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. Stabilization of the interaction between 14-3-3 target protein partner, a series of diphosphorylated CFTR pep- and the mutated CFTR, for example through binding of a small tides were synthesized to study a potential dual-binding mecha- molecule, could improve the trafficking of CFTR to the cell nism for CFTR. These peptides each consisted of two adjacent surface and hence increase chloride transport over the plasma binding motifs, including the native interconnecting peptide se- membrane. The design of such therapeutically useful stabilizing quences and an additional six amino acid residues at both the N molecules could be facilitated by solving the complex structure of and C termini (Fig. 1C). The peptides were N-terminally labeled the 14-3-3–CFTR interaction. In this study, we disentangle this with FITC, and results from an FP assay showed that most of complex but important PPI on a chemical biology and structural them bound to 14-3-3β with significantly stronger affinity than biology basis. Here, we use the 14-3-3 tool compound fusicoccin-A the singly phosphorylated peptides (Fig. 2B and Fig. S1A), with

Stevers et al. PNAS | Published online February 17, 2016 | E1153 Downloaded by guest on September 26, 2021 Fig. 2. Binding between peptides representing segments of the CFTR R domain and 14-3-3β.(A) FP assay of nine singly phosphorylated FITC-labeled peptides (sequences are depicted in Fig. 1B) with 14-3-3β. Background polarization was subtracted from all values. Mean of three experiments; SD error bars are smaller than the data point symbols. pS753 contained five additional C-terminal amino acids, based on the natural sequence, to aid with solubility (asterisk). (B)FP assay of eight doubly phosphorylated FITC-labeled peptides representing segments of the CFTR R domain (sequences are depicted in Fig. 1C). Background polarization was subtracted from all values. Mean of three experiments; SD error bars are smaller than the data point symbols. (C–E) ITC results of the binding of CFTR_R6, CFTR_R7, and CFTR_R8 to 14-3-3β. Experiments were performed in triplicate, as shown in Fig. S2.

Kd values typically in the low micromolar range. The strongest 14-3-3 protomer to be close to 0.5 (Fig. S2D). This suggests that binder in this assay was CFTR_R7, which features the pS768 and one peptide containing two phosphorylated binding motifs is pS795 epitopes, followed by CFTR_R6, featuring pS753 and pS768. binding to one 14-3-3 dimer. Interestingly, both of these peptides contain the pS768 binding The intrinsically disordered nature of the CFTR R domain motif, which by itself showed the strongest individual binding to could potentially also allow the R domain to bind with two non- 14-3-3β. However, peptides not containing the pS768 binding adjacent binding motifs to one 14-3-3 dimer. Therefore, peptide motif also showed a stronger affinity for 14-3-3β than the singly probes featuring two nonadjacent binding motifs, the pS768 motif phosphorylated peptides, especially CFTR_R8, which features and a more distant motif, were synthesized. These two motifs were pS795 and pS813. During these experiments, one of the physi- connected by a glycine linker to reduce the peptide length and cally most relevant isoforms, 14-3-3β, was used. However, the overcome synthesis problems while mimicking the flexibility of other six isoforms were also tested to investigate the isoform the R domain (CFTR_R9 and CFTR_R10). ITC measurements dependence of the CFTR R-domain peptides. Hence, FP assays of these two peptides showed binding to 14-3-3β comparable to of peptides CFTR_R1–CFTR_R8 were performed on all seven CFTR_R6 and CFTR_R7 featuring adjacent binding motifs isoforms. The results showed that in all cases 14-3-3γ binds the (Fig. S3 A–C and H). strongest, followed by η, β, ζ, e, τ,andσ, respectively (Fig. S1A). To confirm that these potential phosphorylated sites are The results from this experiment also showed that CFTR_R7 and relevant for 14-3-3 binding, the wild-type R domain and the R CFTR_R6 exhibit the strongest binding to all seven 14-3-3 isoforms. domain containing a S700A, S753A, or S768A mutation were Data from isothermal titration calorimetry (ITC) measure- coexpressed with PKA. These mutations were constructed to ments on the three strongest-binding peptides showed the same prevent phosphorylation of the mutation site to study the in- trend in binding to 14-3-3β as observed in the FP assay. The fluence of each site on the binding of the CFTR R domain to binding affinities of CFTR_R6, CFTR_R7, and CFTR_R8 for 14-3-3β. Although the coexpression with PKA resulted in only a 14-3-3β were 75.8, 24.0, and 370 μM, respectively (Fig. 2 C–E partially phosphorylated R domain (a variation of three to nine and Fig. S2 A–C). Additionally, the ITC measurements show in phosphoryl groups was attached to the R domain; Dataset S1, all cases the stoichiometry of the CFTR peptide versus the W-AA), eliminating the serine at position 768 shows a significant

