Molecular Dynamics Simulations for the Michaelis Complex of Ectoine Synthase (EctC)
Round 1
Reviewer 1 Report
Starting from crystal structures of Ectoine Synthase (EctC) the authors employ classical MD simulations to better resolve the active site Fe and catalytic residues. Based on 100 ns simulations, the authors propose a refined active site model and suggest possible routes to a form a near attack conformation (NAC) affording catalysis with an octahedral 6-coordinate Fe+2. The writing is clear in most cases and the figures provide clear images that relate directly to the text. A few items need attention. The text could probably be shortened, especially if the refined structure is deposited so others can examine the results directly.
There should be some discussion of the limitations of a 100 ns MD simulation. In particular, starting on line 459 the authors state “Here we show that Trp21 does not spontaneously switch from the A to B conformation within the simulated time ranges, but it does switch in the opposite direction (from B to A). Furthermore, we show that the B conformation of Trp21 is most likely non-productive by contributing to the occurrence of an energetic barrier associated with the formation of NAC.” Presumably the switch from A to B would occur on a longer time scale unless the transition is energetically unfavorable, which seems unlikely.
The model presented for substrate inhibition starting on line 463 would not, by itself account for substrate inhibition. Substrate inhibition observed in the steady state is due to the binding of a second substrate at a second binding site that inhibits turnover, after an initial substrate has bound to the catalytic site. Perhaps a model based on an alternating site mechanism would account for substrate inhibition if the weaker binding of substrate to the second subunit slowed the rate of turnover at the other site in the dimer.
The sentence beginning on line 41 is overly complex in attempting to convey multiple thoughts in a single long sentence. It should be split into two sentences to make it more readable.
The sentence beginning on line 91 is weak:
“Concerning the geometry of the substrate, in the crystal structure it adopts a conformation close to the near attack conformation (NAC) with the alpha-amino nitrogen located 3.8 Å away from the carbonyl carbon of the acetyl moiety.”
A better option is:
“In the crystal structure the substrate adopts a conformation close to the near attack conformation (NAC) with the alpha-amino nitrogen located 3.8 Å away from the carbonyl carbon of the acetyl moiety.”
Include the PDB ID in the legend to figure S1.
The refined enzyme structure should be deposited in the database modelarchive.org so that it will be accessible. The PDB does not accept models, so this database is an important alternative.
Author Response
We would like to thank the Reviewers for their time and effort to review our manuscript and for constructive suggestions on how to improve it. We have taken them into account in the revision. Our detailed responses are listed beneath.
There should be some discussion of the limitations of a 100 ns MD simulation. In particular, starting on line 459 the authors state “Here we show that Trp21 does not spontaneously switch from the A to B conformation within the simulated time ranges, but it does switch in the opposite direction (from B to A). Furthermore, we show that the B conformation of Trp21 is most likely non-productive by contributing to the occurrence of an energetic barrier associated with the formation of NAC.” Presumably the switch from A to B would occur on a longer time scale unless the transition is energetically unfavorable, which seems unlikely.
We absolutely agree with the Reviewer that not observing an event during a 100 ns simulation does not mean the event cannot occur in longer time ranges; in the revised manuscript we have added the following sentence: “As the simulation time is limited (100 ns) it does not mean the Aï‚®B transition is not possible at all, but, based on the very rough estimate from transition state theory for the simulation time, it is endoergic by at least 7 kcal/mol.” (lines 457-459).
The model presented for substrate inhibition starting on line 463 would not, by itself account for substrate inhibition. Substrate inhibition observed in the steady state is due to the binding of a second substrate at a second binding site that inhibits turnover, after an initial substrate has bound to the catalytic site. Perhaps a model based on an alternating site mechanism would account for substrate inhibition if the weaker binding of substrate to the second subunit slowed the rate of turnover at the other site in the dimer.
Indeed, after a thought we agree with this remark of the Reviewer, a conformational change within a single protein monomer cannot account for substrate inhibition within this monomer. On the other hand, we did not notice any signs of cooperation between the two monomers forming a dimer. Hence, we removed this sentence from the manuscript.
The sentence beginning on line 41 is overly complex in attempting to convey multiple thoughts in a single long sentence. It should be split into two sentences to make it more readable.
Indeed, this sentence was too complex. We have split it into two and slightly reformulated the second sentence (lines 41-46).
