Characterization and small-molecule stabilization of the multisite tandem binding between 14-3-3 and the R domain of CFTR Loes M. Steversa,b, Chan V. Lama,b, Seppe F. R. Leysena,b, Femke A. Meijera,b, Daphne S. van Scheppingena,b, Rens M. J. M. de Vriesa,b, Graeme W. Carlilec, Lech G. Milroya,b, David Y. Thomasc, Luc Brunsvelda,b, and Christian Ottmanna,b,d,1 aLaboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands; bInstitute for Complex Molecular Systems, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands; cCystic Fibrosis Translational Research Centre, Department of Biochemistry, McGill University, Montreal, QC, Canada, H3G 1Y6; and dDepartment of Chemistry, University of Duisburg-Essen, 45141 Essen, Germany Edited by Robert Huber, Max Planck Institute of Biochemistry, Planegg-Martinsried, Germany, and approved January 26, 2016 (received for review August 22, 2015) Cystic fibrosis is a fatal genetic disease, most frequently caused by cosylated, misfolded, and retained in the endoplasmic reticulum the retention of the CFTR (cystic fibrosis transmembrane conduc- (ER) (9). The immature protein is recognized by CHIP/Hsc70 tance regulator) mutant protein in the endoplasmic reticulum (ER). cochaperones, which results in the retrotranslocation of CFTR The binding of the 14-3-3 protein to the CFTR regulatory (R) from the ER and its subsequent degradation by the ubiquitin- domain has been found to enhance CFTR trafficking to the plasma proteasomal pathway (10). Nevertheless, when the mutated CFTR membrane. To define the mechanism of action of this protein–pro- is stimulated to be transported to the plasma membrane, for tein interaction, we have examined the interaction in vitro. The example by reducing the temperature of the cell, its chloride disordered multiphosphorylated R domain contains nine different secretion is often still partially functional (11). 14-3-3 binding motifs. Furthermore, the 14-3-3 protein forms a di- Earlier research has shown that 14-3-3 proteins play an im- mer containing two amphipathic grooves that can potentially bind portant role in the biogenesis of the CFTR protein (12, 13). these phosphorylated motifs. This results in a number of possible 14-3-3 is a eukaryotic adaptor protein family that consists of seven binding mechanisms between these two proteins. Using multiple isoforms (β, γ, e, ζ, η, σ,andτ) (14). Isoforms β, γ,ande have biochemical assays and crystal structures, we show that the inter- been shown to be present in human bronchial epithelial cells, action between them is governed by two binding sites: The key whereas other isoforms have been shown to be weak or absent. binding site of CFTR (pS768) occupies one groove of the 14-3-3 Additionally, phosphorylation-dependent binding of 14-3-3β and dimer, and a weaker, secondary binding site occupies the other 14-3-3e to the regulatory (R) domain of CFTR has been shown binding groove. We show that fusicoccin-A, a natural-product tool to significantly increase the CFTR level in the plasma mem- compound used in studies of 14-3-3 biology, can stabilize the in- brane (13). As an explanation, 14-3-3 is considered to reduce teraction between 14-3-3 and CFTR by selectively interacting with retrieval of the CFTR protein to the ER by masking the ER a secondary binding motif of CFTR (pS753). The stabilization of this retention signal and assisting folding and maturation of the protein interaction stimulates the trafficking of mutant CFTR to the plasma membrane. This definition of the druggability of the 14-3-3–CFTR Significance interface might offer an approach for cystic fibrosis therapeutics. protein–protein interaction | disordered protein | multivalency It has been shown that 14-3-3 proteins increase trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) to the plasma membrane by binding to its regulatory (R) ystic fibrosis (CF) is the most common life-threatening re- domain. This paper contains a detailed characterization of the Ccessive genetic disease in the Caucasian population (1). Due 14-3-3/CFTR interaction, showing that multiple phosphory- to mutations in the gene coding for the cystic fibrosis trans- lated binding sites in the CFTR R-domain are necessary for membrane conductance regulator (CFTR), the expression of the significant binding with 14-3-3. We find that one of these CFTR protein in epithelial cells is disturbed. This results in binding sites serves as an anchor, while surrounding weaker dysfunctional anion transport [primarily chloride (2) and bi- sites enhance the interaction. Furthermore, we show the drugg- carbonate (3) anions] across the plasma membrane. The lack of a ability of this interaction using natural-product fusicoccin-A, proper anion flow in epithelial cells causes complications in a which stabilizes the 14-3-3/CFTR interaction by selectively modi- variety of