Single-Molecule Nanopore Profiling of p53-TAD’s Conformational Dynamics, Interactions, and Inhibition
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eLife Assessment
In this potentially useful study, the authors explored the conformational dynamics of the intrinsically disordered protein p53-TAD and a cancer-associated variant by examining its conformation-dependent block of conductance through a MspA nanopore, in conjunction with molecular dynamics simulations. In principle, this combination of single-molecule recordings, hidden Markov modeling, and molecular dynamics simulations can provide a generally applicable platform for studying disordered proteins. While the experimental observations could be described as compelling, there are also issues regarding the apparently low statistics for the observed long-lived pore-blockage events and limited differences between WT and mutant simulations. Therefore, in the current form, the level of evidence is deemed incomplete and requires further validation.
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Abstract
Investigating the conformational dynamics of intrinsically disordered proteins (IDPs) is essential to understanding how their structural heterogeneity underlies function and how their dysregulation contributes to diseases. Here, we utilized an MspA nanopore-based approach for studying the conformational dynamics and interactions of IDPs at the single-molecule level. The platform was demonstrated using the intrinsically disordered transactivation domain of tumor suppressor p53 (p53-TAD), one of the important proteins in cancer biology. We showed that MspA can stably capture p53-TAD and resolve up to six distinct current states with frequent interconversions, revealing a rich conformational landscape. The nanopore also detected the effect of a cancer-associated double mutational variant, N29K/N30D. Combining experiments with steered molecular dynamics simulations, we showed that the mutant sampled compact conformational states more frequently than wild type, consistent with previous NMR studies. Importantly, the MspA platform enabled direct monitoring of E3 ligase MDM2 binding to p53-TAD and resolved how this interaction is inhibited by anti-cancer compound epigallocatechin gallate (EGCG). Notably, EGCG stabilizes one of the six states sampled by p53-TAD, providing a mechanistic explanation for its inhibitory effect. Together, these findings demonstrate the promise of the nanopore platform for label-free monitoring of IDP conformational dynamics, modulation, binding and inhibition at single-molecule resolution.
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eLife Assessment
In this potentially useful study, the authors explored the conformational dynamics of the intrinsically disordered protein p53-TAD and a cancer-associated variant by examining its conformation-dependent block of conductance through a MspA nanopore, in conjunction with molecular dynamics simulations. In principle, this combination of single-molecule recordings, hidden Markov modeling, and molecular dynamics simulations can provide a generally applicable platform for studying disordered proteins. While the experimental observations could be described as compelling, there are also issues regarding the apparently low statistics for the observed long-lived pore-blockage events and limited differences between WT and mutant simulations. Therefore, in the current form, the level of evidence is deemed incomplete and requires …
eLife Assessment
In this potentially useful study, the authors explored the conformational dynamics of the intrinsically disordered protein p53-TAD and a cancer-associated variant by examining its conformation-dependent block of conductance through a MspA nanopore, in conjunction with molecular dynamics simulations. In principle, this combination of single-molecule recordings, hidden Markov modeling, and molecular dynamics simulations can provide a generally applicable platform for studying disordered proteins. While the experimental observations could be described as compelling, there are also issues regarding the apparently low statistics for the observed long-lived pore-blockage events and limited differences between WT and mutant simulations. Therefore, in the current form, the level of evidence is deemed incomplete and requires further validation.
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Reviewer #1 (Public review):
The manuscript combines MspA nanopore measurements with molecular simulations to investigate p53-TAD, its cancer-associated N29K/N30D variant, and its interactions with MDM2 and EGCG. The experiments demonstrate a promising approach for distinguishing sequence- and ligand-dependent single-molecule signals. The manuscript is clearly written, and the use of multiple simulation trajectories is a strength. However, the structural and thermodynamic interpretations extend beyond what the current simulations establish. I recommend addressing the following points.
