Lipid-protein interactions modulate the conformational equilibrium of a potassium channel

Cell membranes actively participate in the regulation of protein structure and function. In this work, we conduct molecular dynamics simulations to investigate how different membrane environments affect protein structure and function in the case of MthK, a potassium channel. We observe different ion permeation rates of MthK in membranes with different properties, and ascribe them to a shift of the conformational equilibrium between two states of the channel that differ according to whether a transmembrane helix has a kink. Further investigations indicate that two key residues in the kink region mediate a crosstalk between two gates at the selectivity filter and the central cavity, respectively. Opening of one gate eventually leads to closure of the other. Our simulations provide an atomistic model of how lipid-protein interactions affect the conformational equilibrium of a membrane protein. The gating mechanism revealed for MthK may also apply to other potassium channels.

2. More details should be given of how the initial state of the system was prepared and equilibrated. E.g., whether protein was fixed while lipids and water were equilibrated, when the pressure coupling was switched on, etc. Also, whether the same initial state was used in all replicas of the same system? 3. What was the electric field applied to the system (in units like V/m)? It is not enough to say "to mimic a transmembrane voltage of 300 mv" since this can be interpreted in different ways.
4. Location of double bond in unsaturated lipids should be given (like POPC 16:0/18:1(n-9) ) Reviewer #3 (Remarks to the Author): In this manuscript, the author's describe the regulation of MthK channel ion conductance by a variety of membrane lipids. The experiments were well performed, and I particularly appreciated the validation of results by using 2 unrelated force-fields. The authors conclude that in MthK, membrane thickness alters ion conductance in part through affecting the equilibrium of bent to kinked conformations of the inner helix. The authors' data suggests that kinking of this inner helix affects both the opening degree of the selectivity filter and the dehydration degree of the central. The authors propose specific atomic interactions that enable the inner helix to impact the structure of the selectivity filter. Therefore, the authors provide specific molecular details about how the lipid conditions examined can alter ion conductance in MthK channels.
Overall, this paper provides novel insights into the molecular details of lipid regulation of MthK channels. Experiments are appropriate, detailed, and well executed. The manuscript is well written.
I offer only these minor points to address.

Minor Points:
Lines 111 -116: Results on Cholesterol. Since cholesterol can have multiple effects on protein function through both changes in bilayer properties and through direct interactions with the protein, it would be useful to briefly mention in this paragraph whether or not cholesterol was interacting with any of the channel subunits in any of your simulations, and whether or not such interactions may contribute to the observed changes in MthK's conductance in that group of simulations. The authors do finally addressed in the section of lines 387 -388, however, there it is somewhat of a late display of important results.
Line 12: "Membranes do not only provide a matrix for a variety…" should say "Membranes not only provide a matrix for a variety…" Lines 52 and 53: Did you mean "responds" instead of "response" in this sentence "However, it remains unclear how the channel structure response to the membrane environment at the atomistic level."??
Line 53: The sentence "This is where MD simulations can provide the missing information." seems a bit awkward. Perhaps something like "This is where MD simulations can provide valuable insights." Lines 75-77: This sentence is poorly structured. Please reword. "More importantly, the conformational changes of the inner helix revealed by our simulations define two gates at the selectivity filter and the central cavity, respectively, and mediate a crosstalk between these two gates, suggesting a complex way of channel gating." Line 77: "Our work provided" should say "Our work provides" Line 392: "The thickness mismatch is mainly resolved by the distortion of the membranes in simulations." I think it's worth noting that this is similar to what was observed for hydrophobic mistmatch in KcsA by Callahan et al., 2019. Supp. Doc.

