Evolution of affinity between p53 transactivation domain and MDM2 across the animal kingdom demonstrates high plasticity of motif‐mediated interactions

Abstract

The 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 (K D around 0.1 μM). The affinity of the bay mussel p53TAD/ MDM2 complex was lower (K D = 15 μM) and those from a placozoan, an arthropod, and a jawless vertebrate were very low or non-detectable (K D > 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 motifmediated 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.Filip Mihalič and Emma Åberg contributed equally to this study.

Conservation of Affinity Rather Than Sequence Underlies a Dynamic Evolution of the Motif-Mediated p53/MDM2 Interaction in Ray-Finned Fishes

“…The previous reconstruction of the p53TAD canonical binding motif from the common ancestor of fishes/tetrapods ( Mihalič et al 2023 ) suggested the presence of all three hydrophobic residues (in bold) that point into the binding pocket and make interactions with MDM2 in human p53TAD: S 15 Q 16 E 17 T 18 F 19 S 20 D 21 L 22 W 23 K 24 L 25 L 26 ( Fig. 1 ).…”

“…See Table 1 and supplementary table S1, Supplementary Material online for errors. The affinity at the node representing the divergence of fishes and tetrapods, marked with a star, was determined previously ( Mihalič et al 2023 ).…”

“…1 ). While the Phe 19 and Trp 23 are strictly conserved among Actinopterygii, there is considerable variation with regard to the third hydrophobic residue (Leu 26 ) and the following position 27, which was not included in the previous reconstruction of p53TAD fishes/tetrapods ( Mihalič et al 2023 ). Here, we included both residues in the experiments since residue 27 contributes to affinity in p53TAD peptides derived from extant D. rerio ( Mihalič et al 2023 ).…”

“…Bioinformatic and experimental analyses suggested that the p53–MDM2 regulation is an ancient mechanism, already present in the common ancestor of all metazoans ( Lane et al 2010 ; Lane and Verma 2012 ; Siau et al 2016 ; Zhang et al 2022 ). We recently investigated the deep evolution of affinity between p53TAD and MDM2 and found that the interaction between the conserved binding motif in p53TAD and MDM2 SWIB is relatively weak in invertebrates, ranging from not measurable to K D = 15 μM in the mollusk bay mussel ( Mytilus trossulus ) ( Mihalič et al 2023 ). Furthermore, the affinity was very low ( K D = 250 μM) in the jawless vertebrate arctic lamprey ( Lethenteron camtschaticum ), whereas in other vertebrates such as human ( Homo sapiens ) and chicken ( Gallus gallus ), the affinity of the interaction is in the 100 nM range.…”

“…To facilitate comparison, the site numbering used here follows the human p53TAD-binding motif, from Ser 15 to Pro 27 . The previously reconstructed maximum likelihood sequence for the most recent common ancestor of bony fishes and tetrapods ( Mihalič et al 2023 ) is indicated in the alignment as “fish versus tetrapods.”…”