Peer review of: TrkA undergoes a tetramer-to-dimer conversion to open TrkH which enables changes in mem (2020)

Reviewers' comments: 
 
Reviewer #1 (Remarks to the Author): 
 
Based on three new structures of the TrkAH complex in the presence of ADP, ATP and 
ATPgammaS, respectively, Zhang et al suggest a novel mechanism of nucleotide-dependent gating 
of an RCK protein-regulated K+ channel. This is particularly interesting as for many of those 
channels a mechanism of gating has been suggested, but sufficient evidence from structural and 
functional experiments is missing. Here, for TrkAH Zhang et al. propose that ATP binding leads to 
the dissociation of the tetrameric TrkA ring into two TrkA dimers, which consequently opens the 
pore subunits by widening the pore-lining helices and displacing the intramembrane loop. In 
contrast, two interaction sites of TrkA and TrkH in the ADP-bound conformation stabilize the 
channel in a closed conformation. This new hypothesis will be of high interest for the K+ channel 
community and the membrane transport field in general. However, to me the manuscript needs a 
significant improvement in data representation to justify the drawn conclusions and additional 
functional experiments are required to prove the raised hypothesis. Most important are the 
following aspects: 
 
1. For all relevant areas (HN1 interface, HN2 interface, intramembrane loop, selectivity filter, 
ligands) the density maps should be represented along with the models to visualize the reliability 
of the models. In particular, the authors mention that residues 155 – 173 of TrkH and 34 – 39 of 
TrkA in the ADP-bound state were not resolved (although at least residues 37 – 39 of TrkA are 
shown in figure 4 and – strangely – are helical and should be better resolved than the successive 
loop). Hence I wonder how well the hydrogen bond between Thr175 from TrkH and Gln40 from 
TrkA and the salt bridge between Arg177 from TrkH and Asp44 from TrkA are resolved. 
A clear HN1/HN2 figure at all three conditions (similar to Fig. 4a) with density maps is required in 
the main manuscript. Instead, I suggest to remove Fig. 4b as it is repetition of Fig. 1a. The 
distances as indicated in Fig. 4b could be indicated there. 
 
2. In support of the hypothesized role of the HN1 interface the authors should provide a 
comparison with the available TrkH structure. How does the TrkH structure compare to the ATP-
bound TrkAH structure? One would assume that the pore-lining helices adopt similar conformations 
because both are not restricted. The intramembrane loop on the other hand could adopt a different 
conformation because the “pulling element” is missing. Or is the assembled ring rather pushing on 
the intramembrane loop (cf. line 157)? How do the patch clamp measurements of the T175A 
mutant compare to TrkH in the absence of TrkA? 
 
3. The authors suggest that N2 binds ADP, ATP and ATPgammaS, while N1 only binds ATP and 
ATPgammaS (lines 190ff). If so, this finding would finally provide an explanation on how TrkAH 
selectively binds ATP over ADP and why under physiological conditions activation could actually 
happen. However, the low resolution of N1 in the ATP-bound conformation did not allow resolving 
the bound ligand. Further, the binding affinities of the N1 and N2 sites for the different nucleotides 
are not known. The manuscript lacks an explanation on why ADP would not bind to N1. I suggest 
the performing of ITC measurements to determine N values and binding affinities for the different 
nucleotides. The R100A mutant likely would even allow the determination of affinities for the N2 
site only, while a similar mutation in the N2 site would allow the determination of N1 binding 
affinities. In fact, those experiments could be performed on the TrkA subunit only to further 
simplify the experiments. 
 
4. Further, one should ask which role nucleotide binding to the N2 site has. Here, a mutation 
similar to the R100A mutation in N1 should be inserted to hinder ATP binding to the N2 site. 
 
5. There is no evidence from any functional data for the suggested role of TrkAH (lines 297ff.). 
Further, to me it does not sound very plausible. Why would it be useful for the cell to pump even 
more protons, if the ATP concentration is already high? This in consequence would actually lead to 
a drastic alkalization of the cell, which most likely would be problematic. In contrast, TrkAH and 
KtrAB have been suggested to be involved in pH regulation. I suggest to remove this far-fetched 
hypothesis on the physiological role of TrkAH or to develop it better. 
 

In addition to these five main points I have several smaller comments/questions: 
 
6. Line 10: Explain RCK 
 
7. Lines 116f.: It is known that at least in Gram-positive bacteria c-di-AMP binding to RCK-C closes 
TrkH. Thus the speculative sentence about C1 and C2 should be removed. 
 
8. The order of the figures in the text (1,5,4,3,2,6,7) is very confusing 
 
9. Figure S3, panel d: The resolution of the map is impossible to judge. Show a slice through the 
map. Panel i: Show densities of important areas (intramembrane loop, selectivity filter, AN1, AN2, 
ligands) and not of any random area. 
 
