Characterization and small-molecule stabilization of the multisite tandem binding between 14-3-3 and the R domain of CFTR

Abstract

Cystic fibrosis is a fatal genetic disease, most frequently caused by the retention of the CFTR (cystic fibrosis transmembrane conductance regulator) mutant protein in the endoplasmic reticulum (ER). The binding of the 14-3-3 protein to the CFTR regulatory (R) domain has been found to enhance CFTR trafficking to the plasma membrane. To define the mechanism of action of this protein–protein interaction, we have examined the interaction in vitro. The disordered multiphosphorylated R domain contains nine different 14-3-3 binding motifs. Furthermore, the 14-3-3 protein forms a dimer containing two amphipathic grooves that can potentially bind these phosphorylated motifs. This results in a number of possible binding mechanisms between these two proteins. Using multiple biochemical assays and crystal structures, we show that the interaction between them is governed by two binding sites: The key binding site of CFTR (pS768) occupies one groove of the 14-3-3 dimer, and a weaker, secondary binding site occupies the other binding groove. We show that fusicoccin-A, a natural-product tool compound used in studies of 14-3-3 biology, can stabilize the interaction between 14-3-3 and CFTR by selectively interacting with a secondary binding motif of CFTR (pS753). The stabilization of this interaction stimulates the trafficking of mutant CFTR to the plasma membrane. This definition of the druggability of the 14-3-3–CFTR interface might offer an approach for cystic fibrosis therapeutics.

Fusicoccin Activates KAT1 Channels by Stabilizing their Interaction with 14-3-3- Proteins

“…Indeed, binding of FC to a non-MODE III motif has been reported also for the cystic fibrosis transmembrane conductance regulator (CFTR). However, in this case, FC binds in its standard conformation, which is made possible by the specific global architecture of the CFTR:14-3-3 complex (Stevers et al, 2016).…”

Small‐molecule stabilization of the p53 – 14‐3‐3 protein‐protein interaction

“…More recently it has been shown that fusicoccane derivatives can also stabilise internal or ‘mode 1 or 2’ partner protein recognition motifs. For example FC‐A has been shown to stabilize the 14‐3‐3 interaction with CFTR , the semi‐synthetic derivative ISIR‐005 stabilizes the interaction with Gab2 and related natural product cotylenin the interaction with C‐Raf .…”

A Binary Bivalent Supramolecular Assembly Platform Based on Cucurbit[8]uril and Dimeric Adapter Protein 14‐3‐3

“…with previous examples, [23,27] by connecting two more weakly binding monovalent ExoS motifs with aflexible linker,which resulted in over 100-fold affinity enhancement (see Figure 2 in the Supporting Information). In the absence of Q8, the FGG-ERa peptide was observed to displace the ExoS peptide with an IC 50 value of 1.0 mm ( Figure 2B,b lack line).…”

“…[21] They are dimerized proteins that typically bind their partner proteins through short, phosphorylated motifs [22] either at two single-motif binding sites or at atandem binding site,which greatly enhances the binding affinity. [23] Whereas PPIs with the 14-3-3 monomer can be modulated by natural products, [23] peptide derivatives, [22,24,25] synthetic molecules, [26] or supramolecular ligands, [3] molecular approaches to control the valency of 14-3-3-protein complexes are currently absent. Therefore,werationally designed abinary bivalent supramolecular assembly platform based on cucurbit [8]uril and dimeric adapter protein 14-3-3.…”