Ordering a Dynamic Protein Via a Small-Molecule Stabilizer

Abstract

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.

Prepaying the entropic cost for allosteric regulation in KIX

“…Overall, we found that binding of MLL and/or c-Myb resulted in an increase in stability (or a reduction in dynamics) of the α 3 C terminus and the L 12 -G 2 loop. This is consistent with observations made in previous NMR and computational studies, which have led to the suggestion that the increased structural stability of KIX may play a role in allosteric signaling (4,20,(28)(29)(30)(31)(32). For example, NMR backbone order parameter measurements of KIX have demonstrated that the L 12 -G 2 loop and the C-terminal residues of the α 3 helix are more rigid when MLL is bound to KIX (29).…”

“…Previous studies have observed changes in the KIX structure resulting from transcription factor binding, and both MLL and c-Myb have been shown to allosterically affect the affinity of KIX for the complementary peptide (4,20,(28)(29)(30)(31)(32). However, a connection between the changes in structural dynamics in KIX and how this directly leads to shifts in transcription factor binding affinities remain tenuous.…”

“…Contained within CBP is an 87-residue-long domain called kinase-inducible domain interacting (KIX) that is critical for regulating transcriptional activity (for review, see ref. 11) and can be cooperatively targeted by a diverse set of transcription factors (12)(13)(14)(15)(16)(17) and small molecules (18)(19)(20)(21)(22)(23). The structure of KIX ( Fig.…”

“…The binding of either MLL or c-Myb to one of these promiscuous sites can stabilize the C terminus of the α 3 helix of KIX and/or decrease the mobility of the L 12 -G 2 loop (4,20,(28)(29)(30)(31)(32), but the functional role of these changes remains to be delineated. Furthermore, in vitro, KIX alone demonstrates a ∼2-fold lower affinity for c-Myb and ∼1.6-fold lower affinity for MLL compared with KIX bound to MLL or c-Myb, respectively (13).…”

“…Although numerous efforts have been put forth to clarify the molecular mechanism for cooperative binding in KIX, the current allosteric picture detailing the thermodynamic and structural effects of MLL and/or c-Myb binding remains incomplete. Although all-atom molecular dynamics (MD) simulations complement experiment and have provided valuable insight into the allosteric process (20,30,33), they are computationally too expensive to explore timescales where multiple binding events or protein folding/unfolding are necessary to understand the underlying mechanism of action. To address these limitations, we use a topology-based Go-like model (34,35) that has shown success in examining coupled protein folding and binding (36)(37)(38)(39)(40)(41)(42)(43)(44) as well as modeling ligand-induced structural transitions, and allosteric communication (45)(46)(47).…”