Structural Divergence Exceeds Sequence Divergence for a Family of Intrinsically Disordered Proteins

Several sequence based evolutionary analyses have concluded that many intrinsically disordered proteins and disordered regions evolve faster than ordered proteins. However, there are only a couple of studies that demonstrate the physical consequences of this rapid evolution. The transactivation domain of the tumor suppressor protein p53 (p53TAD) is an intrinsically disordered protein with some transient helical structure that is stabilized by binding to partners like the E3 ubiquitin ligase, MDM2, and the 70 kDa subunit of replication protein A, RPA70. The secondary structure and backbone dynamics of five mammalian orthologues of p53TAD were investigated using nuclear magnetic resonance (NMR) spectroscopy. Significant differences in secondary structure and dynamics were observed in the binding sites of MDM2 and RPA70 between the orthologues. The degree of transient helical structure observed in the MDM2 binding site was directly related to amino acid substitutions occurring on the solvent exposed side of the amphipathic helix that forms in the bound state, with dog, cow, and mouse orthologues having the lowest fraction of transient helical structure and the fastest backbone dynamics. Differences were also observed in the RPA70 binding site with the guinea pig and rabbit orthologues showing significantly higher propensity for transient helical structure than the other orthologues. Clustering analysis shows greater divergence in secondary structure than expected based on sequence divergence. However, strong correlations were found between the backbone dynamics and the sequence identity of the orthologues. This result is consistent with a previous study from our group and suggests that the dynamic behavior of IDPs is under positive selection.