Oligomerization of BAK by p53 utilizes conserved residues of the p53 DNA binding domain

E. Christine Pietsch, Erin Perchiniak, Adrian A. Canutescu, Guoli Wang, Roland L. Dunbrack, Maureen E. Murphy

Research output: Contribution to journalArticlepeer-review

81 Scopus citations

Abstract

Genotoxic stress triggers a rapid translocation of p53 to the mitochondria, contributing to apoptosis in a transcription-independent manner. Using immunopurification protocols and mass spectrometry, we previously identified the proapoptotic protein BAK as a mitochondrial p53-binding protein and showed that recombinant p53 directly binds to BAK and can induce its oligomerization, leading to cytochrome c release. In this work we describe a combination of molecular modeling, electrostatic analysis, and site-directed mutagenesis to define contact residues between BAK and p53. Our data indicate that three regions within the core DNA binding domain of p53 make contact with BAK; these are the conserved H2 alpha-helix and the L1 and L3 loop. Notably, point mutations in these regions markedly impair the ability of p53 to oligomerize BAK and to induce transcription-independent cell death. We present a model whereby positively charged residues within the H2 helix and L1 loop of p53 interact with an electronegative domain on the N-terminal alpha-helix of BAK; the latter is known to undergo conformational changes upon BAK activation. We show that mutation of acidic residues in the N-terminal helix impair the ability of BAK to bind to p53. Interestingly, many of the p53 contact residues predicted by our model are also direct DNA contact residues, suggesting that p53 interacts with BAK in a manner analogous to DNA. The combined data point to the H2 helix and L1 and L3 loops of p53 as novel functional domains contributing to transcription-independent apoptosis by this tumor suppressor protein.

Original languageEnglish
Pages (from-to)21294-21304
Number of pages11
JournalJournal of Biological Chemistry
Volume283
Issue number30
DOIs
StatePublished - Jul 25 2008

Keywords

  • Animals
  • Apoptosis
  • Caenorhabditis elegans
  • Cell Line, Tumor
  • Fibroblasts/metabolism
  • Humans
  • Mice
  • Mitochondria/metabolism
  • Models, Biological
  • Mutagenesis, Site-Directed
  • Protein Binding
  • Protein Conformation
  • Protein Structure, Tertiary
  • Tumor Suppressor Protein p53/chemistry
  • bcl-2 Homologous Antagonist-Killer Protein/chemistry

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