Role of Graphene Oxide in Inhibiting the Interactions between Nucleoside Diphosphate Kinases -B and -C
Round 1
Reviewer 1 Report
Zaznaev and Macwan here propose the presense of graphene oxide (GO) affect the interaction and dynamic of NDPK-B and NDPK-C. Overall, the study is scientific-sound and only need mild improvement for publication. There're some typos in the manuscript, such as "NPDK-C" in the line 10 in abstract section. In addition, the authors should add more description and rationale for studying the effects GO on NDPKs to strengthen the significance of the manuscript.
Author Response
We thank the reviewer for their constructive comments. Based on their feedback, we have added a paragraph in the introduction section (page 2, lines 63 to 68) to better describe the rationale for studying the effects of GO on NDPK. Please find below the excerpt:
“Studying the effects of GO on NDPK-BC complex would uncover the theoretical considerations for using an agent capable of suppressing the unwanted GDP(i) phosphorylation pathway during a heart failure. The findings would potentially suggest new directions for future research on the topic and highlight some key points on the molecular basis of the problem.”
Reviewer 2 Report
This manuscript presents an interesting analysis of the interaction between NDPK-B and Graphene Oxide and how this affects the interaction between NDPK-B and NDPK-C. This analysis supports the ongoing exploration of Graphene Oxide as a possible therapy for certain types of heart failures. However, to be published, the authors need to answer the following questions/suggestions to support their claims better.
In the manuscript presentation, I have minor suggestions:
1. All the graphs (except in figure 2) order is a, c, b, d (reading from left to right, top to bottom). This is highly confusing and requires clarification and consistency. The natural order would be to have a, b, c, d, as readers usually read from left to right, top to bottom. I suggest following the same order as figure 2 for all the figures.
2. I suggest including a grid or guidelines for the essential values in the graphs. It helps the readers to follow the authors' claims.
I also have several questions:
3. Page 3, lines 94-95: the authors state that the His118 end of the NDPK-B is 10 A away from the GO. What is the center of mass distance between NDPK-B and GO for this initial condition?
4. Page 4, lines 151-165: For the center of mass (COM) analysis, how the initial condition affects the final distance between the center of mass? Would a different initial distance change the final COM between NDPK-B and GO?
5. Page 5, lines 166-169: The authors state a milestone at 150 ns is attained for the COM distance between NDPK-B and GO. Is the variation of ~1 A (or even less) relevant enough to be considered? If yes, they should also comment on the slight COM increase between 25 ns and 150 ns.
6. Page 5, lines 197-198: The authors state that the energy is increased by 25 kcal/mol after 150 ns. However, from figure 3d, it seems to come back to -375 kcal/mol at 200 ns. Does it mean that NDPK-B will return to its original interaction with NDPK-C?
7. Page 9, lines 319-321: the authors state that "~600 atoms of NDPK-B adsorbed on the surface of GO". However, for the optimum adsorption distance (~3 A), much fewer atoms are involved. The authors seem to support their claim with the number of atoms involved in the interaction for ~5 A. Why choose 5 A and not 3 A (which is the optimum adsorption distance)?
8. Page 10, lines 341-342: the authors state that the "optimal distance between NDPK-B and NDPK-C, as can be seen from figure 7b and 7c, it is 2 A". Although, I can see this clearly from figure 2c, I am not sure I understand how figure 7b supports this claim.
9. Page 10, lines 344-346: the authors conclude that the interaction between NDPK-B and GO "is largely due to non-binding energies such as Van der Waals". In the whole manuscript, the authors only showed the Van der Waals interaction between NDPK-B and GO (figure 4d). To support their claim, the authors should add a figure with the electrostatic interaction of NDPK-B and GO.
10. Finally, if initially the GO is not placed on the His118 side of NDPK-B, how would be the adsorption of NDPK-B to GO? Is His118 the preferable bonding place for GO?
Author Response
This manuscript presents an interesting analysis of the interaction between NDPK-B and Graphene Oxide and how this affects the interaction between NDPK-B and NDPK-C. This analysis supports the ongoing exploration of Graphene Oxide as a possible therapy for certain types of heart failures. However, to be published, the authors need to answer the following questions/suggestions to support their claims better.
In the manuscript presentation, I have minor suggestions:
- All the graphs (except in figure 2) order is a, c, b, d (reading from left to right, top to bottom). This is highly confusing and requires clarification and consistency. The natural order would be to have a, b, c, d, as readers usually read from left to right, top to bottom. I suggest following the same order as figure 2 for all the figures.
We thank the reviewer for their suggestion and have modified all the figures in the correct order a, b, c, d (reading from left to right, top to bottom).
- I suggest including a grid or guidelines for the essential values in the graphs. It helps the readers to follow the authors' claims.
We thank the reviewer for providing this helpful insight. We have now added arrowheads on the plots for figures 2 and 3, specifically for the essential values described in the writeup.
I also have several questions:
- Page 3, lines 94-95: the authors state that the His118 end of the NDPK-B is 10 A away from the GO. What is the center of mass distance between NDPK-B and GO for this initial condition?
The initial (at time t = 0) center of mass distance between NDPK-B and GO was ~38.3Å as seen from figure 3A.
