Native Mass Spectrometry Reveals Binding Modes of the Tumor Suppressor Protein p53 to Different DNA Response Elements

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• The tumor suppressor protein p53 plays a critical role in cellular responses to DNA damage.
• p53 binds to specific DNA sequences called DNA response elements (DNA-REs).
• Understanding the precise DNA requirements for p53 binding is crucial for deciphering its regulatory mechanisms.

Purpose of the Study:

• To investigate how the architecture of the p21 DNA response element (DNA-RE) influences the binding mode of p53.
• To define the minimal DNA sequence and structural features necessary for stable p53:DNA complex formation.
• To compare the DNA binding behavior of wild-type tetrameric p53 with a dimeric variant (p53_L344A).

Main Methods:

• Native mass spectrometry (MS) was employed to analyze p53:DNA interactions.
• A comprehensive set of 37 DNA constructs derived from the p21 DNA-RE were synthesized and tested.
• Comparative analysis of p53 binding to full-site sequences, isolated half-sites, and sequence variants was performed.

Main Results:

• The complete 20-bp p21 DNA-RE is necessary for both wild-type p53 and the p53_L344A variant to form stable tetrameric complexes.
• Flanking regions outside the core 20-bp site do not affect the initial p53:DNA complex formation.
• Binding of a single p53 dimer to an isolated half-site is insufficient to stabilize a tetrameric complex.

Conclusions:

• Dimer-dimer interactions within the p53 tetramer are critical for stabilizing the binding to the 20-bp DNA response element.
• The specific architecture of the DNA-RE, particularly the full 20-bp site, dictates the formation of stable, functional p53-DNA assemblies.
• These findings provide insights into the structural basis of sequence-specific DNA recognition by p53.