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Unraveling the Allosteric Mechanism and Mechanical Stability of Partial and Complete Loss-of-Function Mutations in p53 DNA-Binding Domain

Han Zhou, Tao Zhou, Shiwei Yan

TL;DR

This work investigates how two classes of p53-DBD mutations—non-hotspot cooperative mutations (E180R) and hotspot DNA-contact mutations (R248W)—alter allosteric regulation and mechanical stability of p53 dimers bound to DNA. Using all-atom MD and steered MD on WT, E180R, and R248W p53 dimers, it shows that E180R disrupts the dimer interface via L2/L3 rearrangements and salt-bridge loss, while R248W weakens L3/L1–DNA interactions without grossly changing overall conformation. Both mutations accelerate dimer dissociation under mechanical load, with $F_{max}$ ranking WT > R248W > E180R, and reveal distinct sets of key interfacial contacts that act as mechanical clamps. These results delineate mutation-type–specific mechanisms and point to tailored therapeutic strategies—stabilizing the dimer interface for cooperative mutations and enhancing DNA-binding compensation for DNA-contact mutants—informed by the mechanical and allosteric landscape of p53-DBD.

Abstract

TP53 is the most frequently mutated tumor suppressor gene in human cancers, with mutations primarily in its DNA-binding domain (p53-DBD). Mutations in p53-DBD are categorized into hotspot mutations (resulting in complete loss-of-function) and non-hotspot mutations (inducing partial loss-of-function). However, the allosteric mechanisms underlying non-hotspot mutations remain elusive. Using p53 dimer as models, we constructed p53-WT, non-hotspot p53-E180R, and hotspot p53-R248W dimer-DNA complexes to compare the structural and functional impacts of these two mutation types. Our results reveal that both mutations weaken intramolecular interactions in p53-DBD and enhance structural flexibility. Specifically, E180R perturbs dimer interface interactions, impairing dimer stability and cooperative DNA binding; R248W disrupts interactions between the L3/L1 loops and DNA, leading to the loss of DNA-binding capacity. Steered molecular dynamics (SMD) simulations further confirm that both mutations accelerate p53 dimer dissociation, with E180R exerting the most prominent disruptive effect on the mechanical stability of the dimer interface.

Unraveling the Allosteric Mechanism and Mechanical Stability of Partial and Complete Loss-of-Function Mutations in p53 DNA-Binding Domain

TL;DR

This work investigates how two classes of p53-DBD mutations—non-hotspot cooperative mutations (E180R) and hotspot DNA-contact mutations (R248W)—alter allosteric regulation and mechanical stability of p53 dimers bound to DNA. Using all-atom MD and steered MD on WT, E180R, and R248W p53 dimers, it shows that E180R disrupts the dimer interface via L2/L3 rearrangements and salt-bridge loss, while R248W weakens L3/L1–DNA interactions without grossly changing overall conformation. Both mutations accelerate dimer dissociation under mechanical load, with ranking WT > R248W > E180R, and reveal distinct sets of key interfacial contacts that act as mechanical clamps. These results delineate mutation-type–specific mechanisms and point to tailored therapeutic strategies—stabilizing the dimer interface for cooperative mutations and enhancing DNA-binding compensation for DNA-contact mutants—informed by the mechanical and allosteric landscape of p53-DBD.

Abstract

TP53 is the most frequently mutated tumor suppressor gene in human cancers, with mutations primarily in its DNA-binding domain (p53-DBD). Mutations in p53-DBD are categorized into hotspot mutations (resulting in complete loss-of-function) and non-hotspot mutations (inducing partial loss-of-function). However, the allosteric mechanisms underlying non-hotspot mutations remain elusive. Using p53 dimer as models, we constructed p53-WT, non-hotspot p53-E180R, and hotspot p53-R248W dimer-DNA complexes to compare the structural and functional impacts of these two mutation types. Our results reveal that both mutations weaken intramolecular interactions in p53-DBD and enhance structural flexibility. Specifically, E180R perturbs dimer interface interactions, impairing dimer stability and cooperative DNA binding; R248W disrupts interactions between the L3/L1 loops and DNA, leading to the loss of DNA-binding capacity. Steered molecular dynamics (SMD) simulations further confirm that both mutations accelerate p53 dimer dissociation, with E180R exerting the most prominent disruptive effect on the mechanical stability of the dimer interface.
Paper Structure (11 sections, 7 figures)

This paper contains 11 sections, 7 figures.

Table of Contents

  1. Introduction
  2. Methods
  3. System Preparations and MD Simulations Setup
  4. Steered molecular dynamics (SMD) simulations
  5. Analysis Method
  6. Results and discussion
  7. Mutant p53 Dimers Exhibit Reduced Structural Stability and Increased Loop Flexibility Compared with the p53-WT Dimer
  8. The E180R Mutation Drives Conformational Rearrangement at the p53 Dimer Interface
  9. The R248W Mutation Disrupts Interactions Between the L3/L1 Loops and DNA, Leading to Loss of DNA-Binding Capacity
  10. Steered Molecular Dynamics (SMD) Simulations Reveal the Effects of Two Mutations on p53 Dimer Dissociation
  11. Conclusion

Figures (7)

  • Figure 1: (a) Crystal structure of the p53-DBD in complex with a DNA duplex. The p53-DNA interaction interface is highlighted, including the L3 loop (green), L2 loop (yellow), and loop-sheet-helix (LSH) motif (magenta). Key DNA contact residues for are shown as sticks. (b) Initial structures of the p53-E180R and p53-R248W mutant dimers. (c,d) Probability density distributions of (c) backbone hydrogen bond counts and (d) backbone atomic contact counts in p53 dimers. (e,f) Probability density distributions of (e) solvent-accessible surface area (SASA) and (f) hydrophobic SASA of p53 dimers.
  • Figure 2: (a,b) Average root-mean-square-fluctuation (RMSF) of p53 (a) chain A and (b) chain B. Regions with enhanced flexibility relative to the p53-WT dimer are highlighted in the (c) p53-E180R and (d) p53-R248W mutant dimers.
  • Figure 3: (a) Structure of the p53 dimer interface, comprising the L2-H1-L2' loop and L3 loop. (b, c) Structural alignments of (b) the L2-H1-L2' loop and (c) the L3 loop at 500, 600, 700, and 800 ns frames from three independent simulation replicas.
  • Figure 4: (a-c) Contact maps of the p53 dimer interface in (a) p53-WT, (b) p53-E180R, and (c) p53-R248W dimers.(d, e) Conformations of H1 helix pairs in (d) p53-WT and (e) p53-E180R dimers (for three independent simulation replicas). The $C_{\alpha}$ atoms of residue P177 are represented as yellow sphere.
  • Figure 5: (a) Probability density distributions of the contact numbers between p53 dimer and DNA. (b, c) Contact conformation of L3 loop with the DNA minor groove in the (b) p53-WT dimer and (c) p53-R248W dimer. (d-f)Contact maps of residues in the L1 loop and H2 helix: (d) p53-WT, (e) p53-E180R and (f) p53-R248W dimers. (g) Structural alignments of the L1 loop (chain B) at 500, 600, 700, and 800 ns frames from three independent simulation replicas. The representative structure of loop--sheet--helix motif (chain B) in (h)p53-WT and (i) p53-R248W dimers.
  • ...and 2 more figures