(1) The assignment of low-current states to compact conformations near the constriction, and intermediate-current states to more expanded conformations higher in the vestibule, is plausible but not directly validated (Figures 1-2; main text, pp. 4-9). …
Reviewer #1 (Public review):
The manuscript combines MspA nanopore measurements with molecular simulations to investigate p53-TAD, its cancer-associated N29K/N30D variant, and its interactions with MDM2 and EGCG. The experiments demonstrate a promising approach for distinguishing sequence- and ligand-dependent single-molecule signals. The manuscript is clearly written, and the use of multiple simulation trajectories is a strength. However, the structural and thermodynamic interpretations extend beyond what the current simulations establish. I recommend addressing the following points.
(1) The assignment of low-current states to compact conformations near the constriction, and intermediate-current states to more expanded conformations higher in the vestibule, is plausible but not directly validated (Figures 1-2; main text, pp. 4-9). Current blockage can depend on protein position, orientation, charge distribution, and pore contacts as well as compactness. The simulations do not calculate ionic conduction, and the four structural clusters in Figure 2f do not have an established correspondence to the six experimental current states. Could the authors provide a validated conductance calculation for representative configurations, or explicitly present these structural assignments as hypotheses? In particular, an EGCG-associated current level overlapping S4 does not establish that the ligand stabilizes the same structural ensemble sampled by apo p53-TAD.
(2) Figure 2c-d presents occupancy-derived profiles and regional differences using free-energy terminology. However, the simulations describe finite-duration, constant-force capture trajectories initiated outside the pore, rather than demonstrated equilibrium sampling. Could the authors establish stationarity, evaluate dependence on the analysis window, and explain how the applied bias and normalization are treated? Otherwise, these results should be described as driven occupancy profiles, without interpreting their differences as equilibrium stabilization energies. Similarly, Figure 5 should be clearly identified as a schematic reconstruction rather than a measured molecular free-energy landscape.
The apparent EGCG affinity of 0.43 {plus minus} 0.49 μM also requires cautious interpretation. This value comes from event-frequency measurements at +75 mV and is poorly constrained. Event frequency depends on capture efficiency as well as solution binding populations. At +200 mV, manual voltage reversal censors spontaneous pore-escape times for long-lived complexes; moreover, pore escape and molecular dissociation are distinct processes. Please clarify which kinetic and thermodynamic quantities the assay can establish. The proposed confinement-mediated reduction in the entropic cost of EGCG binding is reasonable as a hypothesis, but is not demonstrated by these measurements.(3) The SMD protocol distributes a constant force over acidic residues, restrains the pore, and confines p53-TAD within a cylindrical potential (Figure S12; Supplementary Methods, lines 174-198). These approximations are acknowledged, but their influence on the WT-mutant comparison remains unresolved. The double substitution approximately preserves net charge while changing its distribution, and the force prescription treats acidic and basic residues differently. Do the positional and conformational differences persist across pulling strengths, restraint widths, and alternative force assignments? Please also confirm that the simulated pore contains the mutations present in the experimental M2-MspA-NNN construct.
The selected p53-TAD and MDM2 fragments omit full-length p53 regulation and interdomain coupling. This is a construct limitation, rather than a necessary consequence of the narrow constriction, because capture occurs within the wider vestibule. The existing discussion of wider pores and tethering could be extended to clarify this distinction. Phosphorylation-dependent TAD dynamics would be an interesting future application, although the present states cannot be assigned to modification-sensitive conformers without additional evidence.(4) The mutant exhibits increased intramolecular contacts, but the pore-resident radius-of-gyration differences are only approximately 0.2-0.3 Å, with small standardized effects (Figure 2e; Table S1). Several other descriptors have confidence intervals spanning zero, including the bulk Rg difference. Consequently, the simulations support modest sequence-dependent changes more strongly than a pronounced shift in global compactness. Figure S15 also identifies altered protein-pore contacts, which could contribute directly to positional occupancy and current differences. Could the authors provide convergence analyses, trajectory-level uncertainty, and sensitivity to starting ensembles, and distinguish intrinsic compaction from altered pore association?
