Dynamics of ternary complex formation of an IDP and two binding partners and their allosteric effects studied by three-color single-molecule FRET.

Abstract

Intrinsically disordered proteins (IDPs) are highly flexible and can bind multiple binding partners by folding into different structures. This promiscuity of IDPs is especially important for their function as hub proteins in the formation protein-protein interaction networks, which is a key regulatory mechanism of complex biological processes such as gene transcription and signal transduction. IDPs often have more than one binding domain, suggesting they can interact with multiple binding partners simultaneously. Ternary complexes formed by an IDP and two binding partners have been observed by NMR, but it is difficult to obtain more detailed kinetic information from ensemble measurements. In this work, we used three-color single-molecule FRET to directly visualize the formation and dissociation of a ternary complex. We studied binding of an IDP (p53 transactivation domain (TAD)) and two competitive binding partners (TAZ2 of CBP and MDM2). By labeling TAD with Alexa 488 (donor) and Alexa 647 (acceptor 1) and TAZ2 with CF680R (acceptor 2), it is possible to detect the formation of not only two binary complexes (TAD-TAZ2 and TAD-MDM2) but also the ternary complex (TAD-MDM2-TAZ2), which can be distinguished by the fraction of acceptor 2 intensity. From the kinetic analysis, we found that the TAD-TAZ2 binary complex is the most stable and the ternary complex is formed only transiently. Interestingly, the presence of MDM2 induces more rapid dissociation of TAZ2 in the ternary complex whereas the dissociation rate of MDM2 is not affected by TAZ2. This asymmetric allosteric effect results in the more effective replacement of CBP on p53 with MDM2, which is a novel regulatory mechanism beyond the simple competition between multiple binding partners.