Gamma-Hemolysin Components: Computational Strategies for LukF-Hlg2 Dimer Reconstruction on a Model Membrane

Round 1

Reviewer 1 Report

The Manuscript submitted by Lattanzi and coworkers attempts to analyze the dimerization process of two proteins involved in the formation of an octameric pore-forming toxin. To this end, the authors use state of the art and advance computational techniques within molecular dynamics and docking.

In my opinion, this is an outstanding manuscript about protein-protein interactions with several fine details which will attract the attention of the readers of International Journal of Molecular Sciences. Therefore, I support the acceptance of this manuscript after minor revisions:

1. Page 5, line 194: I think there is a typo in “peristencies”

2. To help the readers, I suggest the authors to repeat the color code of the functional domains in Figure 1.a and Figure 2. Maybe the authors could also add the structure of the protein next to the RSMD figures (like that in Figure S3). This will give consistence to the color code used along the manuscript.

 

3. Page 8, line 299: What do the authors mean by “electrostatic contacts”? Are they specific interactions?

Page 7, line 277: “a particularly persistent electrostatic contact between K65 in LukF and D181 in Hlg2” Are they ionic hydrogen bonds?

 

4. Following query 3. What do the authors mean by hydrophobic contacts?

For instance, in page 8, line 320: “hydrophobic contacts between V12 and A137, and between V16 and A137.”

As a reader, I would like to see a Figure of this contact.

 

Some hydrophobic contacts were described as hydrogen bonds between C-H bonds and pi rings (C-H⋯π). See for instance:

10.1016/j.bpc.2021.106627 

 

Author Response

The Manuscript submitted by Lattanzi and coworkers attempts to analyze the dimerization process of two proteins involved in the formation of an octameric pore-forming toxin. To this end, the authors use state of the art and advance computational techniques within molecular dynamics and docking.

In my opinion, this is an outstanding manuscript about protein-protein interactions with several fine details which will attract the attention of the readers of International Journal of Molecular Sciences. Therefore, I support the acceptance of this manuscript after minor revisions.

REPLY: We are extremely grateful to the Reviewer for the appreciation of our work and for the pertinent corrections and suggestions. In the following we provide a point-by-point reply to all his/her comments.

1. Page 5, line 194: I think there is a typo in “peristencies”

REPLY: We thank the Reviewer for pointing out the typo; we have corrected it in the revised version of the manuscript.

2. To help the readers, I suggest the authors to repeat the color code of the functional domains in Figure 1.a and Figure 2. Maybe the authors could also add the structure of the protein next to the RSMD figures (like that in Figure S3). This will give consistence to the color code used along the manuscript.

REPLY: As suggested by the Reviewer, we have changed the colors of the protein domains in Figure 1.a to match those used in the RMSD and RMSF plots. We have also placed the RMSD and the RMSF plots in a single figure (Figure 2) and we have added the structures of the proteins of Figure 1.a inside the RMSF plot. 

3. Page 8, line 299: What do the authors mean by “electrostatic contacts”? Are they specific interactions?

Page 7, line 277: “a particularly persistent electrostatic contact between K65 in LukF and D181 in Hlg2” Are they ionic hydrogen bonds?

REPLY: We thank the reviewer for raising this point, which allows us to define in a clearer way the non-bonded interactions mentioned in the manuscript. 

By electrostatic contacts, we mean salt bridges between side chains of opposite charge. Specifically, a salt bridge is formed between two different residues if at least one pair of atoms belonging to the charged groups of the two residues is found within a distance of 4.5 Å. In Asp and Glu residues, the atoms forming the carboxylic group are considered, while for Lys, Arg, and His (if protonated) residues the NH3+, the guanidinium group, and the imidazole ring are employed, respectively. More in general, for the definition of electrostatic contacts (or salt bridges), hydrogen bonds, and hydrophobic contacts we have adopted the default criteria employed in the PyInteraph tool (https://doi.org/10.1021/ci400639r).

4. Following query 3. What do the authors mean by hydrophobic contacts? For instance, in page 8, line 320: “hydrophobic contacts between V12 and A137, and between V16 and A137.” As a reader, I would like to see a Figure of this contact.

Some hydrophobic contacts were described as hydrogen bonds between C-H bonds and pi rings (C-H⋯π). See for instance: 10.1016/j.bpc.2021.106627

REPLY: By hydrophobic contacts - following the default criteria employed in PyInteraph - we refer to the case in which the centers of mass of the side chains of two hydrophobic residues are found at a distance shorter than 5 Å. The residues considered for hydrophobic interactions are Ala, Ile, Val, Leu, Phe, Met, Trp, and Pro. According to this definition, it is in principle possible that some hydrogen bonds between C-H bonds and π-rings might have been classified as hydrophobic contacts. 

The hydrophobic contacts V12-A137 and V16-A137, which we mention in the manuscript, correspond to standard hydrophobic interactions between aliphatic side chains. In addition, we observe the transient interaction between V16 and F118; this corresponds, instead, to a C-H⋯π type of interaction. 

