PONE-D-25-04770Bioinformatics and modelling studies of FhuD, the periplasmic siderophore binding protein from the plant pathogen Erwinia amylovoraPLOS ONE

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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: Partly

Reviewer #2: Yes

Reviewer #3: Partly

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

Reviewer #1: N/A

Reviewer #2: N/A

Reviewer #3: Yes

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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: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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

Reviewer #3: 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: In this manuscript entitled “Bioinformatics and modelling studies of FhuD, the periplasmic siderophore binding protein from the plant pathogen Erwinia amylovora”, the team led by Stefano Benini used AlphaFold to model the FhuD protein and performed sequence analyses and structural comparison (including molecular docking) against its homologs from eight organisms with available PDB entries, along with sequence alignment and phylogenetic analysis of 145 orthologs. Their findings revealed some structural features of siderophore binding in gram-negative and gram-positive bacteria, i.e., a similar binding pocket for various ligands that is constructed by variable key residues. The information is useful and will contribute to our understanding about the structural basis of ligand binding.

To improve on the clarity, please address the following concerns/suggestions:

Ile 88 was singled out as a key residue (Abstract and Discussion), but the supporting evidence and argument is missing. The authors need to present their logic clearly and potentially label Ile 88 in all relevant figures (Fig 1a-c, 2a-d, 4a-c, 5a-f) to support their argument.

Please correct the disagreements between text and figures listed below:

Page 9, line 143: “19 residues highlighted in cyan” is not shown in Fig 1a.

Page 10, line 156: “Fig 1d” is missing (not submitted for figure)

Page 12-14: All discussion and legend related to “Figure 2” doesn’t include 2e and 2f as shown. Were the latter two necessary for this study?

Page 16, line 230-231: “The conserved residues are highlighted in red .. in Fig 5A” is not the case.

Please correct other issues listed below:

Page 9, line 149-150: rephrase the sentence to avoid potential confusion

Page 16, line 226: “Glu 90” should be “Glu 86”

Page 16, line 231-232: Figure 5A is mentioned before Figure 4A. It is better to reverse the order.

Page 16, line 236: take out “Ile 88” from “Other residues …”

Page 19, Table 3: remove extra # on top right

Reviewer #2: The manuscript “Bioinformatics and modelling studies of FhuD, the periplasmic siderophore binding protein from the plant pathogen Erwinia amylovora” is dedicated to the sequence and structural comparison of the high score AF FhuD model with its structural and sequence homologs. By sequence alignments and residue conservation analysis, the authors revealed FhuD siderophore binding site, which was followed by the ligand docking targeted to the site. Knowledge of structur and functional details for EaFlXD, which plays a vital role in transporting iron-loaded siderophores to the inner periplasmic membrane

so as details on its ligands specificity may help to develop the treatment against Erwinia amylovora, causing fire blight disease affecting apples and pears.

Below are suggestions which benefit the paper.

Use the same AlphaFold version everywhere in the text when referring to how your model was generated for consistency.

It would be beneficial to have a figure with chemical details of possible classes of siderophores, as mentioned in the introduction to the general reader (page 3, line 53).

Removing redundant entries from the tables 1 and 2 will benefit both tables.

The binding partner for FhuB interacting with FhuD is missing from the table 1.

Removal of non-relevant to siderophore binding site ligands like ACT (which is acetate, not acetone as mentioned in the figure 2 caption), EDO (ethylene glycol) or Cl from the figure 2, table 2,3 and discussion will benefit the paper.

The text is not easy to follow due to many unimportant details. In the chapter dedicated to the comparison of three-dimensional structures of homologs and analysis of their ligand binding pockets, it will be beneficial to follow the same structure description used for E.coli homolog (N-lobe, C-lobe and the linker ).

Include the references to the subfigures of Figure 2a,b,c, etc, in the text.

Figure 2 doesn’t have enough details on the ligands and ligand binding residues and their interactions; residue numbers are missing, as are the ligand residue interactions. Residue labels with numbers are also needed. The subpanels of the figures could follow the same style centered around the ligand binding pocket. The figure caption is far from the journal quality.

Table 3 doesn’t have the bonding details either, which makes you wonder about the roles of the multiple residues mentioned in the table.

Figure 3. The figure caption for needs uniport IDs, not PDB IDs, it is showing sequence alignments.

Figure 4. You already introduced the siderophore binding site in Figure 2; you can color the molecule by residue conservation and show it in 2 different orientations (chimera has this option).

Table 3, please place the units associated with each row for binding energy, ligand efficiency etc.

Create a supplementary figure with details of the ligand protein interactions for each ligand in table 3.

EaFluD has 53% similarity with E.coli FhuD, which was recently characterised as part of a ferrichrome importer FhuCDB from E. coli (https://doi.org/10.1038/s42003-021-02916-2). Aplhafold 4 is reasonable with protein complexes. Could you generate the Erwinia amylovora FhuCDB model? Will it be any differences with E.coli one?

Reviewer #3: Overall Summary:

Bharti et al. present a manuscript that evaluates predicted protein structure for an E. amylovora siderophore binding protein FhuD and binding analysis to a set of known siderophores. The manuscript uses alpha fold for structural predictions and then uses docking through autodock to compare FhuD to 6 siderophores. While the manuscript does a thorough analysis of the predicted structure of FhuD and the phylogenetic differences across various homologs, the docking predictions to the set of siderophores is not validated by binding analysis in vitro. Further, there is also a lack of the evaluation of a larger set of proteins from the E. amylovora genome for binding potential to the set of siderophores tested. These sets of information will be necessary to validate the docking predictions. Comments are specifically provided below.

•Table 3/Figure5: While these are predicted binding for the different ligands to FhuD, it needs to be validated by the recombinant expression of the FhuD and binding assays. Currently these are just predictions.

•Figure 2/ Figure 5: Since the manuscript does not include data about the validated binding of FhuD to these ligands, another exercise would be to selectively modify the protein sequence to alter the potential binding pockets and rerun this through alpha fold and Autodock. To check if this can alter the binding as per the docking analysis.

•Page 15 and Page 19 both have a Table 3. This might be a typo that needs to be modified.

•Figure 4: While FhuD is the main transporter of interest in this study, due to the restricted nature of docking to a set group of siderophores, the data is mainly predictive and needs to be validated by binding studies. However, this data could be also supplemented by reverse docking analysis where the alpha fold predictions of the E. amylovora CFBP1430 genomic sequence can then be the protein database that each of the ligands get reverse docked into. This helps provide an unbiased outlook of the best candidates out of the E. amylovora genome that can bind to these siderophores.

•Table 4: due to just the evaluation of FhuD docked against 6 targets, the binding energy and efficiency is present without any context. These either need to be supplemented with binding assays or reverse docking, otherwise it is unclear how this interaction potential will vary if a larger group of proteins were evaluated.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Bioinformatics and modelling studies of FhuD, the periplasmic siderophore binding protein from the plant pathogen Erwinia amylovora

PONE-D-25-04770R1

Dear Dr. Benini,

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.

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Kind regards,

Abhijeet Shankar Kashyap

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

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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

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

Reviewer #1: Yes

Reviewer #2: N/A

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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

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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 all my concerns and suggestions in this R1 manuscript.

Two minor corrections are needed:

Line 75: Use comma before "which" or use "that" instead of "which".

Table 2: "Seq%" (the far0right column): concert all values to %, e.g., 0.5373 to 53.7 and 0.188 to 18.8, and use one decimal point across the board.

Reviewer #2: The manuscript has been significantly improved, the authors have addressed all reviewer's comments, made the text of the paper clearer, and improved the content of the tables and the figures

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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: No

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