Sequence Properties of An Intramolecular Interaction That Inhibits p53 DNA Binding

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.