Some mechanistic underpinnings of molecular adaptations of SARS-COV-2 spike protein by integrating candidate adaptive polymorphisms with protein dynamics

Reviewer #1 (Public Review):

Summary:
The authors are developing differences in the dynamics and allostery of the SARS-COV-2 spike protein for several of the variants. They consider mainly the delta, omicron, and Omicron XBB, and show major differences in the dynamics of the open forms. In the most compelling step, they go further and compare against experimental values of IC50 and KD.

Overall, this is an important application of methods that were developed in the senior author's lab.

Strengths:
The paper presents a strong case for the difference in the dynamical behavior of these sequence variants and relates this to available experiments.

Weaknesses:
The work does not drill down to the effects of individual mutations, which might be possible and would improve our understanding of the effects of single mutations and would dissect the contributions of each single difference in sequence.

Reviewer #2 (Public Review):

The authors set out to identify CAPs (Candidate Adaptive Polymorphyisms), i.e., simply put mutations that carry a potential functional advantage, and utilize computational methods based on the perturbation of C-alpha positions with an Elastic Network Model to determine if dynamics of CAP residues are different in any way.

In my opinion this manuscript *may* suffer from fundamental flaws in the detection of CAPs, and does not provide enough analysis and discussion to determine if the methodology is applicable. A highly expanded and rewritten manuscript may help clarify the results. Lastly, the authors severely ignore the vast literature and results already in the public domain, not only with respect to the use of normal-mode analysis methods as well as the detection of functionally relevant mutations in general and to understand the evolution of the SARS-CoV-2 Spike protein in particular.

Reviewer #3 (Public Review):

Summary:
The manuscript uses a combination of evolutionary approaches and structural/dynamics observations to provide mechanistic insights into the adaptation of the Spike protein during the evolution of variants.

Strengths:
Very well-written text, pleasant and well-described pictures, and didactical and clear description of the methods.
The citation of relevant similar results with different approaches is of note.
Comparing the calculated scores with previous experimentally obtained data is one of the strongest points of the manuscript.

Weaknesses:
A longer discussion of how the 19 orthologous coronavirus sequences were chosen would be helpful, as the rest of the paper hinges on this initial choice.
The 'reasonable similarity' with previously published data is not well defined, nor there was any comment about some of the residues analyzed (namely 417-484).
There seem to be no replicas of the MD simulations, nor a discussion of the convergence of these simulations.
A more detailed description of the equilibration and production schemes used in MD would be helpful.
Moreover, there is no discussion of how the equilibration procedure is evaluated, in particular for non-experts this would be helpful in judging the reliability of the procedure.