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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.