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Introduction

In their research, the authors investigate the phenomenon of biased signalling using vasopressin V2 receptor (V2R) subtype of class A GPCRs as an example combining experimental techniques such as NMR spectroscopy as well as MD simulations. The authors apply a set of differently acting  ligands such as tolvaptan (TVP, antagonist), arginine-vasopressin (AVP, endogenous unbiased agonist), deaminated lysine-vasopressin (dLVP, agonist), MCF14 (Gs-biased agonist) and paramagnetic derivatives of MCF14 and dLVP. In addition to the wild-type (wt) V2R receptor, the authors investigate the gain-of-function mutant I130N, characterised by an intrinsic bias towards the Gs protein.

This work advances knowledge on structural and dynamics aspects of biased signalling, particularly in V2R. We would like to offer a series of suggestions and questions, the resolution of which could make the narrative even more comprehensible. Additionally, addressing some of the proposed questions may help readers better understand the rational and logical deduction of the experiments  of the conducted research.

Clarifying questions

1. MD simulation. What is the reason for choosing the REST2 protocol for conducting enhanced molecular dynamics simulations? What are its advantages within the scope of the authors' task compared to other enhanced sampling methods?

2. MD simulation. How may be explained the choice of a modelling time of 60 ns per replica? What serves as the criterion for the sufficiency of simulation time?

3. MD simulation. In the text, the authors provide the following conclusions: “R-^parMCF14 turned out to be highly mobile in the pocket during the simulations (Figure S5A), indicating low affinity” and “S-^parMCF14 was stable in the pocket and adopted the same binding pose as the untagged MCF14, suggesting that this enantiomer was the active one”. We would like to address the authors with a request for providing more explanations why the conducted MD simulations are sufficient for formulating accurate conclusions about the affinity of MCF14 enantiomers. Could it be definitely concluded, based on the analysis conducted, that the R-^parMCF14 enantiomer is inactive and should not be considered? Answers to these questions could complement the narrative and provide more information about the study.

4. MD simulation. The following conclusion is given in the article: “The paramagnetic ligands were slightly more mobile than their untagged counterparts during the simulations (Figure S5A), in agreement with their lower affinity in the cell assays”. Why can the observed differences in ligand mobility from the results of MD simulation be considered statistically significant?

Comments on figures

• Suggestion. “The paramagnetic tag position in ^parMCF14 was determined based on the presumed binding pose of TVP-like V2R ligands“. A figure depicting the model of the ligand position in the receptor binding pocket and the presumed spatial location of the tag would more vividly illustrate the reason for choosing a specific position for tag insertion.

• Figure S5A. To enhance the comprehensiveness of the information, it is recommended to include explicit details indicating the reference point for calculating RMSD in Figure S5A, either within the figure legend or the main text.

• Figure 2: Data representation may be unclear due to the selected set of colours, which may be hard to differentiate by colour-blind persons.

• Figure 2A: The figure requires a more detailed legend. To enhance clarity, information regarding the conducted experiments and data analyses should be briefly introduced in the main text of the article, rather than solely in the Supplementary Material.

• Figure 2C. Spatial localisation of MCF14 is not obvious compared to the position of ^parMCF14 in Figure 2C. In the current version, this creates some confusion, as it seems like only ^parMCF14 is visualised, partially in a spherical model coloured by atom type.

• Figure 2D. It appears that Figure 2D duplicates information from Table 2. Therefore, is this subfigure truly necessary?

• Figure 3A. Binding poses of E-5R-TVP and E-5S-TVP are poorly distinguishable in the figure. We would suggest enlarging the scale of this subfigure and using contrasting, vivid, easily distinguishable colours to represent E-5R-TVP and E-5S-TVP.

• Figure 4. In the current version, Figure 4 does not explicitly illustrate conformational changes of H8. For better clarity, we suggest adding a label “H8”, as was done for TM7.

• Figure 5A. In the text of the preprint is stated: “H8 exhibited more constrained conformations and was closer to TM1 than in V2R-AVP (Figure 5A)”. For illustrative purposes, it would be helpful to add labels for H8 and TM1 to Figure 5A (near to structural models) and explicitly show the measured distance between these structural segments.

• Suggestion. In the provided Supplementary Material it is written: “The initial models of AVP V2R in inactive state were built using Modeller v9.15 based on the cryo-EM structures of AVP-V2R-Gs (PDBs 7DW9 ^[17] and 7BB6 ^[1]) and the X-ray crystal structure of the oxytocin receptor in inactive state (PDB 6TPK ^[18])”. For a more convincing assertion of the reliability of the initial models, we would recommend creating a figure showing the comparison between the model constructed using Modeller (AVP V2R in inactive state), the structure of V2R in the active state, and the structure of the oxytocin receptor in the inactive state.

• Table 1: It should have a better explanation about the table contents. Their primary data are not shown in both main and supplementary text which should be included. The authors should mention about the assays which they performed in the table and main text.    

Typos

• Page 4, last paragraph, “intracellular end of TM6.Interestingly,”: add space.

• Page 6, first paragraph, “Binding of the antagonist TVP caused no significant change. . By”: remove the extra dot.

Minor Concerns

• We suggest making the Introduction section a bit more detailed, with all main definitions and abbreviations clearly provided. For instance, abbreviations including GPCRs, NMR or dLVP are not introduced in the Introduction part. 

• The Supplementary Section also needs a more detailed narrative to enhance reproducibility and accessibility for understanding the results. This is particularly relevant to the TR-FRET method.

• We also suggest providing more details about conducted experiments in the main text of the article. This may also make Material and Methods parts, given in Supplementary Material, more clear. 

• We also request a more comprehensive discussion of the obtained results in the Results and Discussion section, including insights into potential future directions for research and practical applications of the findings.

Conflicts of interest

There are no conflicts to declare.

Competing interests

The author declares that they have no competing interests.