Enhanced Binding Affinity via Destabilization of the Unbound State: A Millisecond Hydrogen-Deuterium Exchange Study of the Interaction between p53 and a Pleckstrin Homology Domain.

Abstract

The incorporation of intrinsically disordered domains enables proteins to engage a wide variety of targets, with phosphorylation often modulating target specificity and affinity. Although phosphorylation can clearly act as a chemical driver of complexation in structured proteins, e.g., by abrogating or permitting new charge-charge interactions, the basis for enhancement of the hydrophobically driven interactions that are typical of disordered protein-target complexation is less clear. To determine how phosphorylation can positively impact target recruitment in disordered domains, we have examined the interaction between the disordered N-terminal transactivation domain (TAD) of p53 and the pleckstrin homology (PH) domain of p62. Using time-resolved electrospray ionization with hydrogen-deuterium exchange, we demonstrate that phosphorylation has little effect on the conformation of the p53 TAD when it is bound to the PH domain but instead increases the degree of conformational disorder in the unbound state. We propose that this increase in the degree of disorder creates a wider free energy gap between the free and bound states, providing a target-independent mechanism for enhanced binding when the phosphorylated and unphosphorylated p53-target complexes have similar free energies.