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.
Gabizon
Ronen
R
Institute of Chemistry, The Hebrew University of Jerusalem, Safra Campus, Givat Ram, Jerusalem 91904, Israel.
Mor
Michal
M
Rosenberg
Masha M
MM
Britan
Lena
L
Hayouka
Zvi
Z
Kotler
Moshe
M
Shalev
Deborah E
DE
Friedler
Assaf
A
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*
D015696:Gene Products, tat; D010446:Peptide Fragments; D016159:Tumor Suppressor Protein p53; D000409:Alanine