Exploring the Dynamics of Holo-Shikimate Kinase through Molecular Mechanics
Round 1
Reviewer 1 Report
General comments to the authors.
The manuscript entitled "Exploring the dynamics of holo-Shikimate Kinase through molecular mechanics", is well written and presents interesting data and analyzes in the biophysics and medicinal chemistry expertise field because the authors propose an understanding the connection between local and global dynamics of a crystallographic structure of Helicobacter pylori SK. they approach an interesting field to investigate target biomacromolecules.. However, it has some methodological and manuscript layout failures that strongly need to be considered by the authors before being accepted for publication. Which I encourage you to review.
Introduction:
1 - The authors begin by mentioning that the enzyme under study is promising for studies aimed at discovering and developing new drugs, however they do not indicate the focus for which diseases and/or therapies.
2 - The authors only mention the term "frustration" in the introduction, it seems to be interesting and also key to understanding the proposal of this manuscript by those who read the text. I strongly suggest the insertion of a topic addressing this term with possible functionalities, examples and importance for studies of this nature, still in the introduction.
Materials and Methods
3 - 2.1 Simulation setup – Line 83: In the manuscript, authors indicate that dynamic simulations started from the crystallographic structure of Helicobacter Pylori SK, under the PDB ID 3MUF, but it is not clear why they chose this species of organism, nor why they chose the referred crystallographic structure instead of others available. I recommend that authors insert a topic explaining the selection of frameworks used.
4 - Authors do not provide data on resolution of the selected PDB structure, nor XYZ coordinates of spatial location of domains considered important for functions of the enzyme, whether agonist or inhibitory. This information can help readers to better understand the strategy employed in the study.
5 – No type of validation of the protein structure (reference in this study) was displayed under the code PDB ID 3MUF. This analysis is important to confirm the location of function sites and amino acid residues considered key to function or mutation.
Results and discussion
6 - Are the XYZ coordinates the same in Wild-type (WT) and mutants R116A and R116K?
7 - What are the coordinates of the substrates, the Mg 2+ ion, and the flexible tail of the enzyme?
8 - Lines 235 – 238: In the case of investigation of drugability and drug design for Helicobacter pylori SK, what does it mean for an interaction to be in conflict or a neutrally frustrated structure? Recommend rewriting in order to compare and relate.
In conclusion:
9 - Authors do not clearly show a connection between the novelty of this study and the contribution to investigation processes of drugability and drug design for Helicobacter pylori SK. I recommend rewriting in a relatable way.
10 - Supplementary material is not mentioned in the study. no explanation or discussion of the supporting information is displayed, only a figure. This can make it difficult for readers to understand.
Author Response
We thank Reviewer 1 for the insightful comments that without a doubt will improve the quality of our work. We have addressed all these comments and hope that Reviewer 1 is satisfied with the proposed changes.
Introduction:
1 - The authors begin by mentioning that the enzyme under study is promising for studies aimed at discovering and developing new drugs, however they do not indicate the focus for which diseases and/or therapies.
Reply: Some diseases where Shikimate Kinase is involved were mentioned in our previous publication [Biophysica, 1, 334-343 (2021)] and we thought that writing this information in the present manuscript could be seen as redundant. However, as this issue was raised by the reviewer, we have added the following sentence in the Introduction section:
“ … which cause diseases such as tuberculosis and gastric ulcers [1–4]”
2 - The authors only mention the term "frustration" in the introduction, it seems to be interesting and also key to understanding the proposal of this manuscript by those who read the text. I strongly suggest the insertion of a topic addressing this term with possible functionalities, examples and importance for studies of this nature, still in the introduction.
Reply: We clarified this point by changing the sentence in the Introduction section:
“In the context of enzyme dynamics, it has been found that frustration plays a crucial role in modulating the overall behaviour of the system and influencing functional outcomes [14,16].”
to,
“In the context of enzymes’ functioning, it has been found that catalytic sites are usually highly frustrated [25]. In addition to this, frustration in the substrate-product interactions plays a crucial role in modulating the overall behaviour of the system and influencing functional outcomes such as the facilitation of the rate-determining step [14,16].”
where a new reference [25] (Proc. Natl. Acad. Sci. 2019, 116, 4037–4043) was introduced.
