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.

Like many coactivators, the GACKIX domain of the master coactivator CBP/p300 recognizes transcriptional activators of diverse sequence composition via dynamic binding surfaces. The conformational dynamics of GACKIX that underlie its function also render it especially challenging for structural characterization. We find that the ligand discovery strategy of Tethering is an effective method for identifying small molecule fragments that stabilize the GACKIX domain and enables, for the first time, the crystallographic characterization of this important motif. The 2.0 Å resolution structure of GACKIX complexed to a small molecule was further analyzed by molecular dynamics simulations, revealing the importance of specific side chain motions that remodel the activator binding site in order to accommodate binding partners of distinct sequence and size. More broadly, these results suggest that Tethering can be a powerful strategy for identifying small molecule stabilizers of conformationally malleable proteins, thus facilitating their structural characterization and accelerating the discovery of small molecule modulators.

Allosteric binding events play a critical role in the formation and stability of transcriptional activator-coactivator complexes, perhaps in part due to the often intrinsically disordered nature of one or more of the constituent partners. The kinase-inducible domain interacting (KIX) domain of the master coactivator CREB binding protein/ p300 is a conformationally dynamic domain that complexes with transcriptional activators at two discrete binding sites in allosteric communication. The complexation of KIX with the transcriptional activation domain of mixed-lineage leukemia protein leads to an enhancement of binding by the activation domain of CREB (phosphorylated kinase-inducible domain of CREB) to the second site. A transient kinetic analysis of the ternary complex formation aided by small molecule ligands that induce positive or negative cooperative binding reveals that positive cooperativity is largely governed by stabilization of the bound complex as indicated by a decrease in k off . Thus, this suggests the increased binding affinity for the second ligand is not due to an allosteric creation of a more favorable binding interface by the first ligand. This is consistent with data from us and from others indicating that the on rates of conformationally dynamic proteins approach the limits of diffusion. In contrast, negative cooperativity is manifested by alterations in both k on and k off , suggesting stabilization of the binary complex.IDP | protein-protein interaction P rotein-protein interactions (PPIs) underscore all cellular processes, and the mechanistic dissection of PPI networks is thus a high priority (1-4). A particular challenge is defining the mechanism of PPI formation between intrinsically disordered proteins (IDPs) where allosteric changes play a substantive role (5-7). Allosteric communication between binding sites is vital for the proper function of feedback and regulatory circuits in the cell (8, 9). For example, the conformationally dynamic kinase-inducible domain interacting (KIX) domain of the master transcriptional coactivator CREB binding protein (CBP) undergoes a structural shift and stabilization upon binding to a cognate ligand such as the intrinsically disordered transcriptional activation domain (TAD) of mixed-lineage leukemia protein (MLL) (Fig. 1A) (10). The interaction of a second ligand, phosphorylated kinase-inducible domain (pKID) of CREB, is enhanced (up to twofold) by the presence of MLL and several elegant studies have documented allosteric communication between the two binding sites (11-15). However, the relatively modest affinities (micromolar) of the native complexes and the aggregation propensities and the promiscuous binding profiles of the transcriptional activation domains have hampered kinetic dissection of this and other complexes (16,17).The coactivator CBP exists across metazoans (18)(19)(20) and is a transcription hub that interacts with numerous transcriptional activators, using several discrete domains (21,22). The KIX domain within CBP is a 90-residue motif ...

Several lines of evidence suggest that the prototypical amphipathic transcriptional activators Gal4, Gcn4, and VP16 interact with the key coactivator Med15 (Gal11) during transcription initiation despite little sequence homology. Recent cross-linking data further reveal that at least two of the activators utilize the same binding surface within Med15 for transcriptional activation. To determine whether these three activators use a shared binding mechanism for Med15 recruitment, we characterized the thermodynamics and kinetics of Med15⅐activator⅐DNA complex formation by fluorescence titration and stopped-flow techniques. Combination of each activator⅐DNA complex with Med15 produced biphasic time courses. This is consistent with a minimum twostep binding mechanism composed of a bimolecular association step limited by diffusion, followed by a conformational change in the Med15⅐activator⅐DNA complex. Furthermore, the equilibrium constant for the conformational change (K 2 ) correlates with the ability of an activator to stimulate transcription. VP16, the most potent of the activators, has the largest K 2 value, whereas Gcn4, the least potent, has the smallest value. This correlation is consistent with a model in which transcriptional activation is regulated at least in part by the rearrangement of the Med15⅐activator⅐DNA ternary complex. These results are the first detailed kinetic characterization of the transcriptional activation machinery and provide a framework for the future design of potent transcriptional activators.

Fluorinated small molecules play an important role in the design of bioactive compounds for a broad range of applications. As such, there is strong interest in developing a deeper understanding of how fluorine affects the interaction of these ligands with their targets. Given the small number of fluorinated metabolites identified to date, insights into fluorine recognition have been provided almost entirely by synthetic systems. The fluoroacetyl-CoA thioesterase (FlK) from thus provides a unique opportunity to study an enzyme-ligand pair that has been evolutionarily optimized for a surprisingly high 10 selectivity for a single fluorine substituent. In these studies, we synthesize a series of analogs of fluoroacetyl-CoA and acetyl-CoA to generate nonhydrolyzable ester, amide, and ketone congeners of the thioester substrate to isolate the role of fluorine molecular recognition in FlK selectivity. Using a combination of thermodynamic, kinetic, and protein NMR experiments, we show that fluorine recognition is entropically driven by the interaction of the fluorine substituent with a key residue, Phe-36, on the lid structure that covers the active site, resulting in an ∼5- to 20-fold difference in binding (). Although the magnitude of discrimination is similar to that found in designed synthetic ligand-protein complexes where dipolar interactions control fluorine recognition, these studies show that hydrophobic and solvation effects serve as the major determinant of naturally evolved fluorine selectivity.