BPS2026 – Ternary IDP interactions modulated by heterogeneous binding transition paths

Conformational flexibility of intrinsically disordered proteins (IDPs) enables them to interact with multiple partners, making them hub proteins in complex binding networks. Despite the importance, mechanistic 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, the transcriptional adapter zinc-binding domain 2 (Taz2) of the CREB-binding protein (CBP), and the murine double minute 2 (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. To understand the structural mechanism of these complex interactions, we performed high-intensity (× 100) three-color FRET experiment that is required for measuring the transition path (TP) of binding on the microsecond timescale. We found that the binding transition path (TP) of the binary system, TAD and Taz2 binding in the absence of Mdm2 is heterogeneous and can also be clustered into two groups. These two TPs involve the sequential binding and dissociation of the two subdomains (AD1 and AD2) in TAD in different orders. We also found that one of the transition paths of two-component TAD-Taz2 binding resembles the transition path between the TAD-Taz2 complex and the ternary complex along the allosteric pathway in the three-component experiment. This implies that heterogeneous three-component interaction pathways are closely related with diverse transition paths of two-component TAD-Taz2 binding.