Peer review of: Lipid-protein interactions modulate the conformational equilibrium of a potassium channel (2020)

Reviewers' comments: 
 
Reviewer #1 (Remarks to the Author): 
 
In this study, the authors investigated the effect of different membrane environment on the 
channel permeation rates using molecular dynamics simulations. The authors found that the 
thickness of the bilayer, lipid unsaturation as well as cholesterol content all influence the ion 
permeation rate of MthK. Furthermore, from their restrained and unrestrained simulations, they 
identified two gates at the selectivity filter and the central cavity that are linked to each other. In 
the field of molecular dynamics, the influence of different membranes and lipids on the channel 
permeation has been questioned for quite some time. Therefore I highly appreciate this systematic 
investigation by the authors and I believe this study deserves publication in Nature 
Communications. 
 
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? 
 
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? 
 
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? 
 
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? 
 
 
 
Reviewer #2 (Remarks to the Author): 
 
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 convinsingly 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 simular 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. 

 
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. 
 
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…”