E1154 | www.pnas.org/cgi/doi/10.1073/pnas.1516631113 Stevers et al. Downloaded by guest on September 26, 2021 PNAS PLUS decrease in binding affinity to 14-3-3β (56.5–125.9 μM) whereas of 200 μMFC-AfromaKd of 57.8 μMtoaKd of 8.4 μM (9.0- the other positions (700 and 753) resulted in a smaller decrease fold) (Fig. 4 B and C). In contrast, addition of FC-A did not (59.9 and 92.6 μM, respectively) (Fig. S3 D–H). result in a significant increase in the binding affinity of the 14-3-3– CFTR_R7 interaction (Fig. 4 A–C and Fig. S1B). Crystal Structure of CFTR_R6 and CFTR_R7 with 14-3-3. The doubly To determine the structural basis for the observed stabilization phosphorylated peptide CFTR_R6 was cocrystallized with 14-3-3ζ, of the 14-3-3–CFTR_R6 complex by FC-A, the compound was and the protein complex structure was solved to a resolution soaked into the crystals described above. The structure was of 2.1 Å [Protein Data Bank (PDB) ID code 5D2D] (Fig. 3). solved to a resolution of 2.74 Å (PDB ID code 5D3F) (Fig. 4 D–G). Interpretable density for 19 out of 28 amino acids of the Interpretable density was found for 18 of the 28 amino acids CFTR_R6 peptide (R751–G758 and R764–T774) was found present in the CFTR_R6 peptide (R750–L756 and K764–P774) (Fig. 3 A and B). The protein structure shows that both phos- (Fig. 4D), with FC-A present in one binding groove of the 14-3-3 phorylated binding motifs of the peptide are simultaneously dimer (Fig. 4D). The difference density map directly calculated bound to one 14-3-3 dimer. The pS768 site is located in the after molecular replacement with only 14-3-3 and the CFTR_R6 binding groove of 14-3-3 protomer A, and the pS753 site is lo- peptide as search model, but excluding FC-A, showed unam- cated in 14-3-3 protomer B (Fig. 3 A and B). The five residues of biguous, unbiased density for FC-A (Fig. S4B) in 14-3-3 proto- the flexible, connecting linker are not visible in the electron mer B containing the pS753 binding motif but not in 14-3-3 density. The phosphorylated serines are located in the basic protomer A containing the pS768 binding motif. Compared with pocket of 14-3-3, composed of Arg56 and Arg127, consistent the structure of the binary complex, the pS753 peptide motif with earlier published structures of cocrystallized phosphorylated adapts its conformation slightly to enable the FC-A molecule to peptides with 14-3-3 (Fig. 3 C–E) (21–23). Fig. 3E shows more bind between the 14-3-3 protein and the CFTR peptide (Figs. 3D details of the interaction between protomer A and the pS768 and 4F). Although there are some polar contacts between FC-A binding site. The polar contacts are visualized by dashed lines and 14-3-3 residues (Asn42, Lys122, and Asp215), no polar and are more relevant for the binding of the N-terminal part contacts are observed between FC-A in the pS753 binding motif of the peptide, whereas hydrophobic interactions, depicted as of CFTR_R6. In essence, 14-3-3 and the peptide jointly form a semitransparent spheres, are more relevant for accommodation hydrophobic pocket that accommodates the mostly hydrophobic of the C-terminal half of CFTR (pS768). The most prominent FC-A molecule (Fig. 4G). FP assays of CFTR_R6 with both interaction is the basic pocket in 14-3-3 composed of Arg56 and physiologically relevant 14-3-3 isoforms β and e, and 14-3-3ζ Arg127 binding pS768, stabilized by polar contacts with Tyr128. (which is used in the crystal-soaking experiment), show that FC- Further on, polar contacts can be seen of Arg766 and Arg765 of A increases the binding affinity of CFTR_R6 for all three 14-3-3 CFTR_R6 with Glu180 and Glu131 of 14-3-3 (directly or via a isoforms (Fig. S1B). water molecule). These interactions are generally responsible for The CF mutation database (www.genet.sickkids.on.ca/Home.html) the known mode I and mode II binding of 14-3-3. Additionally, contains mutations that lead to CF and could influence the binding Leu770 and Leu772 form important hydrophobic interactions of the CFTR R domain to 14-3-3. Examples