BPS2026 – Role of cation-π interaction and proline-rich region in regulating p53 autoinhibition

P53 is an intrinsically disordered protein and tumor suppressor that orchestrates gene expression for cell-cycle regulation and apoptosis. It is also the most frequently mutated protein in cancer. P53 autoinhibition is regulated by multivalent intramolecular interactions between its transactivation domains (TAD1/TAD2), the proline-rich region (PRR), and the structured DNA-binding domain (DBD), modulating its DNA-binding selectivity. We integrate NMR spectroscopy, fluorescence polarization (FP), and accelerated molecular dynamics (HREX) in an array of p53 constructs to define a coupled dynamic equilibrium between autoinhibitory states. NMR relaxation revealed elevated transverse rates at W53 and multiple W91 indole resonances indicated slow chemical exchange between open and closed ensembles. Our data support two cooperative “hinges” that regulate this equilibrium: a PRR-DBD hinge, where a cation-π interaction, between R174 and W91, frustrates TAD2-DBD nucleotide mimicry, and a ring stacking, between W23 and W53, that frustrates TAD2 access to DBD sites. HREX further indicates that two subpopulations of TAD2 conformers couple W53 to a serine cluster and a cation-π (R181-W53) interaction that is proximal to the DBD dimer interface and transiently mask DNA-binding surface. Our results suggest that mutating W53, biases populations toward more open states, reducing autoinhibition and enhancing DNA affinity, whereas disrupting W23-W53 frustration increases autoinhibition. We further show that PRR stiffness displaces W53 from its aromatic stacking interaction, while altering PRR architecture, favors π-π contacts, reducing autoinhibition. Consequently, following DNA binding, TAD2 losses affinity for DBD and increasingly interacts back to TAD1. Finally, we demonstrate that mutations within the serine cluster allosterically weaken a salt bridge, thereby disrupting the PRR cation-π interaction and promoting open states. Together, these results refine the regulatory mechanism of p53 autoinhibition and guide strategies to modulate p53-DNA binding in p53-mutant tumors.