Unraveling the allosteric mechanism and mechanical stability of partial and complete loss-of-function mutations in p53 DNA-binding domain

Mutations in functional proteins are the core cause of protein misfolding and dysfunction. Different mutation types exert distinct effects on protein structure and function, thus determining the specificity and diversity of targeted therapeutic strategies. In this study, we took the tumor suppressor protein p53 as a model to investigate the impacts of different mutation types on protein structure and function. Mutations in the p53 protein primarily occur in its DNA-binding domain (p53-DBD), and such mutations are classified into hotspot mutations (causing complete loss-of-function) and non-hotspot mutations (inducing partial loss-of-function). However, the allosteric mechanisms underlying non-hotspot mutations remain elusive. We conducted all-atom molecular dynamics simulations for the three systems: p53-WT, non-hotspot p53-E180R, and hotspot p53-R248W dimer-DNA complexes. Our results demonstrate that both mutations weaken intramolecular interactions in p53-DBD and enhance its structural flexibility. In particular, 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. These allosteric effects agree with available experimental data. Steered molecular dynamics simulations further confirm that both mutations accelerate p53 dimer dissociation. We also reveal for the first time the mechanical stability features of intermolecular interactions within p53 dimers. These findings provide atomic-level insights into p53 mutation-associated allostery and offer a basis for distinguishing mutation subtypes in prognosis prediction and targeted therapeutic strategy design.