10. I wonder whether you observed the dissociation of the tetrameic TrkA when adding ATP to 
isolated TrkA. This could be easily analysed by negative-staining EM. 
 
11. How confident are the authors that the K+ is not yet rehydrated when passing the 
intramembrane loop (lines 147ff)? Is the opening of the pore seen really sufficient to allow K+ 
translocation 
 
12. Which effect could the sandwich conformation have on the observed conformational changes? 
Might it hinder a complete dissociation of TrkA and TrkH? 
 
13. Line 177: Specify the term “intermediate” 
 
14. Lines 202f: “feeble HN1 interface”. Show interaction with density map! Figure S6e only shows 
the TrkH subunit, which doesn’t help the interpretation. 
 
15. HN1 interface: What happens if you enforce the interface by a disulfide bond? Is it ATP 
insensitive? 
 
16. What is the role of the relative rotation of the TrkH protomers? 
 
17. The cartoon in Fig. 7 is very difficult to understand. Breaking of tetramer into dimers is not 
clear, HN1 and HN2 sites should be indicated. 
 
18. Several panels particularly in the supplements are invisible/unreadable (e.g. I220C in S5, 
resolution map in S3d) 
 
 
 
Reviewer #2 (Remarks to the Author): 
 
The manuscript entitled “Opening of the TrkH channel by a tetramer-to-dimer conversion of the 
associated TrkA” describes structures of the TrkHA complex. This is a bacterial cation channel 
which belongs to the SKT superfamily of channels and transporters with important roles in K+ 
homeostasis of bacteria, archaea, fungi and plants. The manuscript presents three different 
conformational states of the TrkHA complex, providing a unique view of its mechanism of 
activation. Crucially, it provides the first description of the open state of a SKT channel. This 
manuscript will be of interest for all of those that are interested in ion channel structure and 
function and also for those interested in bacterial physiology. 
 
Major issues: 
1- A conclusion from this paper relates to the impact of ligand binding to the different sites (RCK1 
and RCK2). In particular, ADP binds just to RCK2 while ATPγS binds to both RCK1 and RCK2. The 
authors reasonably conclude that ATP will likely bind to RCK1 and RCK2. The importance of this 
conclusion must rely on solid data and it is surprising that the conformation of ATPγS in RCK2 in 
the new structure at 3.8Å is different from the conformation of the same ligand in a previous 
structure at 3.0Å. These differences occur at the level of the position by the base (the base in the 

new structure is flipped relative to the previous structure) and at the level of the interactions 
established by the phosphates (see description in lines 184 to 190 and confront with previous 
structure, PDB code: 4J9V). Are the authors sure that the low resolution of the new structure 
allows them to make these changes in the ligand? In addition, I would strongly suggest that the 
panels in Figure 5 show the protein structures in identical views to facilitate a comparison between 
the different ligands and binding sites. 
2- I would like for the authors to consider that there are other structural regions that have a role 
in the closure/opening of TrkH besides the HN1 interface. After all their functional data shows that 
mutations at the interface still allow gating since there is still a large reduction in open probability 
(from 80% open with ATP to ~13% open with ADP versus in the WT where it changed from 87% 
to 2%). In addition, a) the authors describe that changes in the intramembrane loop are 
connected with an increase in the distance separating the selectivity filter from the HN2 interface 
(Lines 140 to 145). By the way, this change is not explained; and b) that the helix D3M2b helix, 
which is directly connected to the intramembrane loop, has moved in the ATP versus the ADP 
structure (Figure 2d). 
Without minimizing the importance of the HN1 contact for the stability of the closed state, why are 
the authors ignoring all these other changes as part of the overall mechanism of moving the 
intramembrane loop out of the way? 
3- I was surprised not to see more information about the ion pore of the "partially" open TrkHA-
ATP structure. In particular, the authors have decided not to show any detailed information about 
the intramembrane loop (both the structure and the experimental map). It seems to me that the 
functional importance of this region and good resolution of the TrkHA-ATP structure merits at least 
a figure showing the intramembrane loop structure and the experimental map. 
4- One of the major issues I have found with the manuscript is its organization. The main text 
does not follow the organization of the figures and this means that the data is spread-out across 
different figures, both main and supplementary, making reading the text and looking/thinking 
about the data a fairly cumbersome process. 
Without being to bullish with this request, I would like to suggest that the authors consider a 
reorganization of the main text so that the manuscript starts with a description of the major 
differences between the ADP and ATP structures, followed by a description of the details that 
underlie those major differences and that support their mechanism proposal, including the 
presentation of the ATPγS structure. I am convinced that this will increase the readability of the 
manuscript and its impact. 
Not sure as well that the comparison of the new structures with the previous TrkHA-NADH 
structure should take such a prominent place in the early sections of the manuscript. It takes a 
whole paragraph in the section describing the TrkHA-ADP structure and also in the final paragraph 
of the description of TrkHA-ATP structure and becomes a distraction to the understanding of the 
new data. I suggest that you discuss the differences between the new structures and the TrkHA-
NADH structure in a single paragraph later in the manuscript. 
 
