Modelling the Activation Pathways in Full-Length Src Kinase

Round 1

Reviewer 1 Report

This research focuses on the study of conformational changes of c-Src tyrosine kinase, involved in several cellular processes, which make it an attractive target for drug design. The authors have done a computational study based on molecular dynamic simulations, using an enhanced sampling technique developed by themselves and published in another article. Also, the effect of the inhibitor imatinib was analyzed. The overall report is well structured, the methods are clearly described. Results support the conclusion. However, some improvements are needed.     

• The conclusion is very extensive. Some parts of it can better fit in the section “Results”. The conclusion should be a concise and clear answer to the research question.
• Indeed, the section “Results” can be better changed by “Results and discussion” since it includes discussions.
• Credits to the software used for the elaboration of the figure must be recognized. 
• A video showing the main conformational changes could be very suitable to include as supplementary material.

Minor comments

• Figure 1. The caption is too short. It should describe a bit more each component of the figure.
• Figure 2. The caption should provide clear information about each trajectory. For example, the trajectory represented in green shows a transition at 250 nm, but no information is provided.
• Line 6: “availableexeperimental” data should be “available experimental” data
• Line 7: “mole-cular” should be “molecular”
• Line 49: “if view” should be "of view"

Author Response

We thank the referee for his/her comments and suggestions. 

Accordingly, we modified the manuscript as following:

• the results section was modified in Results and Discussions
• the Conclusions section was reduced
• Video showing the conformational pathways have been added to the Supporting Material
• The caption of figure 1 was extended
• the Fig. 2 and the label have been changed to better clarify that we compared the most representative MD trajectories of the c-Src open states
• The typo errors in line 6, 7 and 49 were corrected

Reviewer 2 Report

Authors have used extended molecular dynamics to study the open and closed states of c-src kinase in the presence and absence of a known small molecule inhibitor imatinib. The computational methods used are vigorous and the results obtained are useful to understand the protein behavior. This modeling shows that ligand binding can have an allosteric effect as well. The study is of interest to researchers in the field. The authors should modify the manuscript by addressing the comments below;

Section 1.1 Why did the authors choose different times scales for apo and holo enzymes? What was the rationale? Was it possible to increase the timescale to 1 microsecond? Would that provided different results?

Abstract: Line 2, 3, 4, 5, 6 and 7, few words are joined together with no space. They should corrected; eg: line 2 "theinactive"

The sections Introduction, Materials and Methods etc should be re-numbered correctly. Now Introduction starts as "0" and Materials and Methods as "1, 1.1" ... Should change to 1, 1.1 Revise this for all the sections

Figure 2: MD simulation for closed and open states. Please include all the color code information in the figure or in the figure legend for the RMSD/time plot to increase clarity

Figure 1: Font size in this figure can be enhanced as they are not visible. The figure size can be increases

Throughout the text while referring to Figures, use upper case, eg: figure 3 should be "Figure 3". This should be fixed at multiple places

Line 165, change compare to compared

Ref section:

Use consistent format - Ref 1, 13, 25, 26, 27. All the words in the article title starts with upper case. Only the first word should start with upper case

Author Response

We thank the referee for his/her helpful comments.

Accordingly, we modified the manuscript as following:

• Concerning the time scales used for the apo and holo enzyme, we have monitored selected structural observables (RMSDs and RMSFs for the whole protein construct or for some specific regions) along the MD trajectories. Accordingly, we have limited the MD simulations when these observables have achieved a reasonable convergence.
• Missing spaces between words have been added to the abstract.
• The section numbering now starts from 1.
•  Figure 1 size was increased.
• the Fig. 2 and the label have been changed to better clarify that we compared the most representative MD trajectories of the c-Src open states
• All the "figure" was changed to "Figure"
• Line 165: the compare was changed to compared

Reviewer 3 Report

The paper can be accepted as it is

However two small comments:

1. the legend to Fig. 2 is to poor
2. the charges taken for ligand from RESP procedure and applied to Amber99sb force field . The charges - were they checked as to the compatibility with the force field used ?

Author Response

We thank the referee for his/her comments.

Accordingly, the manuscript has been modified as following:

• the Fig. 2 and the label have been changed to better clarify that we compared the most representative MD trajectories of the c-Src open states
• the procedure used to provide atomic charges for the inhibitor force field is one of the most used in the field. In our case, we also checked that chemical group charges as obtained by RESP were not significantly different with respect to the charges present in the Amber99SB force field for the same chemical group.