Determinants of Specificity of MDM2 for the Activation Domains of p53 and p65:  Proline27 Disrupts the MDM2-Binding Motif of p53

Abstract

Transcriptional activation and repression via the transcription factors p53 and p65 are mediated by hydrophobic short linear motifs (FXX phi phi) in their activation domains (ADs). To understand the molecular basis for specificity in binding to disparate biological targets, a series of chimeric peptides was synthesized, with sequences derived from the ADs of p53, which binds both the general transcriptional machinery and the repressor protein MDM2, and p65, which is reported to bind the general transcriptional machinery but not MDM2. The FXX phi phi motifs of p53 and p65 differ by only two residues, whereas the flanking sequences have no sequence identity. The affinities of the chimeric peptides to MDM2(25-117) and hTAF(II)31(1-140) were determined. Specificity for binding MDM2 via FXX phi phi motifs derives almost entirely from Trp23 of p53, with a 3.0 kcal mol(-1) loss of binding energy when Trp23 is changed to p65-derived Leu. The identity of the N-terminal flanking sequence did not significantly affect binding to MDM2. In contrast, replacement of the C-terminal sequence of p53 with that of p65 increased the affinity of the chimera for MDM2 by 1.1 kcal mol(-1), contrary to expectations. Replacement of the highly conserved residue Pro27 of p53 with Ser from p65 resulted in a 2.3 kcal mol(-1) improvement in binding to MDM2, generating a ligand (p53-P27S) (Kd = 4.7 nM) that exhibits the highest MDM2 affinity observed for a genetically encodable ligand. The basis for the increased affinity of p53-P27S over p53 was examined by circular dichroism and nuclear magnetic resonance. Pro27 disrupts the recognition alpha-helix of p53, with p53-P27S significantly more alpha-helical than p53.

Intrinsically Disordered Proteins: Where Computation Meets Experiment

“…These MD simulations are in agreement with NMR results suggesting that p53 residues 18-26 sample alpha helical conformations [126], as well as with their UV resonance Raman spectroscopy results summarizing the distribution of backbone psi angles in the peptide [125]. Xiong et al also examined the conformational preferences of a P27S mutation in TAD, which is associated with increased binding affinity to MDM2 [127], using MD simulations. They found that the unbound mutant peptide more heavily populates conformations similar to the MDM2-bound conformation of p53, due to increased alpha-helical content.…”

“…In this regard, it is always important to compare insights arising from these simulations to data obtained on the complete protein sequence. For example, previous work suggests that stability of the full MDM2-p53 complex is similar to that of the complex formed by a truncated TAD (residues 17-29) peptide [127]. Additionally, the N-terminal domain within the full p53 molecule behaves similarly to the isolated N-terminal residue 1-93 peptide and is not stabilized by contacts with folded regions of p53 [24,133].…”

Structural basis for high-affinity peptide inhibition of p53 interactions with MDM2 and MDMX

“…Zondlo et al recently showed that mutation of the highly conserved Pro-27 in (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) p53 to Ser increased its binding affinity for MDM2 by 50-fold (a decrease in K d from 229 to 4.7 nM) (40). The authors attributed the improvement in MDM2 binding to increased ␣-helicity of the peptide in the complex-a thesis largely confirmed in a molecular dynamic simulation study by Dastidar et al (41).…”

Modulation of the p53-MDM2 Interaction by Phosphorylation of Thr18: A Computational Study

“…The role of Asp21 in helping maintain the helix may not be as critical since mutation to Ala does little by way of affecting the interactions of this peptide with MDM2. 17,35 The mouse sequence (Fig. S1B) has a Gly in place of Asp21.…”