- Page 4, lines 151-165: For the center of mass (COM) analysis, how the initial condition affects the final distance between the center of mass? Would a different initial distance change the final COM between NDPK-B and GO?
That is an excellent question. Typically, we would keep the two molecules in question at distances ranging from 10 to 15 Å. The reason behind this is to ensure that we don’t simply spend vital simulation time simply waiting for the two molecules to approach each other. Once the two molecules are within the energy/ force cut-off range (typically 12Å), that is when the adsorption phenomena would initiate. Until that time, all the analysis would not be reliable. To avoid this waiting game, we won’t keep the molecules more than 15Å away from each other and so if the orientation of the two molecules were to be kept as is with a larger distance between the two, most likely, we would see the same kind of COM plot except after a longer time period.
- Page 5, lines 166-169: The authors state a milestone at 150 ns is attained for the COM distance between NDPK-B and GO. Is the variation of ~1 A (or even less) relevant enough to be considered? If yes, they should also comment on the slight COM increase between 25 ns and 150 ns.
We thank the reviewer for this very important observation. The slight increase of the COM between NDPK-B and GO between 25ns and 150ns can be attributed to the conformational changes of the NDPK-B as it starts to adsorb on the surface on the GO. This can be seen indirectly through its RMSD (Fig 2A) and electrostatic energy (Fig 4A). We have added this comment in the revised version, page 5, lines 178 – 181.
- Page 5, lines 197-198: The authors state that the energy is increased by 25 kcal/mol after 150 ns. However, from figure 3d, it seems to come back to -375 kcal/mol at 200 ns. Does it mean that NDPK-B will return to its original interaction with NDPK-C?
We thank the reviewer for this observation also. Looking at figure 3D, it does seem like the VDW energy is increasing slightly beyond 150ns. However, towards the end of the simulation (~250ns), it appears to again dip beyond 400 kcal/mol. In any case, we don’t think that NDPK-B will return to its original interactions with NDPK-C because of what we find in figures 2B and 2D that NDPK-C has deviated much farther away from NDPK-B compared to the control after 150ns and that the distance between the center of masses of the two enzymes should have been ~55Å to initiate meaningful interactions, which now is beyond 60Å. We expected something like this to happen mainly because NDPK-B after its adsorption on GO, would undergo a conformational change, which won’t be conducive for it to interact with NDPK-C. As we know proteins interact based on its conformational state and since NDPK-B is now in a different conformational state after its adsorption onto the GO, it won’t interact with NDPK-C naturally.
- Page 9, lines 319-321: the authors state that "~600 atoms of NDPK-B adsorbed on the surface of GO". However, for the optimum adsorption distance (~3 A), much fewer atoms are involved. The authors seem to support their claim with the number of atoms involved in the interaction for ~5 A. Why choose 5 A and not 3 A (which is the optimum adsorption distance)?
We thank the reviewer for this question. Typically, an adsorption distance of 5Å is chosen simply because that is the distance when the weak hydrogen bonds and salt bridges start to form. However, as interactions become stronger, the atoms within a certain distance would play a more crucial role at the interface. These number of atoms and distance would change depending upon the nature of interacting surfaces. For instance, if the two surfaces are fully hydrophobic, then this distance would be smaller and for a more hydrophilic interface, this distance may be slightly larger. In any case, it won’t go beyond 5Å as that is the theoretical limit for the hydrogen bonds and salt bridges to form.
- Page 10, lines 341-342: the authors state that the "optimal distance between NDPK-B and NDPK-C, as can be seen from figure 7b and 7c, it is 2 A". Although, I can see this clearly from figure 2c, I am not sure I understand how figure 7b supports this claim.
We thank the reviewer for pointing out this typo. Figure 7B shows the interaction energy with respect to distance and not the optimal distance. We have removed this typo in the revised version.
- Page 10, lines 344-346: the authors conclude that the interaction between NDPK-B and GO "is largely due to non-binding energies such as Van der Waals". In the whole manuscript, the authors only showed the Van der Waals interaction between NDPK-B and GO (figure 4d). To support their claim, the authors should add a figure with the electrostatic interaction of NDPK-B and GO.
We thank the reviewer for pointing this out. The GO model we used is a neutral molecule with no net charges. The enzymes on the other hand do have net charges owing to the different amino acid configurations. Because of this, there is no interaction energy between NDPK-B and GO. We have added this comment in the revised version page 10, lines 363-365.
- Finally, if initially the GO is not placed on the His118 side of NDPK-B, how would be the adsorption of NDPK-B to GO? Is His118 the preferable bonding place for GO?
Yes, the His118 site is the binding site in case of NDPK-B as this is the amino acid that facilitates the transfer of phosphate between NDPK-B and the G protein, which then triggers the cardiac events. By using GO close to this binding site and quantifying its interactions around this site is to ensure that GO can block any interactions between His118 on NDPK-B to come in contact with the G-protein. If GO is not placed on the His118 side of NDPK-B, then we anticipate that NDPK-B may still undergo a conformational change which may prevent His118 to bind with the counterpart on the G protein for phosphate transfer. However, this will have to be verified through future simulations between NDPK-B and G protein in the presence of GO.
