Identification of AtHsp90.6 involved in early embryogenesis and its structure prediction by molecular dynamics simulations

Heat-shock protein of 90 kDa (Hsp90) is a key molecular chaperone involved in folding the synthesized protein and controlling protein quality. Conformational dynamics coupled to ATPase activity in N-terminal domain is essential for Hsp90's function. However, the relevant process is still largely unknown in plant Hsp90s, especially those required for plant embryogenesis which is inextricably tied up with human survival. Here, AtHsp90.6, a member of Hsp90 family in Arabidopsis, was firstly identified as a protein essential for embryogenesis. Thus we modelled AtHsp90.6 in its functionally closed ‘lid-down’ and open ‘lid-up’ states, exploring the nucleotide binding mechanism in these two states. Free energy landscape and electrostatic potential analysis revealed the switching mechanism between these two states. Collectively, this study quantitatively analysed the conformational changes of AtHsp90.6 bound to ATP or ADP. This result may help us understand the mechanism of action of AtHsp90.6 in future.

Comments to the Author(s) The paper titled "Computational approaches for the study of Hsp90.6 from Arabidopsis thaliana" investigated the close and open states by using molecular dynamics. The conformational changes of Hsp90 bound to ATP and ADP were studied. I think the paper has potential to be published. However, several points should be improved.
1. Authors performed two kinds of experiments. However, the correlation between the wetexperiments and dry-experiments were not explained well. From the results and discussion, I cannot understand how to use theory model to explain the biochemical results.
2. The family of Hsp should be further studied by using the methods or webservers provided in references (PMID: 26233307; PMID: 29379521; PMID: 23756733).
3. Authors used Swiss-model to remodel the structure of AtHsp90. I know there are many 3-D structure prediction model. Authors may find them from CASP (http://predictioncenter.org/casp13/index.cgi). Many models have displayed very good performance. Why not use them?

21-Mar-2019
Dear Dr Iuo, On behalf of the Editor, I am pleased to inform you that your Manuscript RSOS-190219 entitled "Identification of AtHsp90.6 involved in early embryogenesis and its structure prediction by molecular dynamics simulations" has been accepted for publication in Royal Society Open Science subject to minor revision in accordance with the referee suggestions. Please find the referees' comments at the end of this email.
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02-Apr-2019
Dear Dr luo, I am pleased to inform you that your manuscript entitled "Identification of AtHsp90.6 involved in early embryogenesis and its structure prediction by molecular dynamics simulations" is now accepted for publication in Royal Society Open Science.
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On behalf of the Editors of Royal Society Open Science, we look forward to your continued contributions to the Journal. This manuscript describes the exploration of the nucleotide binding mechanism in the closed "liddown" and open "lid-up" states by molecular dynamics simulations. Free energy landscape and electrostatic potential analysis were used to address the switching mechanism between the closed and open states. The manuscript mainly showed results, but the discussion was not deep enough, not many comparisons were done to previous studies. Thus, I feel that it is not suitable for publishing in Royal Society Open Science. To improve this manuscript, I would like to make the following comments: 1. From the title, it seems to be a review on computational approaches for the study of Hsp90.6. There are some experiments, but I did not see how the experimental results are related to the results in silicon.
2. The authors used homology modeling to generated the models for AtHsp90.6 with SWISS-MODEL, the crystal structure PDB ID 2CG9 was used as a template. However, the sequence identity was not reported, the sequences alignment was shown, the number of generated models was not mentioned, the assessment of the generated models and how the models were selected were not discussed.
