Native mass spectrometry reveals binding modes of the tumor suppressor protein p53 to different DNA response elements

Abstract

We used native mass spectrometry (MS) to investigate how the architecture of the p21 DNA response element (DNA-RE) controls the binding mode of the tumor suppressor protein p53. We analyzed tetrameric full-length wild-type p53 and compared its DNA binding behavior with a dimeric variant, p53L344A. In total, 37 DNA constructs derived from p21 DNA-RE were examined via native MS, including full-site sequences, isolated half-sites, variants differing in length and composition, and random DNA sequences. The aim was to define the minimal DNA requirements for p53 binding using native MS as a robust platform for comparative analysis of p53:DNA assemblies. Our results show that flanking regions of the full 20-bp DNA-RE have no influence on the initial formation of p53:DNA complexes. However, the complete 20-bp site is required for both p53wild-type and p53L344A to bind to DNA as tetramers. Binding of a single dimer to an isolated half-site is insufficient for generating the stable tetrameric p53:DNA complex. These findings indicate that dimer-dimer interactions are crucial for stabilizing the tetrameric p53:DNA complex.