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The authors responded to all the issues raised by the referees.
# PeerJ Staff Note - this decision was reviewed and approved by Jennifer Vonk, a PeerJ Section Editor covering this Section #
No Comment
No Comment
No Comment
The authors have adequately addressed all issues raised by reviewers.
The submitted paper is clearly written and within the aims of the Journal. However there are some issues that should be addressed following the referees' comments.
This work is primarily a modeling study aimed at elucidating the binding of three different AKH peptides of Schistocerca gregaria to its only AKH receptor. While the article in general addresses the modeling aspect, there are several issues which would need to be addressed before the manuscript can be deemed publishable:
1. The English language remains a primary concern and needs to be vastly improved. The target audience is likely from the entomological community so certain terms and abbreviations which are more appropriate for molecular modeling studies have to be adequately explained.
2. The title is a bit strange sounding "...solution structure of agonists...". The authors employed the three AKH peptides that are found in S. gregaria but not agonists that would mimic the AKH peptides, so I am confused why they prefer the term agonists in such a case. I would probably rephrase the tile as "...ligand-receptor binding modeling studies".
3. Table and Figure legends are very brief. More explanations are required. In general, I would expect stand-alone legends which would sufficiently explain what is being depicted without the need to have recourse to the text.
4. What was the hypothesis being tested?
1.One of the concerns I had about the experimental design was that while all three peptides were synthesized and purified, they were not sufficiently soluble in water (Line 105). What was the solubility?
2. Within the insect body, how would these peptides behave? Were these synthetic peptides checked to determine if they actually mobilized lipids in S. gregaria?. Some information on this should be provided otherwise the relevance of this work remains doubtful.
3. The M&M section is replete with terminology which probably would be easier comprehended by specialists in molecular modeling studies but would be difficult for the average entomologist or insect physiologist to understand. The authors would need to provide a more lucid account of the techniques used. For example Line 115: "using the dipsi2esgpph pulse sequence" What does this mean? Also, line 117: "and noesyesgpph". This is incomprehensible.
4. What was the statistical treatment of the data generated?
1. This study is primarily a modeling study with a view to provide information on how putative agonists or antagonists can bind to the AKH receptor with the eventual goal of designing novel pesticides. However, its validity is in doubt because the authors could easily have designed an antagonist (since they already used synthetic ligands) and checked it for binding competitively to the AKH receptor.
2. It is unclear from the studies which of the three different AKH would show preferential binding to the receptor (i.e. competitive binding). If all three bound to the same receptor with the same efficiency, do the authors hypothesize that the constraint is on which of the neuropeptides are being synthesized and released?
3. What is the statistical validity of the findings?
The English language remains a matter of primary concern along with all other points raised above.
1. Line 21-22: "currently discussed as targets to" could be rephrased as "being considered as a novel target to".
2. Line 23 and 26: I do not like the term "AKH system". Why do the authors refer to this neuropeptide as a "system"?
3. Line 30 Replace "Next" with "Further".
4. Line 58 Aedes aegypti should be in italics.
5. Line 63: Rephrase, it sounds clumsy. I suggest : "which occurs not only in insects and crustaceans".
6. Line 64: Codes "an mRNA".
7. Line 76 "are currently fiercely discussed" replace with "are currently being considered".
8. Line 86 is essentially a rehash of line 84-86.
9. The term ligand and agonist are referred to interchangeably. Since these are actually peptides endogenously synthesized, should they not be referred to as such?
10. Line 152 S. gregaria should be italicized.
These are just some of the edits, I suggest a more thorough revision of the manuscript along these lines.
I found this manuscript written in a lucid manner allowing otehr researcher for both understandings and repeating of the reported procedures.
Although the authors refer to structural study (molecular dynamics) on insect adipokinetic hormones (L 617: Zubrzycki IZ. 2000. Molecular dynamics study on an adipokinetic hormone peptide in aqueous solution. Zeitschrift für Naturforschung C, A Journal of Biosciences 55:125-128. ) I think that it should be stressed that the first 3D-structure derived by NMR spectroscopy has already been published: Zubrzycki IZ, Gäde G. Conformational study on an insect neuropeptide of the AKH/RPCH family by combined 1H NMR spectroscopy and molecular mechanics. Biochem Biophys Res Commun. (1994) 198:228–35. 10.1006/bbrc.1994.1032.
The molecular dynamics study section unfolds the three approaches i.e., "NMR restrained molecular dynamic simulations in vacuum, water and DPC." (please put a comma after water...) Here I have the following question to the authors; 1) what is the logic using three different environments. Could the authors justify this decision especially vacuum simulation? 2. How many water molecules were used in the simulation? The authors put there ~700, but they, in my opinion, say explicitly how many: 703 ... 721..
L 188: How many CL- ions were added for zeroing total charge.
L 198: "Berjanskii and Wishart (Berjanskii & Wishart 2006) and Tremblay (Tremblay 2010) have shown that by comparing the measured chemical shifts to literature values for a random coil structure, some idea as to the structure and flexibility of the peptide can be obtained" - I have a problem with "some idea" statement. In my opinion, It is simply too vague. Please state clearly as to the kind of conclusions that can be derived.
L 436: "Our results show that the three endogenous peptides, Schgr-AKH-II, Aedae-AKH and Locmi-AKH-I, all bind to the same receptor binding site and with very similar binding constants. This may be surprising, as the three ligands are very different." - can the last statement be clarified i.e., ligands are very different - structurally or charge wise?
Conclusions; The first statement "In this paper, we have shown that the putative receptor, Schgr-AKHR, is a member of the Class A superfamily of G-protein coupled receptors." which is a derivative of the statement provided at L 245 The sequence identity between the Schgr-AKHR and the h-OR was 26.33% The h-OR structure belongs to the class A... " 26% of sequence identity is, in my opinion not enough to judge on structural and functional similarities between proteins or peptides. I may cover 26% right from the N-terminal amino acid to, let say 26th amino acid, in case of 100 residue peptide. Please provide the overlay of these sequences to justify your statement that the studied structure renders analogous biological functions.
Figure 1. The authors provide a graphical representation of chemical shifts. Although it is a nice picture it is not very informative without knowing, in depth, what are the relations between chemical shifts and randomness of the structure. There are also colors on the picture but without proper description it simply meaningless.
Figure 2: The superposition of so many conformers make it nearly impossible to deduce the overall shape of the peptide. The authors tried to clarify this be providing stick representation. Nevertheless, in my opinion, if the average cartoon representation would be overlayed on the stick representation the reader would get a much better feeling of the shape of the peptide (it is done in figure 4).
Figure 6. The authors show the surface representation of the binding pocket using unknown to the reader coloring scheme. I presume that this does not reflect the charge. If my presumption is correct I would appreciate changing it to charge coloring scheme for both the surface and the molecule.
The research is within the aims and scope of the journal.
The research questions are well-defined and meaningful. There are some problems that should be addressed (listed in the basic reporting).
The study is of the top technical standard. Methods are generally sufficient to replicate (the problems have been addressed in basic reporting)
It is the novel study employing state of the art technology, including both NMR spectroscopy and molecular modeling. Conclusions are well stated although as outlined in basic reporting I have a problem with the non-discussable statement "In this paper, we have shown that the..."
The paper is generally well written. There are some issues with figure legend and this must be addressed. The same applies to the final conclusion. In my opinion, the authors should "weaken" the first sentence of the Conclusion section and indicate it as more speculative.
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