3. In the Material and Methods section, there are three types of molecular dynamics simulations, 1) 100 ns MD simulations using OPLS force field and spc216 water model, performed with Gromacs; 2) 1000 ns CG MD simulations using MARTINI force field, performed with Gromacs; 3) MD simulations using AMBER ff99 force field for MM-GBSA calculations, performed with Amber 11. a. It is not very clear how many systems were simulated from the Methods section, the readers can only guess from the results sections. From Figure 5 and 6, I speculate that two systems were simulated for 100 ns, respectively, closed ATP-AtHsp90.6 and closed ADP-AtHsp90.6. Why the Apo AtHsp90.6 at two different states were not simulated as the authors also wanted to study the binding of ATP? How many systems were simulated with CG model? I suspect only ATP-AtHsp90.6 was simulated, but not ADP-AtHsp90.6, then why not? Section 3.7 (line 29), the authors mentioned "The initial structure of N-terminal AtHsp90.6 was immersed...", did the authors only simulated the N-terminal of AtHsp90.6 or the whole structure of AtHsp90.6, but it was mentioned that the full length of AtHsp90.6 was modeled in Section 3.6? This is confusing.
b. Three force fields were used in this study, how could the authors justify and correlate the results from three different parameter sets? why not just use one? And I did not see the purpose of running CG MD simulations. The authors even did not mention the simulation time with Amber 11, and how many configurations were used for the MM-GBSA calculations. c. The parameters of ADP and ATP for the three force fields were not mentioned in the Methods sections, the authors developed, or used existed parameters? d. Section 3.9 (page 9), the authors stated "Because of the constant contribution of -TΔS for each complex...", this is not correct, the entropy is not constant, and it can be estimated with normal mode analysis, as done in this paper J. Med. Chem. 2006,49,6596-6606. Figure 3C Figure 4A and 4B (left and middle panels). From the right panel of Figure 4A and 4B, Table 1, I see that MD simulations were also performed for the structures PDB IDs 5FWK and 4XCJ, and then MM/GBSA calculations were done, but the simulations were not mentioned in the Method section and these results were not discussed fully in the main text. The authors did not explain why those simulations were performed. Why did the authors show the structure of 2XCM in Figure 4B? 6. In Figure 5, the distance was shown for all the 100 ns, but the SASA was only shown from 70 to 100 ns, why? 7. In Figure 6B, two intermediates were found, and they were separated by a barrier of ~2.5 kBT. Does it agree with the experimental data? 9. There were a lot of missing references: a. OPLS force field, SPC216 water model, PME algorithm, MARTINI force field, Berendsen thermostat and barostat methods, AMBER ff99 force field, Amber 11 package, SPSS19 software, b. MM/GBSA, the reference [26] given was wrong, which is for GB implicit water model, but not for MM/GBSA.  Figure S1 and Table S1 as mentioned in Section 4.1 ; b. Page 11, "no only forms" should be "not only forms" ; c. There should be a blank between digit and units, "1000ns" should be "1000 ns" … ; d. 1000 kJ mol-1 nm-2, superscript should be used ; e. "using a steepest descent method", "a" should be removed ; f. "Conformation dynamics" should be "conformational dynamics" in the Summary, "conformation changes" on page 19 line 46 should be "conformational changes" g. Abbreviations should be consistent, athsp90.6 should be AtHsp90.6, there are also some abbreviations with full names, ABRC, TAIL-PCR, CLSM, Rg, GFP Dear reviewers, Thanks for your comments on our paper. Your comments were highly insightful and enabled us to greatly improve the quality of our manuscript.

In
In the following pages are our point-by-point responses to each of the comments of the reviewers. Revisions in the text are shown using red color for additions, and blue strikethrough font for deletions. In order to account for the reviewer #2's suggestions to adding simulation systems for apo-AtHsp90.6, we now run independent simulations. As a consequence, we entirely reconstructed corresponding parts from the "result" and "discussion" section.
We hope that these revisions in the manuscript and our accompanying responses will be sufficient to make our manuscript suitable for publication in Royal Society Open Science. Responses to the comments of Reviewer #1 Comments 1. Authors performed two kinds of experiments. However, the correlation between the wet-experiments and dry-experiments were not explained well. From the results and discussion, I cannot understand how to use theory model to explain the biochemical results. Response: Thank you for pointing out. In the wet experiments, we identified AtHsp90.6 was essential for early embryogenesis and plant vitality for the first time by screening out a T-DNA insertion athsp90.6 mutant from ~7600 mutants. Since a so important protein is found, we are interested in its structural information for further understanding this protein.
Given experimental attempts to determine the structure of AtHsp90.6 has failed, we used computational approaches to predict conformational dynamics and nucleotide binding mechanism of AtHsp90.6. We identified three functional loops for the N-terminal domain of AtHsp90.6 and observed two intermediate states in ADP-bound AtHsp90.6. We hope these computational studies would be helpful for protein structure prediction.
Comments 2. The family of Hsp should be further studied by using the methods or webservers provided in references (PMID: 26233307; PMID: 29379521; PMID: 23756733). Response: We agree. We now re-evaluated the characteristic of AtHsp90.6 by using iHSP-PseRAAAC provided in the reference (PMID: 23756733). The results showed our sequence is Hsp90, correlating well with our previous conclusion.

Appendix B
Comments 3. Authors used Swiss-model to remodel the structure of AtHsp90. I know there are many 3-D structure prediction models. Authors may find them from CASP (http://predictioncenter.org/casp13/index.cgi). Many models have displayed very good performance. Why not use them? Response: Thank you for suggestions. Given the sequence identity between our model and the crystal structure (PDB ID 2CG9) was 56%, we homologly modelled AtHsp90.6 by our established methods (PMID: 20936826, 21637806 and 22653607). To assess the generated model, we adopted two criteria: Firstly, the structural similarity was measured by RMSD in their best-superimposed atomic coordinates. The RMSD is 0.545 Å, suggesting highly structural similarity between our model and the experimental structure. Secondly, our model was refined and validated by 1000 ps MD simulations [ Fig. S2], as the Sansom team has previously shown that such short MD simulations are useful to assess the quality of structural models (PMID: 16102990). It is a good choice to predict model from CASP and necessary discussion has been made in the revised manuscript.
Again, we would like to thank the reviewer #1 for his/her insights and constructive suggestions!

Responses to the comments of Reviewer #2
Major revision Comments 1. From the title, it seems to be a review on computational approaches for the study of Hsp90.6. There are some experiments, but I did not see how the experimental results are related to the results in silicon. Response: Thank you for pointing out. In the wet experiment, we identified AtHsp90.6 was essential for early embryogenesis and plant vitality for the first time by screening out a T-DNA insertion athsp90.6 mutant from ~7600 mutants. Since a so important protein is found, we are interested in its structural information for further understanding this protein.
Given experimental attempts to determine the structure of AtHsp90.6 has failed, we used computational approaches to predict the structure of AtHsp90.6 and explore its conformational dynamics and nucleotide binding mechanism. Firstly, we identified three functional loops for the N-terminal domain of AtHsp90.6. Secondly, we observed the closed states in apo-and ATPbound AtHsp90.6. Two intermediate states were observed in ADP-bound AtHsp90.6, separated by a barrier of ~2.5 kBT. We hope these computational studies would be helpful for protein structure prediction. To better describe wet and dry experiments in the current study, we would like to change the title to "Identification of AtHsp90.6 involved in early embryogenesis and its structure prediction by molecular dynamics simulations". Necessary discussion has been made in the revised manuscript.
Comments 2. The authors used homology modeling to generate the models for AtHsp90.6 with SWISS-MODEL, the crystal structure PDB ID 2CG9 was used as a template. However, the sequence identity was not reported, the sequences alignment was shown, the number of generated models was not mentioned, the assessment of the generated models and how the models were selected were not discussed. Response: We agree sequence identity and model assessment were not well explained. 1) The sequence identity between our model and the crystal structure PDB ID 2CG9 was 56%, and this was now shown in the methods section. 2) We modeled AtHsp90.6 by using the crystal structure from Saccharomyces cerevisiae (PDB id: 2CG9, sequence identity 56%) as a template through the SWISS-MODEL server by our established methods (PMID: 20936826, 21637806 and 22653607). SWISS-MODEL is a web-based integrated service dedicated to protein structure homology and we used the default parameters to generate the model. To assess the generated model, we adopted two criteria: Firstly, the structural similarity between our model and the crystal structure was measured by the root-mean-square-deviation (RMSD) in their best-superimposed atomic coordinates. The RMSD is 0.545 Å, suggesting high structural similarity between these two structures. Secondly, our model was refined and validated by 1000 ps MD simulations [ Fig. S2], as the Sansom team has previously shown that such short MD simulations are useful to assess the quality of structural models (PMID: 16102990).
