Eukaryotic transcriptional activators are minimally comprised of a DNA binding domain and a separable activation domain; most activator proteins also bear a dimerization module. We have replaced these protein modules with synthetic counterparts to create artificial transcription factors. One of these, at 4.2 kDa, mediates high levels of DNA site-specific transcriptional activation in vitro. This molecule contains a sequence-specific DNA binding polyamide in place of the typical DNA binding region and a nonprotein linker in place of the usual dimerization peptide. Thus our activating region, a designed peptide, functions outside of the archetypal protein context, as long as it is tethered to DNA. Because synthetic polyamides can, in principle, be designed to recognize any specific sequence, these results represent a key step toward the design of small molecules that can up-regulate any specified gene.

Background: Pyruvate kinase M2 controls glycolytic efflux. Results: Novel FRET sensors report PKM2 structural changes in response to activation by metabolites and phosphorylation events in pancreatic ␤-cells. Conclusion: PKM2 activity is oscillatory and synchronized between islet ␤-cells. Significance: This approach is broadly applicable for measuring pyruvate kinase M2 activity in living cells.

Designer molecules that can be used to impose exogenous control on gene transcription, artificial transcription factors (ATFs), are highly desirable as mechanistic probes of gene regulation, as potential therapeutic agents, and as components of cell-based devices. Recently, several advances have been made in the design of ATFs that activate gene transcription (activator ATFs), including reports of small-molecule-based systems and ATFs that exhibit potent activity. However, the many open mechanistic questions about transcriptional activators, in particular, the structure and function of the transcriptional activation domain (TAD), have hindered rapid development of synthetic ATFs. A compelling need thus exists for chemical tools and insights toward a more detailed portrait of the dynamic process of gene activation.

The conformationally dynamic binding surfaces of transcription complexes present a particular challenge for ligand discovery and characterization. In the case of the KIX domain of the master coactivator CBP/p300, few small molecules have been reported that target its two allosterically regulated binding sites despite the important roles that KIX plays in processes ranging from memory formation to hematopoiesis. Taking advantage of the enrichment of aromatic amino acids at protein interfaces, here we show that the incorporation of six 19F-labeled aromatic side chains within the KIX domain enables recapitulation of the differential binding footprints of three natural activator peptides (MLL, c-Myb, and pKID) in complex with KIX and effectively reports on allosteric changes upon binding using 1D NMR spectroscopy. Additionally, the examination of both the previously described KIX protein-protein interaction inhibitor Napthol-ASE-phosphate and newly discovered ligand 1–10 rapidly revealed both the binding sites and the affinities of these small molecules. Significantly, the utility of using fluorinated transcription factors for ligand discovery was demonstrated through a fragment screen leading to a new low molecular weight fragment ligand for CBP/p300,1G7. Aromatic amino acids are enriched at protein-biomolecule interfaces; therefore, this quantitative and facile approach will be broadly useful for studying dynamic transcription complexes and screening campaigns complementing existing biophysical methods for studying these dynamic interfaces.

Multi-protein complexes such as the transcriptional machinery, signaling hubs, and protein folding machines are typically comprised of at least one enzyme combined with multiple non-enzymes. Often the components of these complexes are incorporated in a combinatorial manner, in which the ultimate composition of the system helps dictate the type, location, or duration of cellular activities. Although drugs and chemical probes have traditionally targeted the enzyme components, emerging strategies call for controlling the function of protein complexes by modulation of protein-protein interactions (PPI). However, the challenges of targeting PPIs have been well documented and the diversity of PPIs makes a “one-size-fits-all” solution highly unlikely. These hurdles are particularly daunting for PPIs that encompass large buried surface areas and those with weak affinities. In this review, we discuss lessons from natural systems, in which allostery and other mechanisms are used to overcome the challenge of regulating the most difficult PPIs. These systems may provide a blueprint for identifying small molecules that target challenging PPIs and affecting molecular decision-making within multi-protein systems.