are S753R, V754M, with Val46 and Phe117 of 14-3-3. The distance between G758 and R766M. To test the influence of these mutations on the and R764 (31.5 Å) is too large to be bridged by the five amino binding to 14-3-3, peptides similar to CFTR_R6 (pS753–pS768) acids connecting these two amino acids. However, this can be were synthesized that contain these mutations. After N-terminal explained by the packing of the CFTR_R6–14-3-3ζ crystal (Fig. labeling with FITC, their affinity for 14-3-3β was tested with an FP S4A). The position of the symmetry elements shows tetramer assay (Fig. S3I) showing that these three peptides all bind less formation of the 14-3-3 proteins. The distance between the C strongly to 14-3-3β than the CFTR_R6 wild-type peptide, further terminus of the pS753 binding site and the N terminus of the hinting at the relevance of the 14-3-3–CFTR interaction for CF. pS768 binding site is 15.3 Å, which is short enough to be bridged by five amino acids. However, this tetramer formation is most Influence of FC-A on the Trafficking of F508del-CFTR. To examine the probably due to packing in the crystal lattice, because additional effects of FC-A on the trafficking of F508del-CFTR in the cell, experiments (size-exclusion chromatography, cross-linking, dy- baby hamster kidney (BHK) cells expressing 3HA-tagged BIOCHEMISTRY namic light scattering, and small-angle X-ray scattering) did not F508del-CFTR were treated for 24 h with different concentra- produce evidence for the presence of 14-3-3 tetramers in tions of FC-A. After fixation of the cells, the combination of solution. mouse monoclonal anti-HA antibody and anti-mouse IgG con- The doubly phosphorylated peptide CFTR_R7 was cocrystal- jugated with FITC was used to detect F508del-CFTR that had lized with 14-3-3γ. This protein complex structure was solved to a trafficked to the plasma membrane, as described before by resolution of 2.75 Å (PDB ID code 5D3E) (Fig. S5). Here, in- Carlile et al. (27). Increased fluorescence was detected with in- terpretable density was found for 11 of the 40 amino acid resi- creasing concentrations of FC-A, up to 54% of a positive control, dues present in the CFTR_R7 peptide (R766–L770 and K793– VX-809 [a known F508del-CFTR corrector (28)], at 100 μM P798) (Fig. S5B). The structure of 14-3-3γ–CFTR_R7 shows (Fig. 5A). that, as for the CFTR_R6 structure, both phosphorylated bind- ing motifs are simultaneously bound to the 14-3-3 dimer: pS768 Discussion is located in the binding groove of 14-3-3 protomer A, and pS795 In recent years, the amount of research done on 14-3-3 proteins is located in protomer B (Fig. S5A). has increased significantly. Each year, new protein binding part- ners for 14-3-3 are found and, with them, the number of diseases Stabilization of the 14-3-3–CFTR Interactions with Fusicoccin-A. PPIs and possible therapeutic approaches related to 14-3-3 increases involving 14-3-3 proteins have been shown to be stabilized by small (29). This brings forward a strong need to understand the different molecules (24–26). To examine the principal possibility of stabi- binding mechanisms of this regulatory hub protein. Although a lizing the 14-3-3–CFTR interaction, a number of these known great number of 14-3-3 interactions have been thoroughly quan- 14-3-3 stabilizers were tested on this interaction. The presence tified, analyzed, and structurally elucidated (22, 23, 30–32), the of 100 μMFC-A(Fig.4E)intheFPassaystabilizedthebinding interaction between CFTR and 14-3-3 remains poorly understood. of CFTR_R6 to 14-3-3, increasing the apparent affinity by a The binding of the intrinsically disordered CFTR R domain to factor of 4.0, from a Kd of 15.9 μMtoaKd of 3.98 μM(Fig.4A 14-3-3, relying on multiple-phosphorylated 14-3-3 binding motifs, and Fig. S1B). ITC measurements confirmed this with an in- employs a unique binding mechanism. There are several 14-3-3 creased binding affinity of CFTR_R6 to 14-3-3 in the presence PPIs known where proteins with multiple-phosphorylated serines

