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Stage-dependent role of NEK7 in the inactive-to-active conformational transition of NLRP3 monomer

PLOS Computational Biology

Dear Dr. Sun,

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Dirk Walther, Ph.D.

Section Editor

PLOS Computational Biology

Dirk Walther

Section Editor

PLOS Computational Biology

Additional Editor Comments:

Dear authors, thanks for your submission to PLoS Comp Biol. Your work has now been reviewed by two expert reviewers. Both found the study interesting and of merit, but made a number of relevant suggestions, which I hope can be addressed. I look forward to receiving your revised version. Best regards, Dirk Walther

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: This manuscript uses state-of-the-art computational approaches to characterize how the NEK7 co-factor affects the inactive-to-active conformational transition of monomeric NLRP3. The main claims are that NEK7 has a stage-dependent role: (i) at early stages NEK7 reshapes dynamics of inactive NLRP3 making it more active-like, (ii) at middle stages NEK7 stabilizing a less-productive intermediate state, and (iii) at late stages NEK7 has negligible effect because a high energy barrier separates the active conformation (implying oligomerization is required to complete activation).

The study integrates multiple simulation modalities and attempts to map Cryo-EM structures into the computed landscape. Results are interesting and plausible and address an important mechanistic question for NLRP3 inflammasome regulation.

Major comment 1 — Role of nucleotides (ATP/ADP) during transition simulations

Although incorporating the effect of ATP hydrolysis into MD simulations is nontrivial, run simulations on models bearing gain-of-function mutations around the ATP binding pocket to test whether nucleotide-site perturbations alter the transition landscape. It would provide valuable insights into NLRP3 activation.

Major comment 2 —NLRP3 oligomerization features and NEK7-mediated disassembly

The authors note that NEK7 contributes to NLRP3 activation and that NLRP3 oligomerization is pivotal for the conformational transition to the active state. Both inactive cage and intermediate oligomeric state were reported previously—whose formation and conformational shifts may be NEK7-independent—suggest a possible role for NEK7 in oligomer disassembly, thereby enabling assembly of the canonical inflammasome disk. The authors might therefore provide mechanistic hypotheses on how NEK7 mediates dissociation and indicate which reported oligomeric forms are predicted by their landscape to be most/least susceptible to NEK7-mediated dissociation

Major comment 3 — The choice of initial inactive-state model (PDB 6NPY vs 8SXN)

The authors used PDB 6NPY as the starting structure for the inactive NLRP3 simulations rather than PDB 8SXN, justifying this choice on the grounds that 8SXN represents a dimeric, pharmacologically inhibited state complexed with MCC950. This rationale requires clarification: 6NPY itself was solved in the presence of ADP, MgCl2 and MCC950 (0.3 mM ADP, 5 mM MgCl2, 0.3 mM MCC950; Humayun Sharif et al., Nature 570:338–343, 2019). Moreover, 8SXN resolves both the N‑ and C‑lobes of NEK7 whereas 6NPY lacks part of the N‑lobe. Because these differences (such as oligomeric context and resolved regions) may influence MD and NEK7 interactions, the authors should provide a more plausible justification for choosing 6NPY as the inactive-state model. Or additional simulations (e.g., using 8SXN-derived starting structures or testing the presence/absence of MCC950) to demonstrate that the main conclusions are not dependent on the choice of initial PDB.

Correction may be needed:

NLRP3 does not contain the “N-terminal CARD domain” (Introduction)

Minor specific typos found, for example:

"NKE7" → "NEK7" (Conclusions).

"the diddle stage" → "the middle stage" (Conclusions).

"an high" → "a high" (multiple places).

"outpout density" → "output density" (Methods 5.6).

