Dissecting the Roles of Dynamic Substructures in Beta-Barrel Containing Coactivators

Abstract

Starting off the central dogma of genetics, transcription is an integral process in all of life. Transcription is highly regulated, with many players and moving parts, such as transcription factors that localize transcriptional machinery to the promoter, and regulators that modulate expression levels. Despite being well studied, there lacks a mechanistic understanding of how binding occur, largely due to the difficult nature of studying these interactions. Intrinsic disorder, transient interactions, and highly dynamic, protein-protein interactions at the site of transcription are difficult systems to study mechanistically and structurally. Due to their critical role in all of life, it is crucial to understand the molecular recognition details of these interactions. Molecular recognition models of activator-coactivator interactions were argued to be largely nonspecific, dictated by unstructured and negatively charged transcriptional activators bound to DNA interacting with amphipathic coactivators. However, this model does not accurately represent the critical role of activator-coactivator interactions. Put another way, it is too simplified. The work in this thesis aims to decipher the molecular recognition mechanisms of coactivators recognizing activator binding partners using a beta-barrel containing coactivator activator binding domain termed Activator Interacting Domain (AcID). Recent data has shifted the paradigm of molecular recognition to one that is more specific. Originally demonstrated in KIX, it has been observed that conformational changes are induced upon activator binding to the activator binding domain AcID of the Mediator complex subunit, Med25. Moreover, it was shown that despite overlapping binding sites, unique conformational ensembles were observed for each binding partner. Using a second protein containing two tandem AcID motifs, the work in this thesis aimed to expand upon this conservation of molecular recognition. We show that the AcIDs are capable of recognizing overlapping binding partners in vitro, demonstrating that selectivity is achieved through means other than activator sequence. Using transient kinetics, binding mechanisms of the different AcID motifs were observed. Specifically, despite being paralogs, it was found that different binding modes were observed, suggesting that changes in overall dynamic resulted in conformations specific to each AcID motif without drastically changing the overall binding affinities. Further, it has been demonstrated that an allosteric network exists between the binding faces of activator binding domains within coactivtors. Using a kinetic approach, we demonstrated that there is allosteric communication within the different AcID motifs. Similar to how conformational changes of activator binding domains are mediated through dynamic substructures, allosteric communication is also mediated by loops and helices. These dynamic substructures can be exploited as hotspots for targeting, as these allosteric regions are not as highly conserved in paralogs. We demonstrate that identified allosteric modulators can be used as chemical probes to perturb the dynamic hotspots, providing an opportunity to target homologous proteins with high selectivity. Further, we show that even highly related activators are able to induce differential conformations in activator binding domains, highlighting that these interactions are specific. Using a biophysical and biochemical approach, the work in this dissertation demonstrates that activator binding domains are capable of sharing a conserved binding mechanism. By demonstrating that differences in dynamic substructures flanking the binding faces can induce differential conformational changes, we provide a mechanism by which molecular recognition can occur. We highlight that conformational plasticity can influence allosteric communication and provide an opportunity to selectively target activator-coactivator interactions.