Stevers et al. PNAS | Published online February 17, 2016 | E1155 Downloaded by guest on September 26, 2021 Fig. 3. Crystal structure of the CFTR_R6 peptide (pS753–pS768) bound to a 14-3-3ζ dimer. (A) Cartoon plot with the semitransparent surface of the 14-3-3ζ dimer (green) complexed with the pS768 and pS753 binding sites of CFTR_R6 (yellow rods). (B) Top view of the pS768 and pS753 sites (yellow rods) binding to the 14-3-3ζ binding grooves (green semitransparent surface). The yellow dashed line shows amino acid residues connecting the two binding sites. (C and D)

Close-up of the binding grooves of protomers A and B (green surface) containing the CFTR_R6 binding sites (yellow rods). The final 2FO − FC electron density map of the peptide is shown (blue mesh; contoured at 1σ). (E) Polar contacts (black dashed lines) and hydrophobic interactions (semitransparent spheres) between the residues of 14-3-3ζ protomer A (green rods and semitransparent cartoon) and the pS768 binding site of CFTR_R6 (yellow rods). Water molecules are shown as red spheres. See Table S1 for data collection and refinement details.

E1156 | www.pnas.org/cgi/doi/10.1073/pnas.1516631113 Stevers et al. Downloaded by guest on September 26, 2021 and threonines interact with 14-3-3, for example LRRK2, p53, and from the natural sequence, these results provide valuable insight PNAS PLUS BAD (30, 33–36). However, the CFTR R-domain interaction with into the possibility that nonadjacent binding motifs can bind si- 14-3-3 is exceptional for the large number of interaction sites multaneously to 14-3-3. Additionally, ITC experiments performed (nine). Apart from increasing our understanding of the in- on the full R domain showed that phosphorylation of S768 is more teractions and specificity of 14-3-3 proteins, we have shown important for the binding to 14-3-3 than, for example, phos- that stabilization of this interaction might present a mecha- phorylation of S700 and S753 (Fig. S3 D–H). nism for tackling the trafficking defect of the F508del-CFTR What is the role for the multiple-phosphorylated 14-3-3 mutant protein. binding sites in the CFTR R domain? One explanation could be FP binding assays of the nine singly phosphorylated CFTR that pS768 acts as a main anchor point for the binding between peptides with 14-3-3β show that their binding to 14-3-3 is weak CFTR and 14-3-3. After the binding of one 14-3-3 protomer to (≥high micromolar range) (Fig. 2A). These individual affinities this anchor point, the local concentration of weaker binding sites are too low to explain the biological function of 14-3-3 in CFTR surrounding the other protomer increases significantly, making expression as described in a publication of Liang et al. (13), and the binding of a second protomer more likely to happen (Fig. 6). are significantly lower than the affinity of the whole phosphor- This mechanism is described by Yaffe as the “gatekeeper” model: ylated R domain to 14-3-3 published by Bozoky et al. (Kd based One dominant site functions as an initiator that must be present on Trp fluorescence measurements: 5.4 ± 1.0 μM) (20). The and phosphorylated to promote a 14-3-3 interaction, which is affinities of the doubly phosphorylated CFTR peptides to 14-3-3 then stabilized by a second interaction (38). However, the case are significantly stronger (low–middle micromolar range) (Fig. of CFTR is exceptional for the large number of binding sites, of 2B) and comparable to those of earlier published 14-3-3 in- which at least two must be phosphorylated and one has to be teraction partners (18) as well as the full-length R domain (20). pS768. In this way, a mutation to one of the eight weaker Therefore, the 14-3-3 dimer most probably binds simultaneously binding sites will not significantly affect the functionality of to two monophosphoepitope sites on the same CFTR R domain. CFTR, whereas having one single anchor point minimizes the Bozoky et al. reported a variety of different binding modes in chance of multiple 14-3-3 proteins binding to one R domain. their NMR-based model for CFTR R-domain binding to 14-3-3 Another explanation is more biological. 