Peer review of: Molecular mechanism of light-driven sodium pumping (2020)

Peer Review File – first round comments: 

Reviewers' comments: 
 
Reviewer #3 (Remarks to the Author): 
 
The authors have incorporated all changes requested by me. I only have two minor changes to add 
before the manuscript is ready for publication concerning the Computational part: 
 
-Methods: please give height and width of Gaussians that were placed in metadynamics runs. 
 
-line 253: change "molecular dynamics" to "metadynamics". Furthermore include a sentence that 
you performed these simulations with the intent to find unbinding pathways, but not to determine 
free energies along them. 
 
Steffen Wolf, University of Freiburg, Germany. 
 
 
 
Reviewer #4 (Remarks to the Author): 
 
In this paper, the authors present the structures of the O-state of the light-driven sodium ion-
pumping rhodopsin KR2. The O-state is the key intermediate of Na+ pumping in KR2. The authors 
showed the protein structure for a physiologically relevant pentameric form. They identified the 
transient Na+ binding site inside the rhodopsin in the O-state and structural differences between 
the ground and the O-states. Together with the structures of the mutants, the authors discussed 
the protein structures to elucidate key determinants of cation pumping by KR2. The structural data 
provided in this paper are highly important and well-analyzed to understand the pathway of Na+ 
and the ion-pumping mechanism of KR2. Insights obtained by recent spectroscopic studies are 
closely related to the pathway and the ion-pumping mechanism proposed in the present study. 
Although the discussion in the manuscript is sound, the manuscript will be improved by the 
addition of the relating insights provided the spectroscopic studies. I suggest that I recommend 
the publication of this work in Nature Communications after major revisions indicated as follows. 
 
 
1. Line 145. The authors claimed that the retinal chromophore adopts a distorted all-trans 
configuration in the O-state. However, this is in contrast to the recently published time-resolved 
FTIR data on KR2. The discrepancy should be discussed more. Is the probability that the electron 
density is attributed to the 13-cis form completely ruled out? The authors mentioned the 
chromophore configuration in the O-state of GLR. But, this is a different protein. The discrepancy 
between the data reported for KR2 should be discussed more clearly. 

 
2. Line 152. The authors claimed that relative locations of the RSBH+ and D116 side chain are 
similar to those in the ground state. This conclusion is quantitatively supported by the data of a 
recent resonance Raman study (Ref. #14). Ref. #14 reported that C=N stretching frequencies of 
the RSB are very similar between the ground state (1640 cm-1) and the O-state (1642 cm-1). The 
C=N stretching frequency is a sensitive marker for the hydrogen bond strength of the protonated 
RSB. The similar frequencies of the C=N stretching mode support the authors’ claim that relative 
locations of the RSBH+ and D116 side chain are similar to those in the ground state. I suggest to 
include the above-mentioned discussion on the C=N stretching frequencies in the manuscript. 

 
3. Line 184. The switching between the expanded and the compact conformations is interesting 
and reasonable to understand the ion pumping mechanism. A recent resonance Raman study 
(Otomo et al., Biochemistry 2020, 59, 520−529) showed that the Na+ binding to the 
interprotomer binding site changes the hydrogen bond strength of the protonated RSB in the 
ground state of KR2, suggesting an allosteric communication from the interprotomer binding site 
to the protonated RSB. If the interprotomer binding site and the RSB are allosterically coupled, the 
transition from the expanded to the compact conformation can induce a structural change in the 

interprotomer binding site and, thus, a change in the dissociation constant of the Na+ binding. 
This promotes the release of the Na+ from the interprotomer binding site. Accordingly, a scenario 
that the “expanded”-to-“compact” switch regulates the Na+ transport channel through pIRC2 is 
possible. I suggest to include the above-mentioned discussion on the allosteric coupling in the 
manuscript.