Thermodynamic resilience of wild-type p53 DNA-binding domain and its disruption by the R273H hotspot mutation: insights from REMD simulations

Abstract

The p53 protein is a cornerstone of tumor suppression, yet its functional integrity is frequently compromised by mutations in the DNA-binding domain (DBD). While R273H is conventionally classified as a DNA-contact mutation, recent evidence suggests it may harbor latent structural effects. In this study, we employed enhanced-sampling replica exchange molecular dynamics to delineate the structural-thermodynamic landscape of wild-type and R273H p53DBD. We identified robust thermal stability in the wild-type H2 helix, governed by a molecular mechanics-solvation free energy compensation (MSC) mechanism. In this regime, the attenuation of enthalpic molecular mechanical interactions at elevated temperatures is offset by an enhanced solvation effect, a process orchestrated by a network of salt bridges (R273, R282), H-bonds and a buried hydrophobic core. Conversely, the R273H mutation dual-destabilizes the DBD: locally, it abrogates critical electrostatic anchors (R273-E285/D281), impairing the H2 helix's MSC efficacy; globally, it triggers an allosteric rigidification of distal loops (L2, L3). This loss of conformational dampening renders the entire DBD scaffold susceptible to thermal fluctuations. Network analysis reveals that the R273H mutation triggers a global topological reorganization of the p53DBD, characterized by the decoupling of L2–L3 inter-loop coordination and an allostery-driven shift in community dynamics that bridges local DNA-contact disruption with distal structural instability. These findings refine the classification of R273H and provide a physicochemical framework for understanding how hotspot mutations reshape the protein's dynamic stability, offering potential leads for therapeutic stabilization.