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

Abstract

The transcription factor p53 is under negative regulation by the ubiquitin ligase MDM2 and its close homologue MDM4. In the bound complex between MDM2 and p53, the transactivation domain of p53 adopts an amphipathic helical conformation which optimizes the spatial organization of three key hydrophobic residues (Phe19, Trp23, Leu26) for maximum interactions. The interaction with MDM2 is known to be abrogated by phosphorylation of Ser/Thr residues in the MDM2 N-terminal domain and in the p53 transactivation domain. In the latter, phosphorylation of Thr18 has been attributed to destabilize a key hbond between Thr18 and Asp21. This interaction has been thought to be critical for the formation of the helical conformation of the p53 transactivation domain. Molecular dynamics simulations of the p53 transactivation domain suggest that phosphorylation of either Thr18 or Ser20 does not disrupt its helical structure but does result in reduced affinities for MDM2. While interactions between the Thr18 and Asp21 are indeed broken due to charge-charge repulsions, the peptide has enough inherent flexibility to form alternate patterns of hbonds, resulting in the maintenance of helicity. Electrostatics of MDM2 reveal local anionic patches in the region where Thr18 docks. These suggest that repulsions will arise because the MDM2 surface will force the p53 to bind in a manner that will place the negatively charged phosphorylated Thr18 near this anionic region. A similar, albeit somewhat attenuated pattern of electrostatic modulations, is seen for a model of MDM4 that has been built. Mutants of MDM2 and MDM4 have been designed to attenuate this anionicity and have been computationally demonstrated to enhance the binding of the phosphorylated peptides.

A spatiotemporal characterization of the effect of p53 phosphorylation on its interaction with MDM2

“…The suggestion that phosphorylation of these sites destabilizes p53-MDM2 interaction is supported by alanine mutagenesis experiments 23 and computer simulations. 18,24,39,40 The physical rationale for the destabilization of binding is usually ascribed to thermodynamic effects 18 however, kinetic effects have not been studied in either experimental or simulation studies. In this study, we turn our attention to at least one model that describes the kinetic aspects: the structure and dynamics of the p53-MDM2 'encounter complex' before and after p53 phosphorylation.…”

“…The basins of attraction are the regions of localization of the peptides around the N-terminal domain of the MDM2 protein that would in principle promote productive encounters that lead to p53-peptide MDM2 N-terminal domain binding. We show that the disruption of this p53-MDM2 interaction upon phosphorylation is not only due to introducing unfavourable interactions in the p53 MDM2 binding site 18 but also due to reduction of the residence times of the phosphorylated p53 peptides in the basins of attraction around the MDM2 protein away from the binding site, which is concomitant with shifting the dominant basin of attraction away from the MDM2 surface and adopting a reversed peptide orientation compared to wild-type p53 peptide. These factors collectively mitigate against phosphorylated p53 reaching the binding site on the MDM2 surface.…”

“…23 Computational models, partly validated by experiments, suggested that the effect of phosphorylation was attributed to enhancing elecrostatic repulsion in the region where Thr18 docks by placing the negatively charged phosphorylated Thr18 near anionic regions of the MDM2 surface. 18,24 Phosphorylation of Ser20, however, was found to be sufficient to disrupt the p53-MDM2 interaction.…”

“…16 Interestingly, Ser15, Thr18 and Ser20 residues were found to be highly conserved in humans and urochordates 17 and therefore their role in modulating p53-MDM2 interactions has received much attention. [13][14][15][17][18][19] Ser15 phosphorylation alone was reported to be insufficient to block the p53-MDM2 interaction. 20 In fact, the X-ray structure of p53 MDM2 bound complex 21 shows that Ser15 lies outside the p53 binding region while Thr18 and Ser20, despite being within the binding region, are not involved in any direct contacts with MDM2…”

“…Although, it is known from several experiments that upon phosphorylation the binding of p53 to MDM2 is abrogated, 26,27 thermodynamic analysis of the p53-MDM2 bound complex suggests that phosphorylated p53 peptides do bind to MDM2, albeit, with a reduced affinity. 18 This could arise from limitations in experimental readout sensitivities or to a more profound basis that underpins the thermodynamic models used to interpret the interactions.…”