14-3-3 proteins are (20). Our structures of CFTR_R6 and CFTR_R7 with 14-3-3 expected to increase CFTR function in three different ways: clearly demonstrate that the phosphorylated binding sites do (i) supporting the folding of CFTR, (ii) masking the degradation bind in the amphipathic binding grooves of 14-3-3, although the recognition site, and (iii) assisting the trafficking to the plasma connecting linker in-between is not visible in the electron density membrane (13, 39). Binding of certain sites of the R domain to and therefore shows no evidence for a functional interaction with 14-3-3 could support the folding of the protein, whereas bind- 14-3-3 (Fig. 3 and Fig. S5). ing of other sites could, for example, mask or present the COPI In binding assays, 14-3-3β was used because this isoform is recognition sites. These sites are located in the CFTR R do- physically relevant in CFTR expression (13). However, for prac- main at positions 683 (KKQS) and 696 (KRKNS) (40, 41). tical reasons, 14-3-3ζ and 14-3-3γ have been used in crystallogra- Phosphorylation of the CFTR R domain significantly increases phy experiments. Although these isoforms are physiologically less its binding to 14-3-3 (Kd based on Trp fluorescence measure- relevant, the binding grooves of 14-3-3 are highly conserved ments shifts from 34.3 ± 7.4 μM to 5.4 ± 1.0 μM) (20). Therefore, among the different isoforms (37), and should therefore provide a we should keep in mind that the phosphorylation positions of the reliable impression of the binding between the R domain and R domain and the presence of certain kinases exert an important 14-3-3. Additionally, the FP assay of all eight doubly phosphorylated influence on the binding of 14-3-3 to CFTR. A prediction of peptides with the seven different isoforms showed that there are phosphorylation sites shows that different kinases are capable of no specific preferences for the different binding sites within the phosphorylating different 14-3-3 binding sites. For example, isoforms (Fig. S1). Although the different isoforms show different PKA is able to phosphorylate all nine sites, PKC is predicted to total binding affinities, they display the same trend in binding to phosphorylate only eight (all but Ser753), and AMPK is able to the different peptides. phosphorylate only five sites (Ser700, Ser737, Ser768, Ser795, The nine 14-3-3 binding sites in the R domain are all related to and Ser813) (42). This can cause different levels of phosphory- BIOCHEMISTRY the mode I (RSXpSXP) and/or mode II (RX[Y/F]XpSXP) 14-3-3 lation at different sites, varying over time, location, and cell fate. binding motifs (32), but none of the motif sequences present In this sense, the multiphosphorylatable R domain could work in CFTR conform exactly to either of these two modes. The as an integrator of different signal-transduction pathways, with R-domain binding site corresponding best to these published con- 14-3-3 acting as a “reader” element of the different phosphorylation sensus binding sites is pS768 (QARRRQpSVLNLMT) (Fig. 1B). patterns. Johnson et al. characterize this as an “AND” gate, Accordingly, pS768 scored the maximum value in the overview of where two phosphorylated sites must be engaged before the the estimated relative occupancy of the bound state of the nine 14-3-3 dimer can bind, so two inputs (phosphorylation of the R different 14-3-3 binding sites in the CFTR R domain (13). These domain by two different kinases) can lead to one output (binding observations are coherent with our experimental findings that of 14-3-3 to the CFTR R domain) (43). Furthermore, multi- indeed pS768 is the strongest individual binding site of the phosphorylation of the R domain could lead to an ultrasensitive CFTR R domain to 14-3-3. Furthermore, we expected, based on response of binding to the 14-3-3 protein. We discovered with the amino acid sequence and the estimated relative occupancy of this study that at least two phosphorylated binding sites in the R the bound state of Liang et al. (13), that pS795 would bind more domain, of which one should be pS768, are needed for the strongly to 14-3-3 than pS753. Our quantification of the binding interaction with 14-3-3. The “extra” phosphorylation sites are of CFTR_R6 (pS753–pS768) and CFTR_R7 (pS768–pS795) to perhaps not per se essential for binding to 14-3-3 but can in- 14-3-3 in both the ITC and FP assays confirms this prediction crease the sensitivity of the system, resulting in a switch-like (Fig. 2). However, there are further alternative bivalent modes of mechanism. Ferrell and Ha describe this phenomenon in an CFTR binding to 14-3-3 imaginable. To test some of the possible excellent review (44) where the Fus3–Ste5 interaction is used as combinations of two CFTR phosphosites binding to 14-3-3, an example. This interaction is important in yeast mating, where we synthesized CFTR_R9 (pS712–G5–pS768) and CFTR_R10 an ultrasensitive response is generated by multisite phosphoryla- (pS768–G5–pS813), which showed a comparable binding affinity tion, two-stage binding with one docking site, and steric hindrance for 14-3-3 as CFTR_R7 (Fig. S3). Although these peptides are (45). The results reported here provide the first indications, to our not exactly similar to the natural sequence, because both the se- knowledge, toward a potential similar ultrasensitive mechanism quence and length of the glycine linker in the peptide are different for regulation of the 14-3-3–CFTR interaction. Interestingly, the

