Investigation into the Binding Site of (-)-Spirobrassinin for Herbicidal Activity Using Molecular Docking and Molecular Dynamics Simulations

Round 1

Reviewer 1 Report

In this study, the authors have used molecular docking and simulations, along with experimental validation to investigate the molecular basis of inhibitory activity of -(-)Spirobrassinin observed on weeds. This study will contribute to the development of -(-)Spirobrassinin as a  natural herbicide.

Introduction section, line 47. Please add some reference for the " unpublished data)", otherwise refrain form mentioning it.

Figure S2, please mention in the legend, which PDB ID is used for which protein and from which species.

In the methods section, "DHDA (PDB code: 5ZE4) or DHAD?" I don't see DHDA being mentioned before.

Please expand the acronyms when they are used for the first time. I don't see any expansions for ACC, DAD2, etc. too.

Under the Introduction section, D13-D3-ASK1 has been mentioned in the text, but under the Methods section, it says D14-D3-ASK1 (PDB code: 5HZG). Please recheck and edit accordingly.

I do not see any reference for the Autodock tool, H++3 tool, Gromacs, PME? No citations for The Lamarckian genetic algorithm (LGA) and the Solis and Wets local search? Please cite responsibly.

For Grid point spacing in the Methods section, the units should be Angstroms, not just A. Please insert the correct symbols.

Results section: the sentence "Using Autodock for virtual screening (Figure S1)". This is not a virtual screening that has been done in this work. Virtual screening is used to search libraries of small molecules in order to identify the hit structures binding to a drug target. In this work, only one compound was studied. NO screening was performed. 

Table 1: Please update the units of binding energy. It should be Kcal/mol. Please mention clearly in the legend that these are predictions from Autodock docking calculations.

Figure 3 snapshots don't give any insight. The author can show specific binding interactions at the pocket (or 2D interaction plots?) to show the interactions are present throughout the simulation.

Figure 4C, please explain in the text, the shift of protein RMSD observed at ~ 25 ns.

In Figure 4, the legends are totally off of what is shown in the figure. A is not RMSD and C is not the radius of gyration!

What information RMSF plots are generating? which residues of the protein were showing high fluctuation? Is that consistent with the proposed binding mode? I would like to see that information to be added in section 3.4

Figure 5: The showed interactions, are they from the last frame? Were all these interactions present throughout the simulations or were stabilized at the end of simulation? If possible, frequency information can be added against each interaction shown in the 2D plot to show how frequently these were observed. (for e.g. 100 % means the interaction was present throughout the simulation). This can be easily done using SID tool of Schrodinger.

Figure 5 legend: Grey representing as ligand? doesn't look true.

Figure 6A seems to be generated using the Schrodinger package. I expect to see the citation. 

The section 3.6 talks about "yellow color in the figure". which figure?

Section 3.7: Which binding energy has been talked about? How is that different from the one listed in Table 1. The whole section needs to be rewritten for better English. what do you mean by "moderate binding strength?".Table 1: please clearly mention that what is listed here is the Autodock scores i.e. binding energy values generated from docking so that the reader can differentiate between the MM/GBSA scores (mentioned here) and Autodock scores (which doesn't seem to be correlating well).

Discussion section: "Docking was considered successful when the RMSD value was below 2 Å". I would like to see a citation for that.

Line 365 "We performed a re-docking". This is not redocking, we use "redocking" term when a ligand is docked again to the co-crystal structure of its bound form with the receptor. Please update accordingly.

Throughout the paper, a moderate issue with English grammar usage and sentence formation was observed. I have listed a couple of sentences to highlight that.

Please rewrite the section "2.4 Cultivation and bioassay of C. vulgaris". Stay consistent with the use of grammar/tenses.

section 3.3: "...simualtion to examine.." should be "simulations were performed to examine..." and "Snapshots were taken". Please recheck the English sentence formation. There is a bit of inconsistency.

The Discussion section, line 333: It should be  "we assessed the binding energies of (-)-Spirobrassinin to the receptors".

Many issues with formatting. Some section headings are ending with a period and some are not. With the text also, spacing is inconsistent. Please make sure a space is added after a period and not before it.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

This study focused on investigation of the mechanism of action of the Spirobrassinin, a secondary metabolite produced form Brassicaceae plants and finding its molecular target to develop Spirobrassinin as herbicide product. The manuscript is well written, and the hypothesis resulting from the computational studies are supported by experimental evidence. However, I found some room for the improvement of this manuscript. Please find the following comments:

This study aimed to investigate the mechanism of action of Spirobrassinin, a secondary metabolite derived from Brassicaceae plants, and identify its molecular target for the development of Spirobrassinin as a herbicide. The manuscript is well-written, and the results presented support the hypothesis. However, there are areas in which this manuscript can be improved. Please find the following comments:

Major Concerns:

1.       The molecular dynamics simulation of 50 nanoseconds is insufficient. The RMSD plot for the protein indicates that the protein had not reached equilibrium even at 30 nanoseconds. Moreover, a 50 nanoseconds simulation may not allow for sufficient sampling of molecular complex conformations if the system is trapped in local energy minima. I suggest that the authors refer to the following study for a better understanding (DOI: 10.1039/D2MD00409G).

2.       I have doubts regarding the basis on which the author claimed that the dissociation of Spirobrassinin and PSBD1 was not observed. Was the distance between the compound and the protein binding site measured throughout the simulation?

3.       The author should provide clarification on which conformation was used as a reference for calculating the RMSD of the protein and its binding pocket. It is advisable to use the most stable conformation as a reference for calculating the RMSD of the binding pocket, considering that the starting structure did not appear to be equilibrated. This approach would help avoid any potential bias in the interpretation.

4.       In section 3.5, the author mentions that Spirobrassinin interacts with a water molecule near the protein's binding site. Does the original crystal structure show the presence of a water molecule in the binding pocket? This point would emphasize the significance of the water molecule for the interaction.

5.       The data presenting the growth inhibition assay should be included in the main text.

6.       To some extent, I hold a different opinion than the author's statement that computational methods cannot be employed to identify new binding sites. While it is true that uncovering novel binding sites can be a challenging task, it is by no means an impossible endeavor (doi:10.1186/1471-2105-10-168).

Minor Concerns:

1.       The full names of the protein targets were not provided in the manuscript.

2.       The author should have linked the figure numbers in the text when explaining them. For instance, manuscript line number 299 explains the figure but does not specify which figure they are referring to.

3.       Use an appropriate writing style as suggested by MDPI to present the equation showing MMGBSA free energy calculation, and terms such as "bind, ele, vdw, surf, solv, int" should be written in subscript font, and terms EGB and APBS are not defined.

4.       Did the author run multiple sets of MD simulations and molecular docking to conduct an F-test and provide statistical significance for the computational study too?

5.       What do the different colors of bars and labels on bars in Figures 2A to 2E represent?

6.       The Spirobrassinin molecule is not clearly visible in Figure 3. Increasing the transparency of the protein structure might help. Additionally, the author should mention that Spirobrassinin and PSBD1 are represented by spheres and cartoon representations, respectively.

7.       The author should highlight the key residues in the RMSF plot to evaluate binding events.

8.       A different color scheme should be used to distinguish the amino acid index number and bond distance in Figure 5C.

9.       The author did not mention what the blue curve represents in Figure 6B.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf