Molecular Insights into Substrate Binding of the Outer Membrane Enzyme OmpT
Round 1
Reviewer 1 Report
The manuscript “Molecular insights into substrate binding of the outer membrane enzyme OmpT” by Yubo Zhang et al. employed in silico methods to investigate how tetrapeptides interact with an outer membrane aspartic protease OmpT from E. coli. This is a regular modeling study which does offer some insights at an atomic level. I think this manuscript may publish in Catalysts, but after the authors address the following concerns.
1. My major concern is that the authors have not clearly presented the rationale of their study.
1) The authors state that their study is “to gain detailed insights into the molecular recognition between ligand and receptor” (page 2, line 71). The community has modeled the holo-OmpT (e.g., Ref. 18). Haven’t these studies provided any insight into the recognition? Which essential information is missing that necessitates the current study?
2) The authors state that their simulations allow them to answer “What mechanism does OmpT apply in recognizing its substrate? Does water play a role in attacking the cleavable peptide bond of the substrate? How does OmpT interact with the lipid bilayer compared to other bacterial outer membrane proteins?” (page 2, lines 73–76). I think the current study answers open question #1. For question #2, I am afraid the current study provides only some hints but no convincing evidence demonstrating how water molecules may be involved in the catalysis, which requires QM simulations. For question #3, I think the current study cannot address it. The authors state that their finding of how OmpT may impact lipid mobility agrees with a previous study of OmpA (page 11, lines 328–329). But this comparison is quite preliminary. I am afraid I cannot extract meaningful results to help me understand how “bacterial outer membrane proteins” interact with lipid bilayers similarly differently.
Having said this, I don’t think I get the necessity or novelty of the current research as this manuscript was written. Also, the authors have somewhat oversold their modeling results. I strongly recommend that the authors revise the Introduction section for a better presentation. If I were the author, I would focus on the ligand–protein recognition part.
2. I have some technical concerns:
1) The authors ran 100 ns for holo-OmpT, and 20 ns for apo-OmpT. I must say these simulations were too short. In membrane proteins simulations, the lipid bilayer may take at least 50–100 ns to equilibrate, given that the protein is restrained when the bilayer equilibrates and that the bilayer is not a mixture of lipids. Note that embedding the protein into a pre-equilibrated bilayer is not exceptional. Unless the authors can demonstrate that the bilayer has been equilibrated by analyzing bilayer thickness, area per lipid, and deuterium parameters, I think neither the protein nor the lipid bilayer has been equilibrated in the simulations. I would suggest the authors rerun the simulations and stepwise equilibrate the systems: I) equilibrate lipid bilayers with OmpT restrained, 50 ns at least; II) 100 ns or longer without any restraints.
2) I don’t understand why the authors have used DMPC lipids with SPC water in their simulations but POPE with TIP4P water in their free energy calculations. E. coli has about 70% POPE lipids in its cell membranes. So, I don’t see any reason that DMPC lipids should be used in the current study. And I don’t understand why the authors have used two sets of simulation systems with different components.
3) Figure 1. My reading is that the differences in either RMSF or SASA are small. I cannot draw solid conclusions about how ARRA peptide may impact the dynamics of OmpT from those plots, especially without any error estimation.
4) Figure 2C and Figure 4 are misleading. I doubt if anyone would take the time to read them. The authors should quantify the number of hydrogen bonds. Note that both the distance and angle should be considered to judge if a hydrogen bond forms. Also, I don’t understand why the authors claim interactions between acidic and basic protein residues (e.g., D85 and R301) hydrogen bonds. Aren’t they ion-pairs (or salt-bridges)?
5) Section 2.7 (free energy calculations). The authors don’t explain what leads to “the strong binding ability of AKKA when recognizing OmpT” (page 8, lines 256–259). The authors should analyze those umbrella trajectories and develop an atomistic explanation.
6) In section 2.8, the authors state that “the orbital distribution of the ARRA observed in extended Hueckel calculations indicates that the cleavable peptide is the region most likely to be attacked” (page 9, lines 285–287). I don’t know what are “Hueckel calculations” and I cannot see any figures or tables to support the authors’ argument here.
7) The authors used WHATIF to determine the pKa values of the OmpT ionizable residues and assign the charge states. Where are the pKa values? I don’t see them anywhere in the manuscript. Also, at which pH did the authors run simulations? pH 7 I guess?
Author Response
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Author Response File: 
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Reviewer 2 Report
Comments for author File: Comments.pdf
Author Response
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Round 2
Reviewer 1 Report
In the revised manuscript, the authors have appropriately addressed all my questions and concerns. I believe the manuscript is in good shape and publishable.