Minor issues: 
1- The size of some of the Figures is too small. For example, all the panels in Supp. Figure 3 and 
Supp. Figures 4g and 4 h are virtually impossible to analyze even after extensive zooming. In 
addition, labels in Figures 4a, and Figure 5 are also too small. 
2- Resolution of left-hand side panels in Figure 5 for ADP and ATPγS is poor. 
3- Supplementary Figure 1c serves no apparent purpose and is very difficult to analyze due to lack 
of contrast between ligand and protein. 
4- There is something wrong with Stereo figures in Supp. Figure 4 g and h. It appears that the 
panels are inverted. 
5- In line 114, the differences in the N2-N2 interface are mentioned when discussing changes 
between the TrkHA-ADP and TrkHA-NADH structures and Supp. Figure 2F is referenced. However, 
the figure only shows the N2-N2 interface of the TrkHA-NADH structure and so these differences 
are not easy to perceive. 
6- Not clear what is being presented in Supp. Figures 4a and 4b, they appear to represent the two 
N2-N2 interfaces from the isolated TrkA-ATPγS structure, but why do the interfaces look different? 
7- The PDB code indicated in panel Supp. Figure 4e is wrong, the TrkA-ATPγS PDB code is not 
4J9U, it is 4J9V. 
8- The Supp. Figure 4F panels are labeled as interfaces when they should be labeled “binding 
sites”. 

9- In Supp. Figure 6 the figures are labeled “Supperimposed” when it should read ”Superimposed”. 
10- In Suppl. Figure 6e, the contrast between D1D2 loop residues and the rest of structure is poor, 
making analysis of the structure difficult. 
11- Supplemental Table 1 should show Ramachandran plot distribution of residues as Favored, 
Allowed and Disallowed as this is a refinement independent parameter. 
 
 
 
 
Reviewer #3 (Remarks to the Author): 
 
The authors studied the structures of TrkH-TrkA complex in the presence of ADP or ATP. The 
structures of TrkHA-ADP and TrkHA-ATPS were solved by X-ray crystallography. TrkH in the 
TrkHA-ADP is in a closed state. The structure of TrkHA-ATP was solved by cryo-EM. TrkH in the 
TrkHA-ATP is in an open state. However, the N1 domain is partially resolved and the C1 and C2 
domains of TrkA are not resolved at all. The major conformational change is the conversion of a 
tetramer to a dimer of TrkA and a loss of constraints on TrkH. Although quite a few local structures 
are presented, the conversion is not clear. It is better to provide a few top views of the TM region 
and the intracellular region separately, and label some of the dimensions. In that way, the 
tetramer to dimer conversion can be seen easily. Also, comparison with the published NADH bound 
TrkA in top view will be helpful. For local structures, it is better to label them in the top views of 
corresponding regions. It might be better to re-organize the figures to focus on the conversion 
from tetramer to dimer. 
 
Here are some detailed comments. 
• In the introduction, it is better to give more information about the difference between TrkH and 
KtrB, and TrkA and KtrA. 
• Fig 1: Are A and B in the same orientation? 
• Fig 2C: It is better to replace yellow with another color. It is easy to confuse it with the yellow 
color in A. 
• Fig S2A: Is there any reason to show D2, D4, D1, and D3 instead of D1-4? 
• Page 6: "Second, the two TrkH protomers have a relative rotation of 12.5 (Supplementary 
Figures 6a and b)." Are the two protomers tilted in the plane perpendicular to the membrane or 
rotated in the plane parallel to the membrane? 
• Page 6: "The N1-N1 interface remains unchanged while the N2-N2 interface adjusts to 
accommodate the changes (Supplementary Figure 2f)." Fig s2f doesn't show the difference or 
similarity at all. 
• Page 7: "In the presence of ATP, TrkAs become two dimers and no longer form a tetrameric ring 
(Figure 3a and b).". It is really hard to see the dimers. A top view of the TrkAs will be helpful. A 
comparison of the whole intracellular dimers/rings will be needed. 
• Page 25: "TrkH-TrkA complex was assembled by mixing TrkH and TrkA in 1.2 to 1 molar ratio." 
Based on the structure, the molar ratio is 1 TrkH to 2 TrkA. Why was 1.2 to 1 molar ratio used? 
• Page 27: ". Each micrograph was collected in 5 frames/s for 10s, with a dose of 8e-/Å/s". Should 
it be 8e-/pixel/s? 
• Page 27: "These particles were reconstituted into a 6 Å map through RELION 3D refinement". 
Shouldn't it be reconstructed instead of reconstituted? 
• Page 27: Was any symmetry used in 3D reconstruction?