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. 2024 Jul 15;15(32):12806-12818.
doi: 10.1039/d4sc03645j. eCollection 2024 Aug 14.

Multiscale simulations reveal the driving forces of p53C phase separation accelerated by oncogenic mutations

Yawei Yu  1 , Qian Liu  1 , Jiyuan Zeng  1 , Yuan Tan  1 , Yiming Tang  1 , Guanghong Wei  1
Affiliations

Multiscale simulations reveal the driving forces of p53C phase separation accelerated by oncogenic mutations

Yawei Yu et al. Chem Sci. .

Abstract

Liquid-Liquid phase separation (LLPS) of p53 to form liquid condensates has been implicated in cellular functions and dysfunctions. The p53 condensates may serve as amyloid fibril precursors to initiate p53 aggregation, which is associated with oncogenic gain-of-function and various human cancers. M237I and R249S mutations located in p53 core domain (p53C) have been detected respectively in glioblastomas and hepatocellular carcinoma. Interestingly, these p53C mutants can also undergo LLPS and liquid-to-solid phase transition, which are faster than wild type p53C. However, the underlying molecular basis governing the accelerated LLPS and liquid-to-solid transition of p53C remain poorly understood. Herein, we explore the M237I/R249S mutation-induced structural alterations and phase separation behavior of p53C by employing multiscale molecular dynamics simulations. All-atom simulations revealed conformational disruptions in the zinc-binding domain of the M237I mutant and in both loop3 and zinc-binding domain of the R249S mutant. The two mutations enhance hydrophobic exposure of those regions and attenuate intramolecular interactions, which may hasten the LLPS and aggregation of p53C. Martini 3 coarse-grained simulations demonstrated spontaneous phase separation of p53C and accelerated effects of M237I/R249S mutations on the phase separation of p53C. Importantly, we find that the regions with enhanced intermolecular interactions observed in coarse-grained simulations coincide with the disrupted regions with weakened intramolecular interactions observed in all-atom simulations, indicating that M237I/R249S mutation-induced local structural disruptions expedite the LLPS of p53C. This study unveils the molecular mechanisms underlying the two cancer-associated mutation-accelerated LLPS and aggregation of p53C, providing avenues for anticancer therapy by targeting the phase separation process.

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Conflict of interest statement

There are no conflicts to declare.

