Using peptides to study the interaction between the p53 tetramerization domain and HIV-1 tat

Peptides are valuable tools for studying protein–protein interactions, especially in cases of isolated protein domains and natively unfolded proteins. Here, we used peptides to quantitatively characterize the interaction between the natively unfolded HIV‐1 Tat protein and the tetramerization domain of the cellular tumor suppressor protein p53. We used peptide mapping, fluorescence anisotropy, and NMR spectroscopy to perform a detailed structural and biophysical characterization of the interaction between the two proteins and elucidate its molecular mechanism, which have so far been studied using cell‐based methods. We show that the p53 tetramerization domain, p53(326–355), binds directly to residues 1–35 and 47–57 in Tat. We have characterized the interaction between p53(326–355) and Tat(47–57) in detail. The p53 residues that are mainly involved in binding to Tat(47–57) are E343 and E349, which bind to the positively charged arginine‐rich motif of Tat by a partly electrostatic mechanism. All oligomerization states of p53(326–355) bind Tat(47–57) without inhibiting p53 tetramerization, since the residues in p53(326–355) that bind Tat(47–57) face away from the tetramerization interface. We conclude that p53 is able to bind Tat as a transcriptionally active tetramer. © 2008 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 90: 105–116, 2008. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com