas β-clade typically lacks such a cage.

They find support for their hypothesis primarily by performing and analysing unbiased all-atom molecular dynamics (MD) simulations; a liposome-binding assay provides additional experimental support.

I find the work reasonable, and aptly executed, and thus likely to be suitable to be published in PLOS Computational Biology. I would, however, like to draw the authors' attention to the following points:

(1) In the Introduction (p5) the authors spell out their hypothesis as:

"[T]he tyrosine and tryptophan residues on the i-face of La_αIB2bi and other α-clade enzymes provides a mechanism to selectively recognize choline-containing lipids as ligands."

However, it appears that none of the performed MD simulations with the aromatic-cage-containing enzymes had other than PC headgroups in their membranes. Therefore, it is not possible to say if the aromatic cage (composed of the said tyrosine and tryptophan residues on the i-face) recognizes SELECTIVELY the PC headgroups. To test the selective recognition, simulations of membranes containing (also) some other headgroup (such as PE) should be performed. If the stated hypothesis holds true, the α-clade enzymes (or more specifically the aromatic cage in them) would not recognize these other headgroups—or would at least bind them much less frequently than the PC headgroups.

(2) The abstract states that: "Here, we confirmed the membrane binding site of α and β clade PLDs on choline and ethanolamine-containing bilayers, respectively." The statement is acceptable concerning the α-clade—although of course 'confirmed' is a bit strong word for a predominantly MD simulation study—where the binding site (for PC) appears to be the aromatic cage; however, it is not quite clear to me what the confirmed binding site for the β clade on ethanolamine is. Could the authors please clarify?

(3) What is the conformation of the PC headgroup bound in the aromatic cage? In particular, is the conformation something typically seen in the other (free) PC heads? Or does the conformation adapt to the binding site, as recently claimed by the proponents of the so-called 'Inverse Conformational Selection Model' [Bacle et al, JACS 143 13701 (2021), https://doi.org/10.1021/jacs.1c05549]?

(4) Please make the raw simulation trajectories available on an open data service. A nice option is using the CERN-run Zenodo (zenodo.org), which is free to use, allows trajectories up to 50 gigabytes, and provides DOIs, which one can then cite directly in the manuscript.

(5) When discussing the system preparation, please (i) mention roughly the size (x/y/z) of the simulation box; (ii) provide the Table S1 in the actual paper; and (iii) include in this table the DOIs for raw MD trajectories, see the previous point.

(6) It is stated that the two replicas "differ by the equilibration step". What is meant by this, that is, how do they differ exactly?

(7) Concerning the ions, was the NB-fix correction of CHARMM36 used?

(8) What was the saving frequency in the simulations?

(9) Concerning the pressure control, was it isotropic, that is, all box vectors were scaled with the same factor?

(10) Was SHAKE applied also in waters?

(11) When determining the depth of insertion, the reference location (of the the upper phosphate plane) was "calculated on the last frame of the simulation". Why? To me it seems that this could introduce a systematic error, especially if the membrane shifts vertically during the simulation. To this end, the location of the upper phosphate plane should be determined for each frame separately. In fact, most natural would be to center the trajetory before analysis such that the center of mass of the upper phosphate plane stays at zero, then the z-coordinate can be directly interpreted as the depth of insertion.

(12) In addition to the protein backbone RMSD shown now in the SI, please show as a function of simulation time (i) the simulation box area in the bilayer plane, and (ii) the minimum distance between the protein and its nearest image.

(13) Please show also the (maximum) depth of insertion as a function simulation time for all the simulations.

(14) On p12 it is written that the "density plots (Fig. 5B) show an anchoring slightly deeper than for the α-clade enzymes". This is rather hard for the reader to see, so it might be better to give this information as numbers—say, list the time averages of the maximum insertion depths for each enzyme.

(15) In Fig. 7D, please rotate the color bar showing the insertion depth, such that it intuitively matches the snapshot (negative numbers bottom, positive top).

(16) In Fig 8, the top panel says "Lar_aIIB2bi", but the caption "La_αIB2bi".

(17) In Fig 8, what do the percentages stand for? Is it correct that for each experiment the "S" and "P" percentages (out of which only the "P" is shown), will add to 100? If yes, then I think it would be easier for the reader, if also the "S" percentages were written on the plot.

