Conformational flexibility and transient structure of the proline-rich domain in p53.

The proline-rich domain (PRD) of the tumor suppressor p53 plays a central role in modulating conformational dynamics and molecular interactions, yet its intrinsic structural behavior remains incompletely understood. Here, we combine extensive all-atom molecular dynamics simulations with biophysical validation to characterize the conformational ensemble of the p53 PRD. The domain behaves as an intrinsically disordered region, sampling a highly heterogeneous ensemble with average end-to-end distance and radius of gyration of 52.5 Å and 21.8 Å, respectively. Despite this disorder, transient local structure is prominent: unordered conformations dominate, followed by substantial polyproline II (PPII) content, with β-bends and turns linking conserved PXXP motifs. Circular dichroism and small-angle X-ray scattering experiments corroborate the largely disordered yet partially structured nature of the PRD. Ramachandran and contact analyses reveal that consecutive prolines, particularly Pro71-Pro72, impose steric constraints that stabilize locally extended conformations and restrict backbone collapse. To approximate the PRD within full-length p53, additional simulations were performed with restrained terminal distances, yielding reduced conformational variability and improved agreement with small-angle X-ray scattering data while preserving secondary-structure propensities. PPII helices emerge as particularly robust features, acting as stiff spacers linking the transactivation domain to downstream regions. Finally, simulations of clinically relevant variants reveal mutation-specific local perturbations: P72R disrupts consecutive proline rigidity and increases flexibility, whereas P82L abolishes a PXXP motif and its associated PPII helix. These results identify proline-mediated rigidity and transient PPII structure as key determinants of the dynamic conformational landscape of the p53 PRD.