(5) The replacement of MDM2-complex events by EGCG-associated events is consistent with inhibition, but differential capture could also change the observed event populations (Figures 3-4). The manuscript acknowledges this possibility on p. 12. Could the authors clarify its implications for the inhibition claim and provide an orthogonal measurement or appropriate controls to distinguish reduced complex abundance from preferential detection? Charge, steric geometry, and pore interactions should be considered without assuming that compaction necessarily explains enrichment.
No new simulations directly investigate EGCG binding or MDM2 competition. Ensemble-based ligand modeling could strengthen the interpretation, but docking poses or short trajectories alone would not establish preferential binding to S4, binding affinity, or conformational selection versus induced fit. The discussion should place the proposed mechanism alongside prior EGCG-p53 studies, including the computational predictions involving Trp23-Lys24 and Pro47-Thr55 reported by Bahena Culhuac and Bello, 2024. If additional modeling is beyond the scope of this work, the claims concerning S4 stabilization, compaction, and MDM2 displacement should be qualified accordingly.
Overall, the experimental platform is promising. The principal revision needed is to distinguish the observed nanopore signal changes from their proposed molecular explanations. Targeted robustness tests and appropriately qualified structural and thermodynamic claims would substantially strengthen the manuscript.
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Reviewer #2 (Public review):
Summary:
This manuscript presents an MspA nanopore-based approach for investigating the conformational dynamics and interactions of the intrinsically disordered transactivation domain of p53. The authors resolve multiple current states associated with trapped p53-TAD, identify differences between the wild-type protein and the N29K/N30D variant, and extend the approach to examine MDM2 binding and the effects of EGCG. The combination of single-molecule recordings, hidden Markov modeling, and molecular dynamics simulations provides a useful framework for studying disordered proteins under nanopore confinement.
Strengths:
The experimental observations are compelling, and the manuscript makes a valuable methodological contribution.
Weaknesses:
The analysis and interpretation could be strengthened.
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Reviewer #3 (Public review):
Summary:
DeCoeur et al. explored the conformational dynamics of the intrinsically disordered protein (IDP) p53-TAD and a cancer-associated variant by examining its conformation-dependent block of conductance through a nanopore. Using this approach, they identified a preferred conformational ensemble stabilized by the anti-cancer compound EGCG. They supplemented their experimental observations with steered molecular dynamics simulations of the IDP's conformational ensembles. Much of the work appears to recapitulate prior observations for this IDP, and as such, this work reads more like a method development paper. Although the use of nanopores to study IDPs as done here is not completely novel, these sorts of applications remain relatively few, and thus this work is still useful. However, there are several …
Reviewer #3 (Public review):
Summary:
DeCoeur et al. explored the conformational dynamics of the intrinsically disordered protein (IDP) p53-TAD and a cancer-associated variant by examining its conformation-dependent block of conductance through a nanopore. Using this approach, they identified a preferred conformational ensemble stabilized by the anti-cancer compound EGCG. They supplemented their experimental observations with steered molecular dynamics simulations of the IDP's conformational ensembles. Much of the work appears to recapitulate prior observations for this IDP, and as such, this work reads more like a method development paper. Although the use of nanopores to study IDPs as done here is not completely novel, these sorts of applications remain relatively few, and thus this work is still useful. However, there are several issues with the data and its interpretation that suggest only incomplete evidence for the conclusions. Namely, what appear to be very low statistics for the observed long-lived pore-blockage events on which much of the experimental conclusions seem to be based, and also conclusions from simulations that do not appear to align with the data. Until these issues are addressed, I find some of the conclusions to be only weakly supported by the data.
Strengths:
The use of nanopore current blockage to interrogate the dynamics of IDPs has the potential to be an ideal approach to investigate both mechanisms of variant pathology and drug efficacy.
Weaknesses:
The apparently small dataset may be limiting, and in several cases the authors appear to also over-interpret their results based on small effects.
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