Figures

Fig. 1
Fig. 1. Initial structures and AA-to-CG mapping of p53C in AA and CG simulations. (a) The structure of the p53C monomer, highlighting loops L1, L2, and L3, helixes H1 and H2, and Zn ion. Residues M237 and R249 are depicted in stick representation. (b) Mapping from the AA structure to CG structure. (c) The initial state of CG simulation for p53C LLPS, which was obtained after a series of equilibrium steps, with each CG molecule colored differently.
Fig. 2
Fig. 2. Impact of M237I mutation on the structural stability of p53C. (a–c) PDF of the (a) total contact number, (b) SASA value, and (c) Rg value of p53C. (d) PDF of the water number around p53C residues with a cutoff of 0.35 nm. (e and f) PDF of (e) backbone HB number and (f) average water number around backbone HB of p53C. (g and h) Time evolution of the backbone RMSD values of (g) L3 and (h) zinc-binding domain. These data suggest that M237I mutation reduces the structural stability of p53C, particularly disrupting the zinc-binding domain.
Fig. 3
Fig. 3. Effects of M237I mutation on the interactions in L2, L3, and zinc-binding domains and the resulting conformational alterations. (a) Differences in L3–L3 contact number map between M237I and WT (i.e., M237I minus WT, L3 residues: M237I–P250). (b and c) Representative snapshots of loop L3 showing reduced interactions in (c) M237I compared to (b) WT (reduced interactions: C238–M246, S240–M246, C242–M246, and S241–R248). (d) Differences in L3–L2 contact number map between M237I and WT (i.e., M237I minus WT, L2 residues: Y163–C176, L3 residues: M237I–P250). Three residue pairs display reduced contact numbers (marked by red rectangles, i.e., M237–R175, R249–H168, and R249–E171). (e and f) Representative snapshots of L3 and L2 showing disrupted interactions in (f) M237I compared to (e) WT (disrupted interactions: M237–R175, R249–H168, R249–E171). (g) A representative snapshot of the zinc-binding domain showing the Zn ion coordination in p53C. Zn ion is shown with a red ball and the coordinated residues are indicated (zinc-binding domain residues: C176, P177, H178, and H179 in L2, C238, N239, S240, S241, and C242 in L3). (h and i) PDF of (h) SASA and (i) Rg values of the zinc-binding domain. (j) PDF of the contact number between the zinc-binding domain and water molecules. These results indicate that M237I mutation induces the disruption of the zinc-binding domain, leading to the global structural destabilization of p53C.
Fig. 4
Fig. 4. The influences of R249S mutation on p53C structure. (a–c) PDF of (a) total contact number, (b) SASA value, and (c) Rg value of p53C. (d) PDF of the water number around p53C residues with a cutoff of 0.35 nm. (e and f) PDF of (e) backbone HB number and (f) average water number around backbone HB of p53C. Time evolution of backbone RMSD value of (g) L3 and (h) zinc-binding domain. These data show that R249S mutation reduces the structural stability of p53C by predominantly disrupting loop L3 and the zinc-binding domain.
Fig. 5
Fig. 5. Analysis of R249S mutation-induced disruption of native interactions and enhancement of hydration on L3 and zinc-binding domain. (a) Differences in L3 contact number map between R249S and WT (i.e., R249S minus WT, L3 residues: M237I–P250). (b and c) Representative snapshots of loop L3 showing reduced interactions in (c) R249S and (b) WT. (d) Differences in L3–L2 contact number map between R249S and WT (i.e. R249S minus WT, L2 residues: Y163–C176, L3 residues: M237I–P250). (e and f) Representative snapshots of L3 and L2 showing the disrupted interactions in (f) R249S and (e) WT. (g) Representative snapshot showing the conformational difference of L3 in WT and R249S mutant (10 representative snapshots were superposed with each other). (h) Time evolution of the distance between the center of mass of Cα atoms in N-terminal (residues M237–M243) and that of Cα atoms in C-terminal (residues G244–P250) of L3. (i) PDF of the contact number between the backbone atoms of L3 residues and water molecules. (j) RDF between the midpoint of Cα–Cα distance of residues C242 and M246 in the central region of L3 and the oxygen atom of water molecules. These results demonstrate that R249S mutation induces more hydrophobic exposure and hydration of L3 and zinc-binding domain.
Fig. 6
Fig. 6. Dynamics of p53C phase separation in CG simulations. (a–c) Representative snapshots illustrating the LLPS process in WT (a), M237I (b), and R249S (c) systems (representative timestamps at 0, 4, 8, and 10 μs). Our data show that both M237I and R249S mutations accelerate the LLPS of p53C.
Fig. 7
Fig. 7. Effect of M237I/R249S mutation on the LLPS behavior of p53C. (a–c) PDF of intermolecular contact number of p53C in WT (a), M237I (b), and R249S (c) systems. (d–f) Time evolution of average intermolecular contact number over two individual trajectories for WT (d), M237I (e), and R249S (f) p53C. (g–i) Intermolecular contact number map of WT (g), M237I (h), and R249S (i) systems, with the enhanced contacts labeled by dashed rectangles. The orange and purple rectangles highlight respectively the L1 region and Turn S3/S4 region (the turn structure between β-strand 3 and β-strand 4). In M237I mutant (h), the green rectangle highlights the zinc-binding domain and part of L3 regions and the red rectangle highlights the L3 region. In R249S mutant (i), the green rectangle highlights the zinc-binding domain plus L2 and the red rectangle highlights the zinc-binding domain plus the L3 region. The regions with enhanced intermolecular interactions are consistent with the disrupted regions with weakened intramolecular interactions observed in AA simulations. Our data show that M237I and R249S mutations hasten the LLPS of p53C.
Fig. 8
Fig. 8. Schematic representation of p53C LLPS and aggregation induced by M237I and R249S mutations. Upper left panel: M237I mutation induces the disruption of the zinc-binding domain, promotes the formation of aggregation-prone molten globule states in p53C. Lower left panel: R249S mutation induces the disruption of both L3 and zinc-binding domain, resulting in the overall structural destabilization of p53C. Right panel: these molecular-level structural disruptions accelerate the LLPS and subsequent fibrillization of p53C in M237I and R249S mutants.
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