Materials and Methods
3 - 2.1 Simulation setup – Line 83: In the manuscript, authors indicate that dynamic simulations started from the crystallographic structure of Helicobacter Pylori SK, under the PDB ID 3MUF, but it is not clear why they chose this species of organism, nor why they chose the referred crystallographic structure instead of others available. I recommend that authors insert a topic explaining the selection of frameworks used.
Reply: This PDB structure (3MUF) has been used previously in studies of others [Chem.Eur.J. 2016, 22,2758 –2768; Chem. Eur.J.2017, 23,16380–16387 ] and ours [Biophysica, 1, 334-343 (2021); Biophysica 2022, 2, 194–202]. Thus, to make a fair comparison of the dynamics of SK with previously reported data, we chose the same structure. We changed the sentence:
“The crystallographic structure of Helicobacter pylori SK was used in our simulations (PDB ID: 3MUF) [23]”
to
“The crystallographic structure of Helicobacter pylori SK was used in our simulations (PDB ID: 3MUF, at 2.3 Å resolution) [23] which was also used in previous studies [17–19,26]”
where a new reference 26 [Chem. – Eur. J. 2016, 22, 2758–2768] was introduced.
4 - Authors do not provide data on resolution of the selected PDB structure, nor XYZ coordinates of spatial location of domains considered important for functions of the enzyme, whether agonist or inhibitory. This information can help readers to better understand the strategy employed in the study.
Reply: The resolution of the structure (2.3 Å) was mentioned in the previous point. The structure considered in the present work was the one with natural products, not the inhibitor.
5 – No type of validation of the protein structure (reference in this study) was displayed under the code PDB ID 3MUF. This analysis is important to confirm the location of function sites and amino acid residues considered key to function or mutation.
Reply: For this organism, the reference structure is displayed in Figure 1 with the important regions denoted with different colors. The conserved residues D33, R57, R116, and R132 were mentioned in a previous publication [Biophysica 2022, 2, 194–202] together with the R116 explicit location (Figure 2 of the mentioned article).
Results and discussion
6 - Are the XYZ coordinates the same in Wild-type (WT) and mutants R116A and R116K?
Reply: Yes, the same initial coordinates were used except for the fact that the amino acid nr. 116 was different. Notice that we performed equilibration steps and therefore the data production started from the last equilibration step structure. However, for the analysis of the trajectories, the crystal structure was used in all cases.
7 - What are the coordinates of the substrates, the Mg 2+ ion, and the flexible tail of the enzyme?
Reply: As it is mentioned in the manuscript the Mg2+ ion was added manually using geometric measurements. The tail was added through the CHARMM-GUI interface.
8 - Lines 235 – 238: In the case of investigation of drugability and drug design for Helicobacter pylori SK, what does it mean for an interaction to be in conflict or a neutrally frustrated structure? Recommend rewriting in order to compare and relate.
Reply: We clarified the concept of a frustrated structure by adding the following sentence:
“In more physical terms, minimally frustrated regions exhibit well-defined structures while highly frustrated regions tend to be more flexible [55]” (lines 252-253)
Here, a new reference 55 [eLife 2020, 9, e54661] was added
In conclusion:
9 - Authors do not clearly show a connection between the novelty of this study and the contribution to investigation processes of drugability and drug design for Helicobacter pylori SK. I recommend rewriting in a relatable way.
Reply: In order to show the connection in a clearer manner we added the following sentence in the Conclusions section:
“We argue that these local conflicts, in the mutants’ case, disrupt crucial contacts around the mutational site (located at the LID domain) resulting in a different local dynamics and that this in turn causes the difference in the global enzyme dynamics w.r.t the WT case.” (lines 350-353)
We also modified this sentence in the Results and Discussion section:
“Thus, we argue that frustration can contribute to the disruption of coordination in the motion of the different parts of the protein structure (Figure S1).”
to
“Thus, we argue that a locally frustrated region, in the mutants’ case, results in different local dynamical motions that disrupt the coordination of the global dynamical motions of the WT protein structure (Figure S1)” (lines 263-266)
10 - Supplementary material is not mentioned in the study. no explanation or discussion of the supporting information is displayed, only a figure. This can make it difficult for readers to understand.
Reply: We added more information for the supplementary figure S1, we change the sentence:
“A directed graph with these results is given in Figure S1 a showing the connection patterns within the protein regions.”
to
“A more pictorial representation of these results is achieved through directed graphs in Figure S1 where the connection patterns among the protein regions are shown.” (lines 219-221)
Reviewer 2 Report
The study aimed to investigate the dynamics at the local and global levels of Shikimate Kinase through molecular dynamics simulations. Which highlights the importance of the study.
