Four domains of p300 each bind tightly to a sequence spanning both transactivation subdomains of p53

Area of Science:

• Molecular Biology
• Protein-Protein Interactions
• Cancer Biology

Background:

• The tumor suppressor p53 is regulated by the transcriptional coactivator p300.
• Both p53 and p300 possess independently folded domains connected by disordered sequences.
• Previous studies showed weak binding between a short p53 peptide and p300 domains.

Purpose of the Study:

• To investigate the binding affinity of a longer p53 N-terminal transactivation domain (p53(1-57)) to p300.
• To determine if p300 binding protects p53 from other interacting proteins.
• To link p53 mutations affecting transactivation to their binding affinity with p300.

Main Methods:

• Used a longer p53 N-terminal construct (p53(1-57)) for binding assays.
• Measured dissociation constants (K(D)) to quantify binding affinity.
• Introduced specific p53 mutations (L22Q/W23S, W53Q/F54S) to assess binding changes.

Main Results:

• The longer p53(1-57) construct exhibited tight binding to all four studied p300 domains.
• The p300 Taz2/CH3 domain showed the highest affinity (K(D) = 27 nM) for p53(1-57).
• p300 binding competed with Mdm2 binding to the p53 N-terminus, suggesting a protective role.
• p53 mutations that impair transactivation significantly reduced binding to p300 domains.

Conclusions:

• p300 tightly binds and protects the p53 N-terminal transactivation domain.
• The binding affinity is linked to p53's transcriptional activity and phenotypic outcomes.
• A model is proposed where tetrameric p53 interacts with four p300 domains.