title: Using peptides to study the interaction between the p53 tetramerization domain and HIV-1 Tat.
abstract: 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.
authors.affiliation: Institute of Chemistry, The Hebrew University of Jerusalem, Safra Campus, Givat Ram, Jerusalem 91904, Israel.
authors.forename: Ronen
authors.initials: R
authors.lastname: Gabizon
authors.forename: Michal
authors.initials: M
authors.lastname: Mor
authors.forename: Masha M
authors.initials: MM
authors.lastname: Rosenberg
authors.forename: Lena
authors.initials: L
authors.lastname: Britan
authors.forename: Zvi
authors.initials: Z
authors.lastname: Hayouka
authors.forename: Moshe
authors.initials: M
authors.lastname: Kotler
authors.forename: Deborah E
authors.initials: DE
authors.lastname: Shalev
authors.forename: Assaf
authors.initials: A
authors.lastname: Friedler
mesh_terms: D000409:Alanine / Q000378:metabolism; D020816:Amino Acid Motifs; D004797:Enzyme-Linked Immunosorbent Assay; D015696:Gene Products, tat / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D015497:HIV-1 / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D055672:Static Electricity; D013696:Temperature; D016159:Tumor Suppressor Protein p53 / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D015696:Gene Products, tat; D010446:Peptide Fragments; D016159:Tumor Suppressor Protein p53; D000409:Alanine