Conservation of coactivator engagement mechanism enables small-molecule allosteric modulators

Abstract

SignificanceTranscriptional coactivators and their partner transcription factors have been labeled as intrinsically disordered, fuzzy, and undruggable. We propose that the identification of conserved mechanisms of engagement between coactivators and their cognate activators should provide general principles for small-molecule modulator discovery. Here, we show that the structurally divergent coactivator Med25 forms short-lived and dynamic complexes with three different transcriptional activators and that conformational shifts are mediated by a flexible substructure of two dynamical helices and flanking loops. Analogous substructures are found across coactivators. Further, targeting one of the flexible structures with a small molecule modulates Med25–activator complexes. Thus, the two conclusions of the work are actionable for the discovery of small-molecule modulators of this functionally important protein class.

Unexpected Specificity within Dynamic Transcriptional Protein-Protein Complexes

“…ETV/PEA3•Med25 interactions represent an ideal system to study dynamic TAD•ABD interactions for several reasons. First, previous studies indicated that interaction of ETV/PEA3 family member ETV5 with the Med25 ABD is typical of a dynamic TAD•ABD complex (30,32); the binding surface is shallow, both acidic and hydrophobic amino acids of the TAD determine affinity and activity, and multiple bound conformational states were detected by both NMR and kinetic analyses. Second, our previous studies of the ETV5•Med25 complex showed that the bound conformational ensemble is directly accessible by transient kinetic analysis.…”

“…Second, our previous studies of the ETV5•Med25 complex showed that the bound conformational ensemble is directly accessible by transient kinetic analysis. (32) The models of TAD•ABD molecular recognition can thus be dissected in this system without the loss of critical conformational information to equilibrium averaging. Third, the ETV/PEA3 family of transcription factors serves as an excellent natural system to test the relationship between TAD sequence and recognition; they contain almost identical arrangements of acidic and hydrophobic residues across the TAD sequence, especially within the helical binding region that undergoes coupled folding and binding with Med25,(30) but the identity of specific residues varies slightly ( Fig.…”

“…This system can therefore be used to test whether TAD•Med25 interactions are truly nonspecific and insensitive to changes in the TAD sequence (i.e., if these PPIs form in a "sequence-independent" manner(11)), or if these interactions are affected by TAD sequence changes and thus have a degree of specificity to formation. Finally, the Med25 ABD is ligandable by small molecules (32), and therefore conclusions from mechanistic studies can be directly applied to guide and assess optimization of small molecule modulators of TAD•Med25 complex formation.…”

“…(4,(27)(28)(29) Here, we take a critical look at activator•coactivator recognition by mechanistically dissecting a representative set of dynamic complexes formed between the ABD of Mediator subunit Med25 and the amphipathic transcriptional activation domains (TADs) of the ETV/PEA3 family of Ets transcriptional activators (ETV1, ETV4, and ETV5). (23,(30)(31)(32) Previous biophysical studies indicated that the interaction of Med25 with family member ETV5 appears to be a prototypical nonspecific TAD•ABD complex: it occurs over a shallow surface, is driven by electrostatic and hydrophobic interactions, and forms a dynamic complex that is recalcitrant to structure determination. (30,32) We utilize a mechanistic and structural approach that combines quantitative data regarding activator•coactivator conformational states obtained via transient kinetic analysis with structural information obtained through mutagenesis and NMR spectroscopy.…”

“…(23,(30)(31)(32) Previous biophysical studies indicated that the interaction of Med25 with family member ETV5 appears to be a prototypical nonspecific TAD•ABD complex: it occurs over a shallow surface, is driven by electrostatic and hydrophobic interactions, and forms a dynamic complex that is recalcitrant to structure determination. (30,32) We utilize a mechanistic and structural approach that combines quantitative data regarding activator•coactivator conformational states obtained via transient kinetic analysis with structural information obtained through mutagenesis and NMR spectroscopy. Our data reveal that the conformational ensembles of ETV/PEA3•Med25 PPIs are strikingly sensitive to slight changes in TAD sequence, despite being dynamic complexes with several well-populated conformational sub-states at equilibrium.…”