Conservation of affinity rather than sequence underlies a dynamic evolution of the motif-mediated p53/MDM2 interaction in teleosts

The transcription factor and cell cycle regulator p53 is marked for degradation by the ubiquitin ligase MDM2. The interaction between these two proteins is mediated by a conserved binding motif in the disordered p53 transactivation domain (p53TAD) and the folded SWIB domain in MDM2. The conserved motif in p53TAD from zebrafish displays a 20-fold weaker interaction with MDM2, compared to the interaction in human and chicken. To investigate this apparent difference, we tracked the molecular evolution of the p53TAD/MDM2 interaction among ray- finned fishes (Actinopterygii), the largest vertebrate clade. Intriguingly, phylogenetic analyses, ancestral sequence reconstructions, and binding experiments showed that different loss-of- affinity changes in the canonical binding motif within p53TAD have occurred repeatedly and convergently in different fish lineages, resulting in relatively low extant affinities ( K D = 0.5-5 μM). However, for eleven different fish p53TAD/MDM2 interactions, non-conserved regions flanking the canonical motif increased the affinity 4 to 73-fold to be on par with the human interaction. Our findings suggest that compensating changes at conserved and non-conserved positions within the motif, as well as in flanking regions of low conservation, underlie a stabilizing selection of “functional affinity” in the p53TAD/MDM2 interaction. Such interplay complicates bioinformatic prediction of binding and call for experimental validation. Motif- mediated protein-protein interactions involving short binding motifs and folded interaction domains are very common across multicellular life. It is likely that evolution of affinity in motif- mediated interactions often involves an interplay between specific interactions made by conserved motif residues and non-specific interactions by non-conserved disordered regions. ### Competing Interest Statement The authors have declared no competing interest.