Allosteric modulation by the fatty acid site in the glycosylated SARS-CoV-2 spike

Reviewer #1 (Public Review):

Summary:

The investigation delves into allosteric modulation within the glycosylated SARS-CoV-2 spike protein, focusing on the fatty acid binding site. This study uncovers intricate networks connecting the fatty acid site to crucial functional regions, potentially paving the way for developing innovative therapeutic strategies.

Strengths:

This article's key strength lies in its rigorous use of dynamic nonequilibrium molecular dynamics (D-NEMD) simulations. This approach provides a dynamic perspective on how the fatty acid binding site influences various functional regions of the spike. A comprehensive understanding of these interactions is crucial in deciphering the virus's behavior and identifying potential targets for therapeutic intervention.

Weaknesses:

The presented evidence is compelling but could be better if this study is supported with sequence analysis to facilitate a complete view of the allosteric networks. The thorough analysis of the simulation results is partially aligned with the discussion because observed in the replicates and the monomers an asymmetry in the perturbations generated by D-NEMD, even when we're using 210 nonequilibrium MD of 10 ns. While the authors claim that the strategy used in this article has been previously validated, the complexity of the spike and the interactions analyzed have required a robust statistical analysis, which is not shown quantitatively. The investigation examines the allosteric modulation within the glycosylated SARS-CoV-2 spike protein, emphasizing the significance of the fatty acid binding site in influencing the structural dynamics and communication pathways essential for viral function, potentially facilitating the development of novel therapeutic strategies. The presented evidence is compelling but needs to be supported by sequence analysis, which will facilitate understanding of the scientific community.

Minor considerations:

Figure S3 shows a discrepancy in the presentation of residue values S325 in the plots of Chains A, B, and C. While chain A shows a value near 0.1, chains B and C plots do not have any value.

Please explain why the plots of figures S6, S7, and S8 show significant changes in several regions, such as RBM and Furin Site. Can these changes be explained?

The flow of the allosteric interaction is complex to visualize just by looking at structures. Could you please include a diagram showing the flow of allosteric interactions (in a sequence diagram or using the structure of the protein)? Or could you include a vector showing how the perturbation done in the FA Active site takes contact with other relevant regions of the Spike protein?

Reviewer #2 (Public Review):

This is a nice paper illustrating the use of equilibrium/non-equilibrium MD simulations to explore allosteric communication in the Spike protein. The results are described in detail and suggest a complex network of signal transmission patterns. The topic is not completely novel as it has been studied before by the same authors and the impact of glycosylation is moderated and localized at the furin site, so not many new conclusions emerge here. It is suggested that mutations are commonly found in the communication pathway which is interesting, but the authors fail to provide evidence that this is related to a positive selection and not simply to a random effect related to mutations at points that are not crucial for stability or function. One interesting point is the connection of the FA site with an additional site binding heme group. It will be interesting to see reversibility, i.e. removal of the ligand at this site is producing perturbation at the FA site?, does it produce other effects suggesting a cascade of allosteric effects? Finally, the paper lacks details to help reproducibility, in particular, I do not see details on D-NEMD calculations. One interesting point is the connection of the FA site with an additional site binding heme group.

Reviewer #3 (Public Review):

Summary:

In a previous study, the authors analyzed the dynamics of the SARS-CoV2 spike protein through lengthy MD simulations and an out-of-equilibrium sampling scheme. They identified an allosteric interaction network linking a lipid-binding site to other structurally important regions of the spike. However, this study was conducted without considering the impact of glycans. It is now known that glycans play a crucial role in modulating spike dynamics. This new manuscript investigates how the presence of glycans affects the allosteric network connecting the lipid binding site to the rest of the spike. The authors conducted atomistic equilibrium and out-of-equilibrium MD simulations and found that while the presence of glycans influences the structural responses, it does not fundamentally alter the connectivity between the fatty acid site and the rest of the spike.

Strengths:

The manuscript's findings are based on an impressive amount of sampling. The methods and results are clearly outlined, and the analysis is conducted meticulously.

Weaknesses:

The study does not clearly show any new findings. The authors themselves acknowledge that the manuscript mainly presents negative results-indicating that glycans do not significantly impact the allosteric network previously reported in other publications. All the results in the paper are based on a single methodology, and additional independent approaches would be needed to confirm the robustness of these findings. Allosteric networks arise from subtle correlations in protein structural dynamics, and it's uncertain whether the results discussed in this manuscript stem exclusively from the chosen force field and other modeling and analysis decisions, or if they indeed reflect something real.