In general, the introduction is well written and addresses the main aspects that cover the study's problematic. The material and methods are clear and adequate. The results and discussion are also well described. Therefore, I consider that the study is suitable for publication in Biophysica after minor corrections.
Author Response
In general, the introduction is well written and addresses the main aspects that cover the study's problematic. The material and methods are clear and adequate. The results and discussion are also well described. Therefore, I consider that the study is suitable for publication in Biophysica after minor corrections.
Reply: We thank Reviewer 2 for the positive comments on our work.
Reviewer 3 Report
The manuscript by May et al delves into the investigation of Shikimate Kinase (SK) dynamics using molecular mechanics over a microsecond time-scale. The study specifically examines the enzymatic activity of the wild-type (WT) and two mutant forms, while analyzing both the local and global dynamics of the enzyme. Interestingly, despite the publication of previous papers by the same authors on the subject (May et al 2021; May et al 2022), this manuscript does not explicitly discuss its contributions in advancing existing knowledge or how it builds upon the findings presented in the earlier reports.
Major comment:
1. The results section of the manuscript could be improved by clearly stating the key findings and highlighting how they contribute to advancing existing knowledge, particularly in comparison to the authors' two previous papers and other relevant research.
2. The conclusion section needs improvement by addressing the gap in the field of Shikimate Kinase (SK) and explaining how this manuscript attempts to fill that gap. It would be helpful to discuss the authors' findings related to SK from their previous report and highlight how the current manuscript advances knowledge and contributes to the significance of the research in this area.
Minor corrections required
Author Response
We thank Reviewer 3 for the insightful comments that we have addressed and we hope that the Reviewer is satisfied with the proposed changes.
1. The results section of the manuscript could be improved by clearly stating the key findings and highlighting how they contribute to advancing existing knowledge, particularly in comparison to the authors' two previous papers and other relevant research.
Reply: We have made a comparison with our previous papers on several parts of the manuscript, for instance on lines: 147, 163, and 179. We added some paragraphs and sentences to the present manuscript to further highlight our findings in our study that we haven’t clearly mentioned previously:
“The different local and global dynamics of the mutants can induce different “chewing” motions on the substrates (including reactant, transition, and product states) than in the WT case which can in turn contribute to their reduced catalytic activity. For instance, in the present study we found that SK opens faster in the mutants than in the WT which affects the product release step. In addition to this, the “chewing” motions in the mutants can create non-optimal orientations for residues during catalysis.” (lines 257-262)
and also:
“As in the apoenzyme case, we found that the choreography of the motions in WT holoenzyme is destroyed for the studied mutant cases [5,19]” (lines 263-264)
2. The conclusion section needs improvement by addressing the gap in the field of Shikimate Kinase (SK) and explaining how this manuscript attempts to fill that gap. It would be helpful to discuss the authors' findings related to SK from their previous report and highlight how the current manuscript advances knowledge and contributes to the significance of the research in this area.
Reply: In order to make the advances in knowledge of SK clearer, we extended the following paragraph:
“In conclusion, our analysis of the dynamics and interactions in the WT and mutant variants of SK provides insights into the structural and functional consequences of specific mutations. These findings contribute to a deeper understanding of the interplay between residue dynamics, conformational transitions, and higher-order interactions in SK. The knowledge gained from this study can facilitate the design of novel strategies for modulating enzyme activity and guide future investigations aimed at optimizing enzymatic performance for various applications.”
to:
“In conclusion, our analysis of the dynamics and interactions in the WT and mutant variants of SK provides insights into the structural and functional consequences of specific mutations. These findings contribute to a deeper understanding of the interplay between residue dynamics, conformational transitions, and higher-order interactions in SK. We found that the choreography of these interactions is disrupted for the mutants considered in this study. The knowledge gained from it can facilitate the design of novel strategies for modulating enzyme activity and guide future investigations aimed at optimising enzymatic performance for various applications. For instance, our study suggests that one needs to consider the dynamics of the enzyme as a major player in enzyme design.”
Round 2
Reviewer 1 Report
The author has not inserted any other the manuscript can be accepted for publication as it is.
Reviewer 3 Report
The author addressed all the comments raised
Very few corrections from the editorial side may required.
