The molecular mechanism of temperature-dependent p53C phase separation accelerated by oncogenic mutations: insights...

The aggregation of mutant p53 generally contributes to loss-of-function and gain-of-function effects, which increase cancer aggressiveness and progression. Recent studies have revealed that the DNA-binding domain of p53 (p53C) undergoes liquid-liquid phase separation (LLPS) in a temperature-dependent manner during the aggregation process. Two hotspot mutants, M237I and R249S, have been shown to accelerate this temperature-dependent phase separation. However, the underlying molecular mechanisms remain poorly understood. Here, we employed all-atom (AA) and coarse-grained (CG) molecular dynamics simulations to investigate the effect of M237I and R249S mutants on structural properties and aggregation propensity of p53C at different temperatures. Our results show that both mutants alter the temperature-dependent behavior of β-sheet content in a manner opposite to that of WT. Compared to the WT p53C, the two mutants, especially R249S, exhibit higher temperature sensitivity in conformational flexibility, intramolecular interactions, and solvent exposure. Temperature-sensitive regions are mainly involved in two specific regions: Region A (containing loop L1 and adjacent structural elements) and Region B (encompassing loops L2 and L3 along with their surrounding regions). CG simulations reveal that intermolecular interactions within these regions are significantly strengthened at elevated temperatures, which may facilitate the formation of liquid-like condensates and promote a transition to solid-like phases under thermal fluctuations. Specifically, M237I mutation enhances the aggregation propensity of p53C at lower temperatures (e.g., 15 °C and 37 °C) compared to the wild type by increasing solvent exposure of aggregation-prone segments and altering crucial inter-interactions. In contrast, R249S mutation resulted in greater water retention, along with the emergence of independent spherical aggregates. These results provide mechanistic insights into how M237I and R249S mutations promote temperature-dependent liquid-liquid phase separation (LLPS) and subsequent aggregation of p53C, suggesting promising avenues for anticancer therapeutic strategies that target phase separation-driven oncogenesis.