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A phosphorylation-dependent switch in the disordered p53 transactivation domain regulates DNA binding.

ABSTRACT: The tumor-suppressor p53 is a critical regulator of the cellular response to DNA damage and is tightly regulated by posttranslational modifications. Thr55 in the AD2 interaction motif of the N-terminal transactivation domain functions as a phosphorylation-dependent regulatory switch that modulates p53 activity. Thr55 is constitutively phosphorylated, becomes dephosphorylated upon DNA damage, and is subsequently rephosphorylated to facilitate dissociation of p53 from promoters and inactivate p53-mediated transcription. Using NMR and fluorescence spectroscopy, we show that Thr55 phosphorylation inhibits DNA-binding by enhancing competitive interactions between the disordered AD2 motif and the structured DNA-binding domain (DBD). Nonphosphorylated p53 exhibits positive cooperativity in binding DNA as a tetramer. Upon phosphorylation of Thr55, cooperativity is abolished and p53 binds initially to cognate DNA sites as a dimer. As the concentration of phosphorylated p53 is further increased, a second dimer binds and causes p53 to dissociate from the DNA, resulting in a bell-shaped binding curve. This autoinhibition is driven by favorable interactions between the DNA-binding surface of the DBD and the multiple phosphorylated AD2 motifs within the tetramer. These interactions are augmented by additional phosphorylation of Ser46 and are fine-tuned by the proline-rich domain (PRD). Removal of the PRD strengthens the AD2-DBD interaction and leads to autoinhibition of DNA binding even in the absence of Thr55 phosphorylation. This study reveals the molecular mechanism by which the phosphorylation status of Thr55 modulates DNA binding and controls both activation and termination of p53-mediated transcriptional programs at different stages of the cellular DNA damage response.

SUBMITTER: Sun X

PROVIDER: S-EPMC7817127 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Intrinsically disordered proteins (IDPs) are frequently associated with human diseases such as cancers, and about one-fourth of disease-associated missense mutations have been mapped into predicted disordered regions. Understanding how these mutations affect the structure-function relationship of IDPs is a formidable task that requires detailed characterization of the disordered conformational ensembles. Implicit solvent coupled with enhanced sampling has been proposed to provide a balance between accuracy and efficiency necessary for systematic and comparative assessments of the effects of mutations as well as post-translational modifications on IDP structure and interaction. Here, we utilize a recently developed replica exchange with guided annealing enhanced sampling technique to calculate well-converged atomistic conformational ensembles of the intrinsically disordered transactivation domain (TAD) of tumor suppressor p53 and several cancer-associated mutants in implicit solvent. The simulations are critically assessed by quantitative comparisons with several types of experimental data that provide structural information on both secondary and tertiary levels. The results show that the calculated ensembles reproduce local structural features of wild-type p53-TAD and the effects of K24N mutation quantitatively. On the tertiary level, the simulated ensembles are overly compact, even though they appear to recapitulate the overall features of transient long-range contacts qualitatively. A key finding is that, while p53-TAD and its cancer mutants sample a similar set of conformational states, cancer mutants could introduce both local and long-range structural modulations to potentially perturb the balance of p53 binding to various regulatory proteins and further alter how this balance is regulated by multisite phosphorylation of p53-TAD. The current study clearly demonstrates the promise of atomistic simulations for detailed characterization of IDP conformations, and at the same time reveals important limitations in the current implicit solvent protein force field that must be sufficiently addressed for reliable description of long-range structural features of the disordered ensembles.

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A phosphorylation-dependent switch in the disordered p53 transactivation domain regulates DNA binding.

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