Stevers et al. PNAS | Published online February 17, 2016 | E1157 Downloaded by guest on September 26, 2021 Fig. 4. Stabilization of the interaction between 14-3-3 and CFTR_R6 by FC-A. (A) FP assay of FITC-labeled CFTR_R6 (pS753–pS768) and CFTR_R7 (pS768–pS795) with 14-3-3β, in the absence (black) or presence (purple) of 100 μM FC-A. Background polarization was subtracted from all values. Mean of three experiments; SD error bars are smaller than the data point symbols. (B) ITC results of the binding of CFTR_R6 and CFTR_R7 to 14-3-3β, in the presence of 200 μM FC-A. The

experiment was performed in triplicate, as shown in Fig. S7.(C) Comparison of the Kd of the interactions between CFTR_R6 or CFTR_R7 and 14-3-3β in the absence or presence of 200 μM FC-A, measured by ITC. The CFTR_R6–14-3-3β interaction is stabilized by a factor of 9.0, whereas CFTR_R7–14-3-3β is stabilized by a factor of 1.6. (D) A top view of the crystal structure of the CFTR_R6 peptide (yellow rods) bound to a 14-3-3ζ dimer (green semitransparent surface), soaked with FC-A (purple spheres). The yellow dashed line shows amino acid residues connecting the two binding sites. (E) Fusicoccin-A. (F) Close-up of the

14-3-3 protomer B binding groove (green surface) containing the pS753 binding site of CFTR_R6 (yellow rods) and FC-A (purple rods). The final 2FO − FC electron density map of FC-A is shown (blue mesh; contoured at 1σ). (G) Polar contacts (black dashed lines) and hydrophobic interactions (green and yellow semitransparent surfaces) between the residues of 14-3-3ζ protomer B (green cartoon and rods with semitransparent gray/green surface), the pS753 binding site of CFTR_R6 (yellow rods with semitransparent gray/yellow surface), and FC-A (purple rods). See Table S1 for data collection and refinement details.

E1158 | www.pnas.org/cgi/doi/10.1073/pnas.1516631113 Stevers et al. Downloaded by guest on September 26, 2021 PNAS PLUS

Fig. 5. Stabilizer FC-A stimulates the trafficking of F508del-CFTR. (A) 3HA-tagged F508del-CFTR–expressing (BHK) cells were treated for 24 h with different concentrations of FC-A (positive control: F508del-CFTR corrector VX-809). After fixation, the cells were treated with mouse monoclonal anti-HA antibody and anti-mouse IgG conjugated with FITC. Fluorescence units were measured with a plate reader and background fluorescence was subtracted; n = 10; error bars represent SD. (B) Cartoon representation of a cell expressing F508del-CFTR. Stabilization of the interaction between 14-3-3 and CFTR stimulates the trafficking of the protein to the plasma membrane.

