Structural Basis for p300 Taz2-p53 TAD1 Binding and Modulation by Phosphorylation

Abstract

Summary Coactivators CBP and p300 play important roles in mediating the transcriptional activity of p53. Until now, however, no detailed structural information has been available on how any of the domains of p300 interact with p53. Here, we report the NMR structure of the complex of the Taz2 (C/H3) domain of p300 and the N-terminal transactivation domain of p53. In the complex, p53 forms a short α-helix and interacts with the Taz2 domain through an extended surface. Mutational analyses demonstrate the importance of hydrophobic residues for complex stabilization. Additionally, they suggest that the increased affinity of Taz2 for p531-39 phosphorylated at Thr18 is due in part to electrostatic interactions of the phosphate with neighboring arginine residues in Taz2. Thermodynamic experiments revealed the importance of hydrophobic interactions in the complex of Taz2 with p53 phosphorylated at Ser15 and Thr18.

Structure of the Taz2 domain of p300: insights into ligand binding

“…5a), explaining the moderately decreased binding affinity of the Taz2 R1731A mutant protein. On the other hand, mutation of Arg1737 to Ala would remove the unfavorable hydrophilic environment for Leu25 of p53 and should increase the binding affinity, which is in agreement with previous results (Feng et al, 2009).…”

“…NMR titration showed a sevenfold and 11-fold increase in affinity for Taz2 upon monophosphorylation at Ser15 and Thr18, respectively. Interestingly, only a sevenfold increase was generated upon diphosphorylation at these sites (Feng et al, 2009). Several findings pointed towards the possibility of two distinct modes of interaction with Taz2 for phosphorylated and unphosphorylated forms of p53-TAD1.…”

“…The interaction surface for binding of protein ligands revealed by the structures of the Taz2 domain complexed with p53-TAD1 (Feng et al, 2009) and with STAT1-TAD (Wojciak et al, 2009) is shown in Fig. 5.…”

“…As is the case with STAT1-TAD, this second amphipathic sequence within p53-TAD is preceded by negatively charged amino acids and its affinity of interaction with Taz2 is not affected by phosphorylation . Evidence for overlapping binding sites for p53 TAD1 and TAD2 peptides was provided by 1 H-15 NHSQC titration experiments on 15 N-labeled Taz2 (Feng et al, 2009). Two binding motifs from p53 TAD are separated by a 20-residue flexible linker.…”

“…The mechanism of this specific recognition and stimulusdependent selectivity is poorly understood. Three-dimensional structures have been determined by NMR for unliganded CBP-Taz2 (De Guzman et al, 2000) and its complexes with peptides derived from the TAD of STAT1 (Wojciak et al, 2009) and from the conserved region 1 (CR1) domain of the adenoviral oncoprotein E1A , as well as for the p300-Taz2-p53-TAD1 complex (Feng et al, 2009). Both STAT1-TAD and p53-TAD1 form short helices upon binding and interact with the Taz2 domain through an extended interface.…”