organs in the human body, including chronic airway fying a weaker binding site. This mechanism of action can serve infection and obstruction, pancreatic insufficiency, and intestinal as a model for the development of new trafficking corrector obstruction (4). Although advances in diagnosis and therapies molecules to treat cystic fibrosis. have significantly improved the life quality and life expectancy of CF patients, the median life expectancy in the United States is Author contributions: L.M.S., L.G.M., D.Y.T., L.B., and C.O. designed research; L.M.S., still below 40 y (38.7 for males and 36.0 for females) (5). C.V.L., S.F.R.L., F.A.M., D.S.v.S., R.M.J.M.d.V., G.W.C., and L.G.M. performed research; More than 1,900 different CFTR mutations have been iden- L.M.S., L.G.M., D.Y.T., L.B., and C.O. analyzed data; and L.M.S., L.G.M., D.Y.T., L.B., and tified to cause CF (6). These mutations can be divided into C.O. wrote the paper. different classes according to the mechanism by which they dis- The authors declare no conflict of interest. rupt CFTR expression (7). Most patients suffer from a class II This article is a PNAS Direct Submission. mutation, including the deletion of a phenylalanine residue at Data deposition: The crystallography, atomic coordinates, and structure factors reported ∼ in this paper have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes position 508, F508del, which is present in 70% of the CF alleles 5D2D, 5D3E, and 5D3F). in patients in Europe, North America, and Australia (8). In cells 1To whom correspondence should be addressed. Email: [email protected]. containing class II mutations, transcription and translation of the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. CFTR protein is successful, but the protein is only partly gly- 1073/pnas.1516631113/-/DCSupplemental. E1152–E1161 | PNAS | Published online February 17, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1516631113 Downloaded by guest on September 26, 2021 PNAS PLUS Fig. 1. Schematic overview of the CFTR protein domains, with a close-up of the intrinsically disordered regulatory domain (RD). (A) Location of the nine phosphorylated 14-3-3 binding sites in the intrinsically disordered R domain of CFTR. NBD, nucleotide-binding domain; TMD, transmembrane domain. (B)Amino acid sequence of the nine singly phosphorylated 14-3-3 binding sites in the CFTR R domain. (C) Amino acid sequence of the doubly phosphorylated peptides used for this study, representing segments of the CFTR R domain. (15, 16). This makes the binding between CFTR and 14-3-3 a highly (FC-A) to stabilize the 14-3-3–CFTR interaction as a basis for the interesting protein–protein interaction (PPI). design of novel therapeutics for CF. The intrinsically disordered R domain of the CFTR protein bears nine different motifs that could, upon phosphorylation, Results bind to 14-3-3 (Fig. 1 A and B) (13, 17). Additionally, 14-3-3 Binding Affinities of the Interaction Between CFTR R-Domain Peptides BIOCHEMISTRY proteins naturally form dimers, which contain two adjacent am- and 14-3-3β. The separate binding motifs in the R domain were phipathic binding grooves capable of simultaneously binding one investigated to resolve the binding mechanism between the in- or more protein partners (18, 19). The numerous binding pos- trinsically disordered CFTR R domain and the 14-3-3 protein. sibilities hereby arising make the interaction between 14-3-3 and Peptides corresponding to the nine possible 14-3-3 binding mo- CFTR an exceptionally complex 14-3-3 PPI. To investigate how tifs were synthesized (Fig. 1B), with the phosphorylated serine in this interaction actually occurs, Liang et al. (13) estimated the the center surrounded by six amino acids of the natural sequence occupancy of the bound state of these different binding motifs to on each side. After N-terminal labeling with fluorescein isothio- 14-3-3β. Furthermore, Bozoky et al. (20) constructed several cyanate (FITC), a fluorescence polarization (FP) assay was models for this interaction based on the chemical shift of the R performed to measure the binding of these labeled peptides to domain upon binding to 14-3-3. However, only minimal quanti- 14-3-3β. Remarkably, none of the nine peptides of 14-3-3 had tative data on the binding interaction of these nine different sufficient affinity to allow determination of the association con- motifs have yet been published, and the role of the multivalency stants (Fig. 2A). However, the results from the assays showed the of the interaction remains unclear. pS768 peptide to be the strongest binder. The increased trafficking to the plasma membrane of CFTR Because a 14-3-3 dimer contains two binding grooves that are by binding to 14-3-3 might offer a therapeutic approach for the known to simultaneously bind two epitope sites on the same treatment of CF.