Comments 3. In the Material and Methods section, there are three types of molecular dynamics simulations, 1) 100 ns MD simulations using OPLS force field and spc216 water model, performed with Gromacs; 2) 1000 ns CG MD simulations using MARTINI force field, performed with Gromacs; 3) MD simulations using AMBER ff99 force field for MM-GBSA calculations, performed with Amber 11. a. It is not very clear how many systems were simulated from the Methods section, the readers can only guess from the results sections. From Figure 5 and 6, I speculate that two systems were simulated for 100 ns, respectively, closed ATP-AtHsp90.6 and closed ADP-AtHsp90.6. Response: We now summarized our simulation systems, shown as Table S2.

Comments 4.
Why the Apo AtHsp90.6 at two different states were not simulated as the authors also wanted to study the binding of ATP? Response: Thank you for pointing out. To provide more quantitative statistic analysis, we now ran three independent 100ns explicit-solvent MD simulations with AMBER ff99 force fields and the TIP3P water model. We analyzed the results in the following parts. 1) We re-evaluated the RMSF of Cα for AtHsp90.6, the distance between the lid and the helix H2, and the solvent accessible surface area of the lid. Our new, extended analysis confirms the conformational changes of the ATP lid, and we have fully revised the corresponding analysis. 2) We re-analyzed the two-dimensional free energy landscape as a function of Rg and RMSD of AtHsp90.6. We found a good agreement between our computational data and previous simulation work of the ATP lid on AdK.
Comments 5. How many systems were simulated with CG model? I suspect only ATP-AtHsp90.6 was simulated, but not ADP-AtHsp90.6, then why not? Section 3.7 (line 29), the authors mentioned "The initial structure of N-terminal AtHsp90.6 was immersed...", did the authors only simulated the N-terminal of AtHsp90.6 or the whole structure of AtHsp90.6, but it was mentioned that the full length of AtHsp90.6 was modeled in Section 3.6? This is confusing. Response: Thank you for pointing out. 1) Given the lower-resolution coarse-grained model, we only used CG MD simulations to preliminarily test if key contacts can stabilize during long simulations. To study the conformational changes, we used 100 ns MD simulations using AMBER ff99 force fields, performed with Gromacs shown on Table S2. To make the article coherence, we now put the results of CG MD simulations to supplementary materials. 2) We modelled the whole structure of AtHsp90.6 based on the crystal structure (PDB id: 2CG9, sequence identity 56%) as a template. This whole structure was named as AtHsp90.6FL shown on Table S2. Given the nucleotide can directly bind to the N-terminal of AtHsp90.6, we select the N-terminal for the nucleotide binding study and conformation dynamics study. The N-terminal of AtHsp90.6 was now named as AtHsp90.6N.
Comments 6. b. Three force fields were used in this study, how could the authors justify and correlate the results from three different parameter sets? why not just use one? And I did not see the purpose of running CG MD simulations. The authors even did not mention the simulation time with Amber 11, and how many configurations were used for the MM-GBSA calculations. Response: Thank you for pointing out. 1) We now ran three independent 100 ns explicit-solvent MD simulations with AMBER ff99 force fields and have fully revised the corresponding analysis.  Table S2 and materials).
Comments 7. c. The parameters of ADP and ATP for the three force fields were not mentioned in the Methods sections, the authors developed, or used existed parameters? Response: We agree the parameters should be mentioned in the Methods sections. 1) The parameters for ADP and ATP were taken from the AMBER parameter database, maintained by The Bryce Group (http://research.bmh.manchester.ac.uk/bryce/amber). The parameters were developed by Carlson HA (PMID: 12759902), and their details have been put in the Supplemental materials. 2) To perform MD simulations with the GROMACS software package, we conduct the conversion to GROMACS compatible topology using ACPYPE (PMID: 22824207).
Comments 8. d. Section 3.9 (page 9), the authors stated "Because of the constant contribution of -TΔS for each complex...", this is not correct, the entropy is not constant, and it can be estimated with normal mode analysis, as done in this paper J. Med. Chem. 2006,49,6596-6606.