CF mutation database contains CF mutations that are present in Furthermore, a possibility is that leucine and alanine of the the 14-3-3 binding sites of the CFTR R domain. We show with an pS795 binding site, and valine and leucine of pS768, do not FP assay of the CFTR_R6 peptide containing a selection of these form the right surface topography to present to FC-A. Another mutations (S753R, V754M, and R766M) that they can decrease explanation could be that the short linker between pS753 and the binding affinity of the CFTR R domain to 14-3-3 (Fig. S3I). pS768 (14 amino acids) is “guided” toward the direction of the This indicates that interruption of this PPI could be the underlying second binding groove by FC-A, which is not essential for the mechanism in a certain mutation class of the disease. longer linker between pS768 and pS795 (26 amino acid resi-

To analyze the possibility of stabilizing the 14-3-3–CFTR in- dues). This would explain why the other doubly phosphorylated, BIOCHEMISTRY teraction, known stabilizers for 14-3-3 PPIs were tested in binding but weaker-binding, peptides containing short linker lengths, assays between 14-3-3 and the different doubly phosphorylated CFTR_R1 (pS660–pS670) and CFTR_R3 (pS700–pS712), show CFTR peptides. Addition of 200 μM FC-A resulted in a ninefold stabilization upon addition of FC-A as well (Fig. S6B), whereas increase of affinity between 14-3-3β and CFTR_R6. Interestingly, peptides with longer linker lengths and the singly phosphorylated until now, FC-A was reported to only stabilize mode III binding peptides do not show a significant increase of affinity through motifs ([pS/pT]X-COOH, where X is not Pro) (22), and yet the R addition of FC-A (Figs. S6 and S7). FP assays of CFTR_R6 domain does not contain any mode III binding sites. The structure with both physiologically relevant 14-3-3 isoforms β and e,and of 14-3-3–CFTR_R6 crystals soaked with FC-A shows that FC-A ζ, showed that there is no isoform specificity in stabilization of is only present in the binding groove containing the pS753 binding the 14-3-3–CFTR interaction (Fig. S1B). In this manner, tool site (Fig. 4D). This is possibly the reason why the FP assays and compound FC-A shows the “ligandability” of the 14-3-3–CFTR ITC measurements showed that FC-A is able to stabilize the interaction in an intermolecular specific manner. The differ- 14-3-3–CFTR_R6 (pS753–pS768) interaction but not the 14-3-3– ence between the mode I/II binding of pS753 to 14-3-3 and the CFTR_R7 (pS768–pS795) interaction (Figs. 4 A–C and 5). The mode III binding of other known 14-3-3 PPIs that can be sta- most important interactions between FC-A and CFTR_R6 are bilized with FC-A could provide the stabilization specificity the hydrophobic contacts with Val754 and Ile755, which form a needed for further drug discovery. hydrophobic pocket together with 14-3-3 residues Leu218, Carlile et al. developed a sensitive F508del-CFTR cell-based Ile219, Ile168, and Val64 (Fig. 4G). However, the pS768 and assay to detect correctors of trafficking (27). This assay showed pS795 binding motifs also display hydrophobic residues at the that FC-A increases F508del-CFTR trafficking to the plasma +1and+2 positions of the phosphorylated serine but are not membrane, up to 54% of the positive control VX-809 (at a con- stabilized by FC-A. A possible explanation why the pS768 motif centration of 100 μM; Fig. 6A), thus confirming the biological is not stabilized by FC-A is that Leu772 contains hydrophobic relevance of the 14-3-3–CFTR PPI and its small-molecule stabi- contacts with both the Phe117 of 14-3-3 and Leu770 of the lization. This makes FC-A an F508del-CFTR trafficking corrector peptide itself (Fig. 4E), which would be broken by addition of with a structurally known mechanism of action, which is rare in FC-A in this pocket, leading to a decreased binding affinity. the field.

