Transient exposure of a buried phosphorylation site in an autoinhibited protein
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Evaluation Summary:
The manuscript describes a metadynamics simulation-based characterization of the binding and unbinding dynamics of a buried phosphorylated residue in the inhibitory module to the functional domain of Vav1, in an effort to shed lights on the autoinhibition mechanism. The work led to a conformation-selection scenario of the event. The authors discussed the inconsistency between the computational findings and the NMR data, in terms of the free energy differences between the bound and unbound states. Further justification is required for the somewhat ad hoc choice of three collective variables for the metadynamics simulations, including two that are highly correlated.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Evaluation Summary:
The manuscript describes a metadynamics simulation-based characterization of the binding and unbinding dynamics of a buried phosphorylated residue in the inhibitory module to the functional domain of Vav1, in an effort to shed lights on the autoinhibition mechanism. The work led to a conformation-selection scenario of the event. The authors discussed the inconsistency between the computational findings and the NMR data, in terms of the free energy differences between the bound and unbound states. Further justification is required for the somewhat ad hoc choice of three collective variables for the metadynamics simulations, including two that are highly correlated.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested …
Evaluation Summary:
The manuscript describes a metadynamics simulation-based characterization of the binding and unbinding dynamics of a buried phosphorylated residue in the inhibitory module to the functional domain of Vav1, in an effort to shed lights on the autoinhibition mechanism. The work led to a conformation-selection scenario of the event. The authors discussed the inconsistency between the computational findings and the NMR data, in terms of the free energy differences between the bound and unbound states. Further justification is required for the somewhat ad hoc choice of three collective variables for the metadynamics simulations, including two that are highly correlated.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
This is essentially a simulation study of how IDPs interacts with a folded protein, or a folding-upon-binding event. This study speaks little to the the specific mechanism of Vav1, although it uses Vav1 autoinhibitory interaction as an example system. While the atomic details of the folding-upon-binding event is generally plausible, several aspects of this study requires clarification.
The first issue is a technical but potentially important one. The authors used three CVs in the metadynamics simulation-based study, which can be sound, except that the 1st and the 3rd CVs (native IDP binding contacts and the native binding contacts of a particular tyrosine of the IDP) seem highly correlated. This is generally less than ideal in sampling. What is the justification of introducing the 3rd CV other than the …
Reviewer #1 (Public Review):
This is essentially a simulation study of how IDPs interacts with a folded protein, or a folding-upon-binding event. This study speaks little to the the specific mechanism of Vav1, although it uses Vav1 autoinhibitory interaction as an example system. While the atomic details of the folding-upon-binding event is generally plausible, several aspects of this study requires clarification.
The first issue is a technical but potentially important one. The authors used three CVs in the metadynamics simulation-based study, which can be sound, except that the 1st and the 3rd CVs (native IDP binding contacts and the native binding contacts of a particular tyrosine of the IDP) seem highly correlated. This is generally less than ideal in sampling. What is the justification of introducing the 3rd CV other than the practical need to observe the binding/folding event in simulations? Was any analysis performed to preclude the possibility that the combination of these two correlated CVs distorts the binding process?
Secondly, the described binding event as an conformational selection process is inconsistent with a very similar process (IDP forming a helix and binding with a protein) as studied using unbiased MD simulation (https://doi.org/10.1021/jacs.0c03217), which showed that the helix formation was more of a consequence of the binding contacts, or in other word, an induced process. Likely the realty is not black and white, but because the cited study was based on unbiased situations and because the concerns with respect to the CVs used in the metadynamics simulations, further investigation and discussion may be needed.
Thirdly, the inconsistency with previous NMR study of the system in terms of binding free energy is concerning and further calculation/simulations seem necessarily. For example, can the simulations reproduce the energetic effect of certain mutations, which was also studied using NMR? Further work to ascertain the degree and depth of the issue would be extremely valuable.
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Reviewer #2 (Public Review):
The manuscript describes a computational characterization of the binding and unbinding dynamics of a buried phosphorylated residue in the inhibitory module to the functional domain of Vav1, in an effort to shed lights on the autoinhibition mechanism. However, as the authors discussed at length in the "binding equilibrium" section (starting from line 269), the inconsistency between the computational findings and the NMR data, in terms of the free energy differences between the two states, is puzzling. While I appreciate very much the honest and insightful discussion about the potential convergence and force field issues that may have contributed to the inconsistency, the advancement that this work brings to our understanding of the mechanism is not obvious. Thus, it appears that this manuscript may fit better …
Reviewer #2 (Public Review):
The manuscript describes a computational characterization of the binding and unbinding dynamics of a buried phosphorylated residue in the inhibitory module to the functional domain of Vav1, in an effort to shed lights on the autoinhibition mechanism. However, as the authors discussed at length in the "binding equilibrium" section (starting from line 269), the inconsistency between the computational findings and the NMR data, in terms of the free energy differences between the two states, is puzzling. While I appreciate very much the honest and insightful discussion about the potential convergence and force field issues that may have contributed to the inconsistency, the advancement that this work brings to our understanding of the mechanism is not obvious. Thus, it appears that this manuscript may fit better to a specialized journal in which the strength and weakness of using metadynamics to investigate IDR can be thoroughly presented and discussed.
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Reviewer #3 (Public Review):
By using enhanced sampling simulations and force fields that ar able to describe both folded and unfolded states, the authors address the timely and important question of autoinhibition regulation in a prototypical system: the Dbl Homology domain in the protein Vav1. Their simulations and free energy landscapes are consistent with a complex and intriguing process, whereby partial folding
the Ac-helix while unbound is followed by its docking to the binding site.
The authors carefully compare their results to available experiments and openly discuss the discrepancies and their possible source.The computational methods used here can be easily adapted to explore other auto inhibition mechanisms.
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