In the revised version of the manuscript we have added the criteria employed to identify the salt-bridges, the hydrophobic contacts and the hydrogen bonds; moreover, we specified the type of hydrophobic interactions observed, as suggested by the Reviewer. 

Reviewer 2 Report

Manuscript ijms-2327587. C. Paternoster, T. Tarenzi, R. Potestio and G. Lattanzi, “GammaHemolysin Components: Computational Stategies for LukF-HIg2 Dimer Reconstruction on a Model Membrane.”

The authors of the manuscript have studied the mechanism of the formation of a functional dimer. This study has been performed by the application of molecular dynamics simulations and proteinprotein docking. The paper is well written and organized. However I present some few suggestions to be taken in consideration by the authors.

1. Page 2, Line 50

Provide an numerical value or range of values for the angle between the axis of nearby subunits in the assembled octamer.

2. Page 2, Line 68

Change: “organism” to “organisms”

3. Page 4, Line 127

Change: “POPC” to “1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)”

4. Page 15, Line 403

Specify in how many degrees is the angle reduced.

Author Response

The authors of the manuscript have studied the mechanism of the formation of a functional dimer. This study has been performed by the application of molecular dynamics simulations and proteinprotein docking. The paper is well written and organized. However I present some few suggestions to be taken in consideration by the authors.

REPLY: We are grateful to the Reviewer for the attentive reading of the manuscript and for the appreciation of our work.

1. Page 2, Line 50: Provide an numerical value or range of values for the angle between the axis of nearby subunits in the assembled octamer.
2. Page 2, Line 68: Change: “organism” to “organisms”
3. Page 4, Line 127: Change: “POPC” to “1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)”
4. Page 15, Line 403: Specify in how many degrees is the angle reduced.

REPLY: We thank the Reviewer for his/her useful comments and corrections. We have included the suggestions provided in the revised version of the manuscript; however, regarding point 3, we maintained the abbreviation “POPC”, since the full name of the lipid molecule and the corresponding abbreviation were defined a few lines before (line 121).

Reviewer 3 Report

The manuscript “Gamma-Hemolysin Components: Computational Strategies for LukF-Hlg2 Dimer Reconstruction on a Model Membrane” from Costanza Paternoster et al. appears interesting and it is well written, but there are some issues that need further attention.

The authors should not introduce abbreviations without resolving them first (e.g., EM, SAXS, etc.) Also, some kind of jargon and simplifications precluded the manuscript from being read easily. They should explain better what is “stem” or “folded prestem,” “rim,” or “rim domain” etc. Some of the things referred to are much specific to be understandable in a general journal like this.

Also, the authors state that “unveiling the steps leading to pore formation from S. aureus PFTs can be instrumental for the structure-based development of new antivirulence agents.” Can they propose how based on their conclusions? Of course, they say “can be” but it will be interesting to whether some of their findings give some clear ideas on it.

In summary, in my opinion should be accepted with minor changes.

Author Response

The manuscript “Gamma-Hemolysin Components: Computational Strategies for LukF-Hlg2 Dimer Reconstruction on a Model Membrane” from Costanza Paternoster et al. appears interesting and it is well written, but there are some issues that need further attention.

REPLY: We thank the Reviewer for his/her appreciation of our work, as well as for the pertinent and constructive comments and suggestions. In the following we provide a reply to all concerns raised, and the description of the modifications implemented in the manuscript.

1. The authors should not introduce abbreviations without resolving them first (e.g., EM, SAXS, etc.). Also, some kind of jargon and simplifications precluded the manuscript from being read easily. They should explain better what is “stem” or “folded prestem,” “rim,” or “rim domain” etc. Some of the things referred to are much specific to be understandable in a general journal like this.

REPLY: We thank the Reviewer for pointing out these issues. In the revised version of the manuscript, we have specified the meaning of all the abbreviations, and we have added a more detailed description of the protein domains and their structural features. We are confident that this will assist the reader for a better understanding of the biological processes that are mentioned in the manuscript.

2. Also, the authors state that “unveiling the steps leading to pore formation from S. aureus PFTs can be instrumental for the structure-based development of new antivirulence agents.” Can they propose how based on their conclusions? Of course, they say “can be” but it will be interesting to whether some of their findings give some clear ideas on it.

REPLY: We have addressed this point by adding in the manuscript a more complete explanation of the experimental/simulation data that might have a practical utility for the therapeutic development of drugs targeting pore-forming toxins. These include the recognition of membrane receptors, the identification of residues crucial for oligomerization, and the analysis of the conformational rearrangements that are required for pore formation. In addition, we have included in the text the example of small molecules that have been found to bind to the monomeric form of the pore-forming toxin alpha-hemolysin, thus preventing the oligomerization that leads to the formation of a lytic pore. This example shows how the knowledge on the mechanism of the formation of a functional dimer can assist in the development of drugs that inhibit the assembly of the toxin components.