Dynamic control of IDP interaction network via diverse binding pathways

Binding promiscuity is a central feature of interactions involving intrinsically disordered proteins (IDPs). IDPs can interact even simultaneously with multiple binding partners, but quantitative characterization of these multi-component interactions is challenging. Here, we characterize the binding pathways of the transactivation domain (TAD) of p53 with two binding partners (Taz2 and Mdm2) using three-color single-molecule Förster resonance energy transfer (FRET) spectroscopy. We show that the interactions of these three proteins occur via two pathways. The first pathway is competitive in that binding of one partner occurs after the other partner completely dissociates. The second is an allosteric pathway via the formation of a ternary complex. High time-resolution FRET using photon-by-photon analysis shows that these heterogeneous three-component interaction pathways are closely related with diverse transition paths of two-component TAD-Taz2 binding. Kinetic analysis shows that the allosteric pathway allows faster exchange of the binding partners with opposite functions. Our work demonstrates how a heterogeneous allosteric binding network can enable a faster response to changes in the external environment.