The αC-β4 loop controls the allosteric cooperativity between nucleotide and substrate in the catalytic subunit of protein kinase A

Reviewer #1 (Public Review):

Summary:
The authors use insights into the dynamics of the PKA kinase domain, obtained by NMR experiments, to inform MD simulations that generate an energy landscape of PKA kinase domain conformational dynamics.

Strengths:
The authors integrate strong experimental data through the use of state-of-the-art MD studies and derive detailed insights into allosteric communication in PKA kinase. Comparison of wt kinase with a mutant (F100A) shows clear differences in the allosteric regulation of the two proteins. These differences can be rationalized by NMR and MD results.

Weaknesses:
The very detailed insights gained by the authors into allosteric regulation require very specialized techniques in this study. This poses a challenge to communicate the methods, the results, and the meaning of the results to a broader audience. In some places, the authors overcome this challenge better than in others.

Reviewer #2 (Public Review):

Summary:
In this study, Olivieri & Wang et.al. probe the role of the conserved alphaC-beta4 loop in the allosteric regulation of the PKA catalytic subunit. The authors employ a combination of NMR-restrained molecular dynamics simulations and mutational analysis to uncover the conformational transitions between distinct excited states and identify a pivotal role for the alphaC-beta4 loop in facilitating these conformational transitions. These studies support previous models proposing the alphaC-beta4 loop as a critical element in kinase conformational regulation. Overall, this is a timely and fitting study.

Strengths:
1. Exciting application of NMR and MD to explore hidden conformation states of kinases.
2. Novel mechanistic insights into the role of the alphaC-beta4 loop in PKA.

Weaknesses:
1. While the alphaC-beta4 loop is a conserved feature of protein kinases, the residues within this loop vary across various kinase families and groups, enabling group and family-specific control of activity through cis and trans acting elements. F102 in PKA interacts with co-conserved residues in the C-tail, which has been proposed to function as a cis regulatory element. The authors should elaborate on the conformational changes in the C-tail, particularly in the arginine that packs against F102, in the results and discussion. This would further extend the impact and scope of the manuscript, which is currently confined to PKA.
2. The MD data and conformational states would be a valuable resource for the community and should be shared via some open-source repositories.
3. The authors state that ES1 and ES2 states are novel and not observed in previous crystal structures. The authors should quantify this through comparisons with PKA inactive states and with other AGC kinases.
4. Based on the results, can the authors speculate on the impact of oncogenic mutations in the alphaC-beta4 loop mutations in PKA?

Reviewer #3 (Public Review):

Summary:
Combining several MD simulation techniques (NMR-constrained replica-exchange metadynamics, Markov State Model, and unbiased MD) the authors identified the aC-beta4 loop of PKA kinase as a switch crucially involved in PKA nucleotide/substrate binding cooperatively. They identified a previously unreported excited conformational state of PKA (ES2), this switch controls and characterized ES2 energetics with respect to the ground state. Based on translating the simulations into chemical shits and NMR characterizing of PKA WT and an aC-beta4 mutant, the author made a convincing case in arguing that the simulation-suggested excited state is indeed an excited state observed by NMR, thus giving the excited state conformational details.

Strengths:
This work incorporates extensive simulation works, new NMR data, and in vitro biochemical analysis. It stands out in its comprehensiveness, and I think it made a great case.

Weaknesses:
The manuscript is somewhat difficult to read even for kinase experts, and even harder for the layman. The difficulty partially arises from mixing technical description of the simulations with structural interpretation of the results, which is more intuitive, and partially arises from the assumption that readers are familiar with kinase architecture and its key elements (the aC helix, the APE motif etc).