AbstractThe interaction between the transcription factor p53 and the ubiquitin ligase MDM2 results in the degradation of p53 and is well‐studied in cancer biology and drug development. Available sequence data suggest that both p53 and MDM2‐family proteins are present across the animal kingdom. However, the interacting regions are missing in some animal groups, and it is not clear whether MDM2 interacts with, and regulates p53 in all species. We used phylogenetic analyses and biophysical measurements to examine the evolution of affinity between the interacting protein regions: a conserved 12‐residue intrinsically disordered binding motif in the p53 transactivation domain (TAD) and the folded SWIB domain of MDM2. The affinity varied significantly across the animal kingdom. The p53TAD/MDM2 interaction among jawed vertebrates displayed high affinity, in particular for chicken and human proteins (KDaround 0.1 μM). The affinity of the bay mussel p53TAD/MDM2 complex was lower (KD= 15 μM) and those from a placozoan, an arthropod, and a jawless vertebrate were very low or non‐detectable (KD> 100 μM). Binding experiments with reconstructed ancestral p53TAD/MDM2 variants suggested that a micromolar affinity interaction was present in the ancestral bilaterian animal and was later enhanced in tetrapods while lost in other linages. The different evolutionary trajectories of p53TAD/MDM2 affinity during speciation demonstrate high plasticity of motif‐mediated interactions and the potential for rapid adaptation of p53 regulation during times of change. Neutral drift in unconstrained disordered regions may underlie the plasticity and explain the observed low sequence conservation in TADs such as p53TAD.