(18) When discussing the Fig. 8 in the text, two numbers are given: 15+/-4% and 28+/-4%. How were these exactly calculated?

(19) Accuracies in tables. The H-bond and cation-pi occupancies are not significant to tenths of promilles; I would expect to see them maximally in the precision of percentages. Same for hydrophobic contacts; probably a hundreth of a contact (as in Table 2) is not really significant?

(20) What is the cyan ball shown in the snapshots of Fig 4? An ion?

(21) Typos:

- p3, 3rd last line: differences in is the active -> differences in the active

- p4, 7th last line: L1_αIII1 has two tyrosines (Y44, Y46, Y62) -> L1_αIII1 has three tyrosines (Y44, Y46, Y62)

- p7, 5th line: The protein were -> The proteins were

- p8, 6th last line: 10ps -> 10 ps

- p10, 10th last line: diffuses away rapidly (Fig. 2 and 3A) -> diffuses away (Fig. 2 and 3A) [The speed of diffusion can not really be reliably determined from two simulations, I would expect?]

- p10, 8th last line: Figure 4A -> Figure 3A

- p10, 7th last line: (EDP) and the depth of insertion below -> (EDP) and Fig. 3D the depth of insertion of Li_αIA1 below

- p10, 6th last line: The two enzymes -> The two α-clade enzymes

- p10, 2nd last line: with the lipids chains -> with the lipid chains

- p11, 9th last line: 27.54 -> 28

- p12, 3rd line: lipids. - The binding -> lipids. The binding

- p12, 4th line: molecular docking and -> molecular docking [CITATION] and

- p13, last line: Fig. 8 -> Fig. 7A

- p16, 10th last line: ammonium which positive -> ammonium whose positive

- p18, 9th last line: MSA indicate that the-terminus -> MSA indicate that the [N?/C?]-terminus

- caption of Table 1: Asterix -> Asterisk

**********

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: No: No deposited input simulation codes and data on liposomal studies lack any data tables for the estimated variance.

Reviewer #2: No: The authors state 'All the MD data are stored locally and are available'.

My understanding was the PLoS Comp Biol required simulation trajectories to be made available in a public repository e.g. zenodo.

Reviewer #3: No: Authors have the MD data stored locally, and available upon request. The data should be made directly openly available, e.g., using the Zenodo server (zenodo.org).

**********

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

Reviewer #3: No

Figure 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. 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 us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example in PLOS Biology see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Attachments

Attachment

Submitted filename: Review of PCOMPBIOL.docx

Dear Prof. Reuter,

Thank you very much for submitting your manuscript "Specificity of Loxosceles α clade phospholipase D enzymes for choline-containing lipids: role of a conserved aromatic cage" 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. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you properly modify the manuscript according to the comments/suggestions of Reviewer #3.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Guanghong Wei

Associate Editor

PLOS Computational Biology

Nir Ben-Tal

Deputy Editor

PLOS Computational Biology

***********************

A link appears below if there are any accompanying review attachments. If you believe any reviews to be missing, please contact ploscompbiol@plos.org immediately:

[LINK]

Reviewer's Responses to Questions

Comments to the Authors:

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

Reviewer #1: Updated revision addresses my comments

Reviewer #2: The authors have addressed my comments in the revised version of this ms.

Reviewer #3: See attached pdf

**********

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

Reviewer #3: Yes

**********

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

Reviewer #3: No

Figure 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. 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 us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example in PLOS Biology see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

 

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References:

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.

Attachments

Attachment

Submitted filename: RevComments_2.pdf

Dear Prof. Reuter,

We are pleased to inform you that your manuscript 'Specificity of Loxosceles α clade phospholipase D enzymes for choline-containing lipids: role of a conserved aromatic cage' 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,

Guanghong Wei

Associate Editor

PLOS Computational Biology

Nir Ben-Tal

Deputy Editor

PLOS Computational Biology

***********************************************************

Reviewer's Responses to Questions

Comments to the Authors:

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

Reviewer #3: The Authors have provided careful responses to all the points raised.

**********

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 #3: Yes

**********

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 #3: No