Reviewers' comments
I have some minor suggestions and questions: 1. The authors showed a strong correlation between the Thr59 O(gamma)-O(gamma) distance and the Ile84-Thr59 side chain distance. Does the interaction between the Ile84-Thr59 also influence the ion occupancy at the S4 in the selectivity filter?
Reply: We thank the reviewer for the constructive suggestions. We indeed found a correlation between Ile84-Thr59 side chain distance and the ion occupancy at S4 and S3 sites in our simulations. However, we propose that this is mainly because the Ile84-Thr59 side chain interactions modulate the Thr59 Oγ-Oγ distance, which in turn regulates ion occupancy. Supplementary Fig. 5 indicated that a larger Oγ-Oγ distance associates with a lower ion occupancy at S4 site and a higher occupancy at S3 site. We discussed this point at the end of the "Opening of the selectivity filter in the two states of MthK" section.
2. The authors stated that conductive states are found for asymmetrical structures when some subunits are in the kinked states whereas the remaining ones are in the bent state. Is the conductance the same when only one of the subunit is in the kinked state or two and three subunits are in the kinked state?
Reply: The ion permeation rates are not the same when different numbers of subunits are in the kinked state.
First, this can be seen from Fig. 3C, which showed different currents for MthK with different bent state fraction. In addition, we performed simulations in which one, two, three, and all four subunits are restrained in the occluded state, respectively, while the remaining subunits are left free. We restrained the subunits in the occluded state (see Supplementary Table 1) so that (a) Ile84 side chain was moved away from Thr59 and (b) Phe87 side chain pointed toward the central cavity. The subunits which are not restrained remained in the kinked state during most of the simulation time. When more subunits restrained in the occluded state, larger opening degree of the selectivity filter is found, but the central cavity is more dehydrated. The optimum current was observed when two subunits were restrained in the occluded state (~10 pA), while ion permeation rates are much smaller when one or three subunits are restrained in the occluded state (~5 pA, and ~2 pA). The channel is non-conductive if all four subunits are in the occluded state. These results are summarized in Supplementary Table 4 and at the end of the "Gating of the central cavity by Phe87 of the inner helix" section of the manuscript.
3. In a recent publication in Nature Communications (Hydrophobic gating in BK channels) a hydrophobic gate was identified in the pore. Since MthK has been considered as a bacterial homologue of the BK channel, do the authors think that both channels share in a way same mechanism in gating?
Reply: We agree with the reviewer that BK channel may share the same mechanism. Actually, BK channels are what we are planning to simulate as a follow-up project. In the revised manuscript, we discussed the results of BK channel simulations by Jia et al. in the last paragraph of the discussion section.
4. Is MthK a mechanosensitive channel? If the channel conductance and open probability are sensitive to the lateral pressure in the membrane as proposed by the authors, I would naively think that the channel would be also sensitive to the mechanical force. Is this the case?
Reply: A mechanosensitive channel refers to a channel gated by the mechanical force of the membrane. Our simulations indicated that the equilibrium between two states of the channel is affected by the lateral pressure of the bilayer. Our results may suggest that the lateral pressure is able to modulate the open probability of the channel in the presence of a gating signal. However, our simulations are not able to tell whether the lateral pressure is a gating signal of the channel. In this regard, we cannot conclude whether MthK is a mechanosensitive channel or not and hence prefer not to speculate in that context. The paper demonstrates, by molecular dynamics computer simulations, that lipid membrane composition modulates conductivity of MthK potasium ion channel, and reveals molecular details of this phenomena. Particularly it was convincingly demonstrated that two states of the transmembrane helix near residues 81-86, "kink" and "bent", are related to lower respectively higher conductivity of the channel, while the equlibrium between these two states are affected by the composition of surrounding lipids. The study is very well designed and the text is clearly written. The main conclusion are supported by the use of different force fields showing similar results, as well as by repeating simulations significant amount of times. The study is also a nice example on how computer modeling can contribute to getting novel insight into membrane protein functioning. I favor publication of this paper, subject to some revision accountings for the points below.
1. The temperature of the simulations, as follows from the SI, was set to 323 K. I guess the reason for his was to provide conditions for some lipids (particularly DPPC) to be in a liquid crystalline phase, but question arise how the temperature difference (compared to the physiological temperature) may affect results of the paper. The authors need to discuss possible effects of the temperature shift.
Reply: Keeping the membrane in the liquid phase state is indeed one reason of using a higher temperature. Another effect is that the higher temperature accelerates conformational transitions in MD simulations, which is essential for sampling as much conformational transition events as possible at given simulation time. This also applies to the observed ion current, our primary and limiting readout from the simulations. We do not expect any adverse effects on the protein as MthK is extracted from Methanothermobacter thermautotrophicus, a thermophile of which the maximum growth rate lies even at a slightly higher temperature. We discussed these points in the section of "Parameters of molecular dynamics simulations" in the supplementary information.
2. More details should be given of how the initial state of the system was prepared and equilibrated. E.g., whether protein was fixed while lipids and water were equilibrated, when the pressure coupling was switched on, etc. Also, whether the same initial state was used in all replicas of the same system? Reply: We first equilibrated the system with gradually removed restraints on the protein and lipids in six steps using the default scheme suggested by CHARMM-GUI. We then performed 0.1-0.3 μs simulations without any restraints. The electric field was not applied at this stage. At last we conducted production simulations with transmembrane voltage applied. For the production simulation, we randomly selected 2-4 snapshots from the equilibrium simulations as initial conformation, depending on the number of replicates we simulated. We included these details in the section of "Parameters of molecular dynamics simulations" in the supplementary information.
3. What was the electric field applied to the system (in units like V/m)? It is not enough to say "to mimic a transmembrane voltage of 300 mv" since this can be interpreted in different ways.
Reply: We applied an electric field of ~0.0325 V/nm. The transmembrane voltage V was calculated by the following equation: = × where E is the electric field strength and L is the box size along the z direction. Because of the low dielectric of the membrane, the vast majority of the applied voltage drops across the membrane. We used approximately the same box size for different systems, so that only minor change of the electric field strength (<3%) is necessary to result in the same transmembrane voltage. We explained this point in the Method section in the revision. 4. Location of double bond in unsaturated lipids should be given (like POPC 16:0/18:1(n-9) ) Reply: We noted the locations of double bonds in unsaturated lipids in the Method section of the manuscript and in Table S2 and S7 of the supplementary information.
In this manuscript, the author's describe the regulation of MthK channel ion conductance by a variety of membrane lipids. The experiments were well performed, and I particularly appreciated the validation of results by using 2 unrelated force-fields. The authors conclude that in MthK, membrane thickness alters ion conductance in part through affecting the equilibrium of bent to kinked conformations of the inner helix. The authors' data suggests that kinking of this inner helix affects both the opening degree of the selectivity filter and the dehydration degree of the central. The authors propose specific atomic interactions that enable the inner helix to impact the structure of the selectivity filter. Therefore, the authors provide specific molecular details about how the lipid conditions examined can alter ion conductance in MthK channels.
Overall, this paper provides novel insights into the molecular details of lipid regulation of MthK channels. Experiments are appropriate, detailed, and well executed. The manuscript is well written.
I offer only these minor points to address.

