p53 Phosphomimetics Preserve Transient Secondary Structure but Reduce Binding to Mdm2 and MdmX

The disordered p53 transactivation domain (p53TAD) contains specific levels of transient helical secondary structure that are necessary for its binding to the negative regulators, mouse double minute 2 (Mdm2) and MdmX. The interactions of p53 with Mdm2 and MdmX are also modulated by posttranslational modifications (PTMs) of p53TAD including phosphorylation at S15, T18 and S20 that inhibits p53-Mdm2 binding. It is unclear whether the levels of transient secondary structure in p53TAD are changed by phosphorylation or other PTMs. We used phosphomimetic mutants to determine if adding a negative charge at positions 15 and 18 has any effect on the transient secondary structure of p53TAD and protein-protein binding. Using a combination of biophysical and structural methods, we investigated the effects of single and multisite phosphomimetics on the transient secondary structure of p53TAD and its interaction with Mdm2, MdmX, and the KIX domain. The phosphomimetics reduced Mdm2 and MdmX binding affinity by 3–5-fold, but resulted in minimal changes in transient secondary structure, suggesting that the destabilizing effect of phosphorylation on the p53TAD-Mdm2 interaction is primarily electrostatic. Phosphomimetics had no effect on the p53-KIX interaction, suggesting that increased binding of phosphorylated p53 to KIX may be influenced by decreased competition with its negative regulators.

Modelling Intrinsically Disordered Protein Dynamics as Networks of Transient Secondary Structure

ABSTRACTDescribing the dynamics and conformational landscapes of Intrinsically Disordered Proteins (IDPs) is of paramount importance to understanding their functions. Markov State Models (MSMs) are often used to characterize the dynamics of more structured proteins, but models of IDPs built using conventional MSM modelling protocols can be difficult to interpret due to the inherent nature of IDPs, which exhibit fast transitions between disordered microstates. We propose a new method of determining MSM states from all-atom molecular dynamics simulation data of IDPs by using per-residue secondary structure assignments as input features in a MSM model. Because such secondary structure algorithms use a select set of features for assignment (dihedral angles, contact distances, etc.), they represent a knowledge-based refinement of feature sets used for model-building. This method adds interpretability to IDP conformational landscapes, which are increasingly viewed as composed of transient secondary structure, and allows us to readily use MSM analysis tools in this paradigm. We demonstrate the use of our method with the transcription factor p53 c-terminal domain (p53-CTD), a commonly-studied IDP. We are able to characterize the full secondary structure phase space observed for p53-CTD, and describe characteristics of p53-CTD as a network of transient helical and beta-hairpin structures with different network behaviors in different domains of secondary structure. This analysis provides a novel example of how IDPs can be studied and how researchers might better understand a disordered protein conformational landscape.