The sentence beginning on line 91 is weak:
“Concerning the geometry of the substrate, in the crystal structure it adopts a conformation close to the near attack conformation (NAC) with the alpha-amino nitrogen located 3.8 Å away from the carbonyl carbon of the acetyl moiety.”
A better option is:
“In the crystal structure the substrate adopts a conformation close to the near attack conformation (NAC) with the alpha-amino nitrogen located 3.8 Å away from the carbonyl carbon of the acetyl moiety.”
Indeed, the sentence proposed by the Reviewer is better; we have changed it in the text accordingly.
Include the PDB ID in the legend to figure S1.
PDB ID (5ONN) has been included in the legend of Fig. S1.
The refined enzyme structure should be deposited in the database modelarchive.org so that it will be accessible. The PDB does not accept models, so this database is an important alternative.
The models have been deposited in the suggested database (modelarchive.org).
Reviewer 2 Report
The paper describes the modeling and MD simulations of the Ectoine Synthetase. The overall evaluation of the work is that it is well presented, enjoys the interest of a wider public researching protein function and catalytic mechanism as well as being a protein that is part of the ectoine synthesis, with pharmacological importance.
I believe the paper is sound and I have not detected relevant issues that will render the work open to further revisions.
Author Response
We would like to thank the Reviewers for their time and effort to review our manuscript and for constructive suggestions on how to improve it. We have taken them into account in the revision. Our detailed responses are listed beneath.
We thank the Reviewer for very positive assessment of our manuscript.
Reviewer 3 Report
Title: Molecular Dynamics simulations for the Michaelis complex of Ectoine Synthase (EctC)
The manuscript reports the details of the structural analysis of Etocoine Synthase enzyme in the presence of its substrate (N-g-ADABA) using molecular dynamics simulation. There are some comments that should be addressed by the authors.
interactions of some phenolic compounds with alpha-lactalbumin using different experimental techniques. The authors have tried to describe the possible involved covalent and non-covalent binding interactions based on the observed results.
1- The authors presented a lot of structural information for the enzyme based on the references [1] and [2]. It is recommended that a main part of these information would be presented in the discussion section.
2- In the discussion section only two references [1] and [2] are provided!!
3- In page 7, lines 215-216: what is the criteria for distinguishing the strong and weak hydrogen bonds? (energy or distance?)
4- In figure 6: there are different figures that must be numbered by a, b, c,…
5- In Page 12: decreasing the mobility of alpha-II-helice was considered as the evidence for increasing the stability according to the RMSF results. Is this evidence sufficient for this conclusion?
Author Response
We would like to thank the Reviewers for their time and effort to review our manuscript and for constructive suggestions on how to improve it. We have taken them into account in the revision. Our detailed responses are listed beneath.
1- The authors presented a lot of structural information for the enzyme based on the references [1] and [2]. It is recommended that a main part of these information would be presented in the discussion section.
The reason the references [1] and [2] are extensively cited in the manuscript is because these are the only currently available papers dedicated to EctC structure, and the structure of the EctC in complex with its substrate is in focus of this work. We thank the Reviewer for the suggestion, but we prefer to present the “background” information in the Introduction, to prepare the reader for the results; repeating these in the Discussion would significantly lengthen the manuscript, which is already quite long.
2- In the discussion section only two references [1] and [2] are provided!!
The Discussion section is rather short and provides a summary of the key findings of this work on the background of previous structural work on EctC, which currently limits to these two publications ([1] and [2]).
3- In page 7, lines 215-216: what is the criteria for distinguishing the strong and weak hydrogen bonds? (energy or distance?)
The strength of H-bonds was provisionally assessed based on the distance, a short explanation was added to the text: “(as judged based on the H-X distance)” (line 219)
4- In figure 6: there are different figures that must be numbered by a, b, c,…
Sub-figures of Fig. 6 have been labeled A, B and C.
5- In Page 12: decreasing the mobility of alpha-II-helice was considered as the evidence for increasing the stability according to the RMSF results. Is this evidence sufficient for this conclusion?
The Reviewer is right, it is rather an evidence of lower mobility of the helix, not its internal stability. In the revised manuscript instead of “stablizes the alpha-II helix” we have written “stablizes the position of the alpha-II helix”
