The R337C mutation in the p53 oligomerization domain affects the regulatory domain and its ability to bind response elements: Evidence based on structural and biophysical studies

Abstract

The homotetrameric form of p53 is critical for performing essential functions like maintaining genomic stability and preventing uncontrolled cell proliferation. In part, these crucial functions are mediated by the p53 C-terminal region (CTR) containing the tetramerization/oligomerization domain (TD/OD) and regulatory domain (RD), responsible for maintaining the protein's oligomeric state and regulating its function. Mutations in the tetramerization domain reduce the transactivation potential and alter the transactivation specificity of p53. This study investigates the effect of high-frequency tetramerization missense mutation p53R337C on protein stability, oligomeric state, and its ability to bind the DNA response elements. For the first time using CD and FTIR spectroscopy, we have shown that the p53 regulatory domain (residues 363–393) and oligomerization domain (residues 327–355) possess a characteristic alpha helix secondary structure, which is enhanced upon binding to DNA, implicating stabilization of the domain. The mutation R337C in the OD impacts the secondary and tertiary structure of p53 CTR, leading to the loss of secondary structure and the formation of unstable tetramers, as shown by CD and DSC thermal studies. Surprisingly, the secondary structure of mutant p53 CTR partially stabilized upon binding to the DNA sequence. Our data suggests that the unstable p53R337C tetramer exhibits weaker binding to the DNA promoter sequence with decreased transcription activity, consistent with previous cell-based assays. Our study conclude that the loss of salt-bridge interactions between Arg337 and Asp352 in the intra-dimer of p53 leads to the formation of unstable tetramers, and the DNA-binding ability of the regulatory domain.