Minor Points:
Lines 111 -116: Results on Cholesterol. Since cholesterol can have multiple effects on protein function through both changes in bilayer properties and through direct interactions with the protein, it would be useful to briefly mention in this paragraph whether or not cholesterol was interacting with any of the channel subunits in any of your simulations, and whether or not such interactions may contribute to the observed changes in MthK's conductance in that group of simulations. The authors do finally addressed in the section of lines 387 -388, however, there it is somewhat of a late display of important results.
Reply: In the revised version, we mentioned in this paragraph that cholesterol are depleted from MthK during the simulations, and proposed the possibility that cholesterol modulate ion conduction by changing the lateral pressure of the protein.
Line 12: "Membranes do not only provide a matrix for a variety…" should say "Membranes not only provide a matrix for a variety…" Lines 52 and 53: Did you mean "responds" instead of "response" in this sentence "However, it remains unclear how the channel structure response to the membrane environment at the atomistic level."??
Line 53: The sentence "This is where MD simulations can provide the missing information." seems a bit awkward. Perhaps something like "This is where MD simulations can provide valuable insights." Reply: The above three corrections suggested by the reviewer are included in the revised manuscript.
Lines 75-77: This sentence is poorly structured. Please reword. "More importantly, the conformational changes of the inner helix revealed by our simulations define two gates at the selectivity filter and the central cavity, respectively, and mediate a crosstalk between these two gates, suggesting a complex way of channel gating." Reply: We rephrased the sentence in the revision as "More importantly, the conformational changes of the inner helix revealed by our simulations define two gates at the selectivity filter and the central cavity, respectively. We also revealed a crosstalk between these two gates mediated by the conformational changes, suggesting a complex way of channel gating." Line 77: "Our work provided" should say "Our work provides" Reply: We revised the manuscript accordingly. Line 33. "We conducted a series of simulations with restraints…" should say "We conducted a series of simulations of the MthK potassium channel with restraints…" Reply: We corrected the supporting information accordingly.

REVIEWERS' COMMENTS:
Reviewer #1 (Remarks to the Author): All questions are answered satisfactory. I have no further comment and therefore recommend this paper to be published in Nature Communications.
Reviewer #2 (Remarks to the Author): In the revised manuscript the authors addressed to all reviewer comments, and I can recommend the paper for publication Reviewer #3 (Remarks to the Author): I am satisfied with all changes made to the manuscript. Congratulations on an excellent study.

EXTENDED COMMENTS: NCOMMS-19-40919A .
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EDITOR'S SUMMARY
Potassium (K+) channels, such as MthK, are essentional for many biological processes, but how lipid-protein interactions regulate ion permeation of K+ channels remained unclear. Here authors conducted molecular dynamics simulations of MthK and observed different ion permeation rates of MthK in membranes with different properties.
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