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Sequence Properties of An Intramolecular Interaction That Inhibits p53 DNA Binding.

Emily Gregory1, Gary W Daughdrill1

  • 1Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, FL 33620, USA.

Biomolecules
|November 11, 2022
PubMed
Summary

Mutations in the p53 transactivation domain 2 (TAD2) reduce autoinhibition, significantly increasing DNA binding affinity. This suggests cooperative roles for acidic, nonpolar, and aromatic residues in p53

Keywords:
DNA bindingcounterion condensation theoryfluorescence anisotropyhydrodynamic radiusintramolecular interactionintrinsically disordered proteinssalt-dependent binding affinitytumor suppressor p53van’t Hoff

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Area of Science:

  • Molecular Biology
  • Protein-DNA Interactions
  • Biochemistry

Background:

  • The tumor suppressor protein p53 plays a critical role in maintaining genomic stability.
  • p53's DNA binding activity is regulated by intramolecular interactions, including autoinhibition.
  • Understanding this autoinhibition is key to deciphering p53's regulatory mechanisms.

Purpose of the Study:

  • To investigate the role of specific amino acid residues in the transactivation domain 2 (TAD2) of p53 in autoinhibition.
  • To quantify the effect of mutations in TAD2 on the DNA binding affinity of p53.
  • To explore the contribution of acidic, nonpolar, and aromatic residues to p53 autoinhibition.

Main Methods:

  • Utilized a p53 fragment (ND WT) encompassing N-terminal transactivation domains (TAD1 and TAD2) and the DNA binding domain (DBD).
  • Introduced mutations in TAD2 targeting acidic, nonpolar, and aromatic amino acids to disrupt interaction with DBD.
  • Measured DNA binding affinity using fluorescence anisotropy across varying ionic strengths.
  • Assessed protein structure using size exclusion chromatography.

Main Results:

  • Mutants exhibited significantly increased DNA binding affinity compared to wild-type p53, indicating reduced autoinhibition.
  • The free energy change (ΔΔG) for DNA binding increased from -3.0 kcal/mol (ND WT) to -1.03 kcal/mol (ND DE) and -1.13 kcal/mol (ND NP).
  • Ionic strength dependence suggested altered counterion release upon DNA binding for mutants.
  • Size exclusion chromatography confirmed that mutations did not alter global protein structure.

Conclusions:

  • Acidic, nonpolar, and aromatic residues in TAD2 cooperate to inhibit p53 DNA binding.
  • Disrupting these interactions enhances p53's ability to bind DNA.
  • These findings provide insights into the molecular mechanisms of p53 regulation and DNA binding.