Conformational Flexibility and Transient Structure of the Proline-Rich Domain in p53

Contains scripts and data for the paper "Conformational Flexibility and Transient Structure of the Proline-Rich Domain in p53" Abstract: The proline-rich domain (PRD) of the tumour suppressor p53 plays a central role in modulating conformational dynamics and molecular interactions, yet its intrinsic structural behaviour remains incompletely understood. Here, we combine extensive all-atom molecular dynamics (MD) simulations with biophysical validation to characterise the conformational ensemble of the p53 PRD. The domain behaves as an intrinsically disordered region (IDR), 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 beta-bends and turns linking conserved PXXP motifs. Circular dichroism and small-angle X-ray scattering (SAXS) 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 SAXS 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. Together, these results identify proline-mediated rigidity and transient PPII structure as key determinants of the dynamic conformational landscape of the p53 PRD.