Reviewer #2: This interesting work uses molecular dynamics simulations to assess the effect of kinase NEK7 binding to NLRP3 protein on NLRP3’s ability to adopt an active state conformation. The authors find that NEK7 binding biases the NLRP3 towards more active-like states initially, but that a significant barrier remains regarding assumption of the completely formed active state. This finding is consistent with previous simulation studies, in particular that of Xu et al. (ref 16) but also provides new insight into possible intermediate states. The methods employed appear soundly executed and simulations suitably analyzed and interpreted. The results should be of interest to PLoS Comp Biol readers and I recommend the work for publication after consideration of the following remarks:

The study makes a couple of significant assumptions. The first is the study of solely monomer, as NLRP3 is generally thought to exist as oligomer in inactive and active forms. Cooperative action of the aggregate is likely to play a significant role in activation. However, interesting mechanical insights can still be derived by a reductionist study here of the monomer. In the Introduction, the discussion of experimental evidence for the (i) existence and importance of monomer and (ii) the latest thinking regarding the role of NEK7 needs to be expanded with more references.

The second assumption is the omission of cofactor for the transition simulations (but not the end point simulations). The state of the cofactor is known to be very important in activation of NLRP3. The authors also argue that omission of cofactor from both NEK7-bound and unbound forms mitigates the assumption of no cofactor, which does not seem a strong assumption. The authors also attempt to explore for a couple of snapshots the effect of this on RMSD and RMSF (quite low sensitivity measures). The two snapshots appear to have the domains at different degrees of rotation of LRR with respect to NACHT domain and thus provides something of a test (what are the angle values and time points of the samples?). What is the effect of this omission on the cofactor binding pocket residues and the adjacent domains which are very important to the hinge transition?

On a related note, it is not entirely clear why at the molecular level the active conformation has lower RMSFs and is more restricted (“intrinsically stable”) than the inactive form. The authors call this observation “remarkable”. Is this due to the presence of ATP to some degree? Some discussion of this would be useful.

Other:

Why is 6NPY far from the sampled region in Fig. 5C and 5D? Were gradually reducing constraints used to carefully equilibrate the protein? A brief comment on the resolution of the cryo-EM structures may also be useful, given their resolution is not that high.

Fig. 3(B) – why is the x-axis showing 3 us and yet 3 replicate simulations appear to be shown? Was it 3x 3 us or 3x 1 us MD simulations?

Section 2.3 - How is “loop” formation defined?

In places the language appears unduly emphatic, eg. “hallmark”, “uniquely” and “reveal”. These are predictions from MD simulations using models. Similarly the word “drive” is used in several places although this is conjecture rather than proven in the work. We note that while there clearly is electrostatic complementarity between NEK7 and NLRP3, the MMGBSA electrostatic terms of NEK7-NLRP3 largely cancel in Fig 6A; the van der Waals term is negative (the opposing nonpolar solvation term is not stated).

“diddle” should be “middle”

NKE not NEK in several places

Fig. 4 zoomed-in box is not obvious

“LINCS” not “LINC”

Cite Lopez-Blanco et al. correctly from text

Covariance equation symbols missing

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes:    Xiaodi Yu

Reviewer #2: No

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Dear Prof. Sun,

We are pleased to inform you that your manuscript 'Stage-dependent role of NEK7 in the inactive-to-active conformational transition of NLRP3 monomer' has been provisionally accepted for publication in PLOS Computational Biology.

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Best regards,

Dirk Walther, Ph.D.

Section Editor

PLOS Computational Biology

Dirk Walther

Section Editor

PLOS Computational Biology

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Dear authors, both reviewers are content with your revised version. Reviewer 2 suggested to reproduce Fig 5C in Fig S5, to allow an easier and direct comparison. I trust that the production process may allow updating Fig 5S accordingly, in case you wish to follow the reviewer's suggestion. Also, any reader can look at both figures side-by-side anyway.

Best regards,

Dirk Walther

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: I have reviewed the revised version of the manuscript and the authors’ point‑by‑point responses. The authors have appropriately addressed all of my previous comments, and the quality of the manuscript has improved accordingly.

I have no further concerns and recommend the manuscript for acceptance.

Reviewer #2: The authors have satisfactorily addressed my comments and I have only one very minor remark:

Please duplicate Fig 5C into Fig S5 for ease of comparison between surfaces with and without mutations.

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Have the authors made all data and (if applicable) computational code underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data and code underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data and code should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data or code —e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes:    Xiaodi Yu

Reviewer #2: No