Evolution of Interactions Involving Intrinsically Disordered Proteins

Abstract: This thesis describes the evolution of intrinsically disordered proteins and their interaction partners. The work presented is a combination of phylogenetic analysis, ancestral reconstruction and biophysical characterization in order to examine the evolutionary trajectory of protein-protein interactions involving disorder. The intrinsically disordered domains, NCBD and CID are both part of transcriptional co-regulating proteins. In evolution, NCBD existed before the emergence of CID and the most ancient domains display a low affinity complex with many weak contacts and high degree of conformational heterogeneity. Later in evolution, when NCBD and CID co-exists, a few mutations have altered the interaction in a way that the affinity is increased 25-fold and the conformational heterogeneity is decreased. In the same manner, the interaction is further optimized in extant species, resulting in a high affinity complex with less contacts of higher strength and less conformational heterogeneity. The intrinsically disordered transactivation domain of the tumour suppressing protein p53 and its negative regulator MDM2 date back to the beginning of animal life. The interacting domains are either lost or conserved in distinct phyla indicating a tight co-evolution. Phylogenetic trees produced by only including phyla with a conserved interaction domain follow the species evolution. Resurrection of p53 and MDM2 in the vertebrate lineage display an evolution of a high affinity complex in the ancestor of fish and tetrapods to a slightly improved affinity in modern tetrapods but a substantially lower affinity in zebrafish. The p53 protein family, which also includes p63 and p73, diverged from a common ancestor. The individual proteins display altered affinities to MDM2 which is a result of the high sequence divergence between them. The ionic dependence for the interactions is small, and not in line with other studies of disordered proteins. In conclusion, the work in this thesis have contributed with evolutionary analysis and experimental data of interactions involving intrinsically disordered proteins.