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Updated: Mar 28, 2026
Purification of Ubiquitinated p53 Proteins from Mammalian Cells
Published on: March 21, 2022
Multisite Phosphorylation Regulates the Structure and Auto-Inhibitory Function of the Intrinsically Disordered
Liang Fu1,2, Beifeier Li3, Kuan Liang1,2
1Interdisciplinary Institute of NMR and Molecular Sciences, Wuhan University of Science and Technology, Wuhan 430081, China.
Abstract:
Multisite phosphorylation plays an important role in essential cellular processes such as cell cycle control and circadian rhythm regulation and is also closely involved in the onset and progression of cancer and neurodegenerative diseases. As one of the most critical human tumor suppressor proteins, p53 is greatly modulated by phosphorylation. It contains numerous phosphorylation sites in its intrinsically disordered terminal domains. Specifically, multisite phosphorylation in the N-terminal domain (NTD) stimulates its autoinhibitory function, thereby repressing its downstream DNA transcriptional activity. However, the molecular mechanisms by which phosphorylation regulates p53 autoinhibition remain largely unclear. In this study, we employed all-atom molecular dynamics simulations combined with enhanced sampling methods to investigate how phosphorylation influences the structural properties of the intrinsically disordered p53-NTD, as well as its interaction with the DNA-binding domain (DBD). Our results show that phosphorylation significantly modulates the structural properties of p53-NTD, including both local structures and long-range residue interactions. T55 phosphorylation promotes the insertion of the aromatic rings of F54 and W53 into the DNA-binding pocket (DBP) of DBD and synergistically stabilizes the NTD-DBD interactions. Although S46 single phosphorylation would not induce NTD binding to the DBP, it can amplify the self-inhibitory ability of pT55 by reducing the dynamic conformational entropy of NTD. This study reveals the detailed molecular mechanism by which phosphorylation on p53-NTD regulates the structure and self-inhibition, providing crucial insights into the molecular basis underlying intrinsically disordered protein functions.
Insights
Phosphorylation of the p53 N-terminal domain (NTD) regulates its function by altering its structure and interactions. This study reveals how specific phosphorylation sites, like T55, control p53
Area of Science:
- Molecular Biology
- Biochemistry
- Structural Biology
Background:
- Multisite phosphorylation is crucial for cellular processes like cell cycle control and is implicated in cancer.
- The tumor suppressor protein p53 is regulated by phosphorylation, particularly in its intrinsically disordered terminal domains.
- The precise mechanisms by which phosphorylation controls p53's autoinhibition and DNA binding remain unclear.
Purpose of the Study:
- To investigate how phosphorylation affects the structural properties of the intrinsically disordered p53 N-terminal domain (NTD).
- To elucidate the impact of phosphorylation on the interaction between the p53 NTD and its DNA-binding domain (DBD).
- To reveal the molecular mechanisms underlying p53 autoinhibition regulated by phosphorylation.
Main Methods:
- All-atom molecular dynamics simulations.
- Enhanced sampling techniques.
- Analysis of structural properties and residue interactions.
Main Results:
- Phosphorylation significantly alters the structural properties of p53-NTD, affecting local structures and long-range interactions.
- T55 phosphorylation promotes the insertion of key aromatic residues into the DNA-binding pocket of the DBD, stabilizing NTD-DBD interactions.
- S46 phosphorylation, while not inducing NTD binding alone, enhances the inhibitory effect of T55 phosphorylation by reducing NTD's conformational entropy.
Conclusions:
- Phosphorylation on p53-NTD is a key regulator of its structure and self-inhibitory function.
- The findings provide detailed molecular insights into how phosphorylation controls the activity of intrinsically disordered proteins like p53.
- This research deepens our understanding of the molecular basis of p53 function in cellular processes and disease.
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