Response:
We agree with the reviewer. The sentences as mentioned in Section 3.9 have been removed completely. We agree entropy effects play an important role in ligand-receptor interactions. Especially, our improved hSMD method (PMID: 23176748) would provide more efficiency in free energy calculation. In this study, we neglected the entropic and focused on the relative binding free energy to rank complexes that are closely related as suggested by (PMID 20936826, 26507522). We are totally open, and would like to seek for more tools for free energy analysis, as publication is clearly not the ultimate goal of our endeavor.
Comments 9. In Figure 3C, the authors showed the number of contacts of L1-ATP, L3-ATP, L2-H2 and L3-catalytic loop, it is not clear how the number of contacts were calculated, what's the cut-off? The y-axis ranges of the four panels are different, this does not looks obvious. Response: We agree. Necessary information have been added in the revised manuscript, where the cut-off value of the number of contacts were 0.7 nm according to Sansom's method (PMID: 20409475, 24204243) and y-axis ranges of the four panels are same now.  Figure 4B)", I don't see how these values can be in agreement with a crystal structure, one can only state that the model have a similar binding mode with the crystal structure, as shown in Figure 4A and 4B (left and middle panels). Response: We agree. Necessary changes in these statements have been made in the Section 4.4(line 52-53).
Comments 11. From the right panel of Figure 4A and 4B, Table 1, I see that MD simulations were also performed for the structures PDB IDs 5FWK and 4XCJ, and then MM/GBSA calculations were done, but the simulations were not mentioned in the Method section and these results were not discussed fully in the main text. The authors did not explain why those simulations were performed. Why did the authors show the structure of 2XCM in Figure 4B? Response: We agree our missing necessary explanations for crystal structures. We reconstruct these sections as following: 1) Simulation details for the structures (PDB code: 5FWK and 4XCJ) have been put in the Methods section of the revised manuscript.
2) The atomic coordinates of HsHsp90 (PDB code: 5FWK) in H.sapiens adopted a ''closed'' conformation while the atomic coordinates of DdHsp90 (PDB code: 4XCJ) in D.discoideum adopted an ''open'' conformation. We performed MD simulations for these two experimental structures to understand if the "open" and "closed" conformations of AtHsp90.6 were in good agreement with the experimental results. We now added this information in the revised manuscript.
3) The atomic coordinates of HvHsp90 (PDB code: 2XCM) in H.vulgare represented a particular "open" conformation of Hsp90, while Rar1 stabilized the lid of Hsp90 into an open state. We now added this information in the revised manuscript.
Comments 12. In Figure 5, the distance was shown for all the 100 ns, but the SASA was only shown from 70 to 100 ns, why?
A major revision was done on this manuscript, new simulations and new analyses were performed, and new results were included. However, there is still one major issue that I am concerned.
For the reply to Comment 13 of Reviewer 2: " Comments 13. In Figure 6B, two intermediates were found, and they were separated by a barrier of ~2.5 kBT. Does it agree with the experimental data?
Our data correlates well with previously published data of adenylate kinase (AdK) protein (PMID: 26244746). In the current study, two intermediate states were observed in ADP-bound AtHsp90.6, separated by a barrier of ~2.5 kBT. Interestingly, AdK contained an ATP-binding domain (LID), can transit between an open conformational state and a closed conformational state. Four intermediate states β, γ, δ and ε corresponded to the semi-open-semi-closed conformations. The intermediates γ and δ had a barrier of ~1.02 kBT, the intermediates δ and ε had a barrier of ~1.7 kBT while the intermediates β and ε had a barrier of 2.72 kBT. Thus, our data agrees well with this published data. " In Figure 6, three states were found in this study, and the energy differences between states were compared in a previous computational study, reference [54]. However, the agreement between the current and previous studies is not convincing. The proteins are different, three states were found in this work, while 4 states were found in the previous study. And the collective variables used for the 2D free energy landscapes, and the free energy barriers between states are also different ( Figure 6 of reference [54]). Are the conformations of the states found in this work similar to the states in the previous study?
Another thing, the reviewer asked for experimental data, while the authors cited computational work, which is not answering the reviewer directly.