Stevers et al. PNAS | Published online February 17, 2016 | E1159 Downloaded by guest on September 26, 2021 Fig. 6. Overview of the interaction between the intrinsically disordered CFTR R domain (blue) and the 14-3-3 dimer (gray) with two amphipathic binding grooves. Binding of the strongest binding site (pS768; red sphere) increases the local concentration of the weaker binding sites of the R domain (orange spheres), followed by a second binding event (e.g., pS712, pS753, pS795, or pS813). All interactions are individually weak. However, the combinationofthe two binding sites creates a stronger interaction. FC-A has been shown to stabilize specifically at the pS753 binding epitope.

In conclusion, we have presented a thorough and quantitative NMP) before resin cleavage. The peptides used for FP assays were labeled biophysical and structural characterization of the interaction with FITC (Sigma-Aldrich) attached to a short polyethylene glycol-based between 14-3-3 and CFTR, and provided clear evidence for a linker introduced via Fmoc-O1pen-OH (Iris Biotech) under the standard SPPS tandem mode-of-action binding event. One anchor binding motif conditions. All peptides were purified by preparative reversed-phase HPLC of CFTR (pS768) occupies one binding groove of the 14-3-3 di- with MS detection (Dataset S1). mer, and a weaker, secondary binding motif (e.g., pS712, pS753, 14-3-3 Expression. His -tagged 14-3-3 isoforms (full-length and ΔC) were pS795, or pS813) occupies the other binding groove. Additionally, 6 expressed in NiCo21(DE3) competent cells with a pPROEX HTb plasmid and we show that the 14-3-3–CFTR interaction can be specifically purified with a nickel column. The His6 tag was cleaved off using TEV pro- stabilized by targeting the secondary binding-motif interaction with tease, and a second purification was performed by size-exclusion chroma- 14-3-3 with the small-molecule stabilizer FC-A, which thus might tography. The proteins were dialyzed against FP, ITC, or crystallization buffers display a possible corrector activity in the trafficking of F508del- before use (procedures are described below). CFTR. The characterization of the 14-3-3–CFTR interaction presented in this paper is an important step in understanding Fluorescence Polarization Assay. The FITC-labeled peptides were dissolved in the purpose of multisite phosphorylation in disordered domains FP buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 0.1% Tween 20, 1 mg/mL BSA) to initiate 14-3-3 PPIs. Furthermore, it provides a promising to a final concentration of 100 nM. Dilution series of 14-3-3β were made approach for CF drug discovery. on Corning black round-bottom 384-well plates, and their polarization was measured on a Tecan Infinite F500 plate reader (excitation 485 nm, emission Materials and Methods 535 nm). During the PPI stabilization experiments, FC-A was dissolved in Reagents. Fusicoccin-A was obtained from Enzo Life Sciences. DMSO (10 mM) before addition to the peptide solution to a final concen- tration of 100 μM [final DMSO concentration 1% (vol/vol)]. Peptide Synthesis. All peptides were synthesized via Fmoc solid-phase peptide synthesis (SPPS), either manually or using an automated Intavis MultiPep Isothermal Titration Calorimetry. The ITC measurements were performed on a RSi peptide synthesizer. Rink amide AM resin (Novabiochem; 0.59 mmol/g Malvern MicroCal iTC200. The cell contained 0.2 mM 14-3-3β and the syringe loading) was used to synthesize the singly phosphorylated peptides, and contained 1.0 mM peptide, both in ITC buffer [25 mM Hepes, pH 7.4, 100 mM TentaGel R RAM resin (Rapp Polymere; 0.18 mmol/g loading) was used for NaCl, 10 mM MgCl2, 0.5 mM Tris-(2-carboxyethyl)-phosphin]. Two repetitions of the synthesis of the diphosphorylated peptides. Pseudoproline dipeptides aseriesof182-μL titrations were performed at 37 °C (reference power, 5 μCal/s; (respectively QS and AS; Novabiochem) were used for the synthesis of initial delay, 60 s; stirring speed, 750 rpm; spacing, 180 s), and the data were CFTR_R2 and CFTR_R7. The peptides used for ITC measurements and for merged with ConCat32 software (MicroCal). For the PPI stabilization experi-

crystallization studies were acetylated at the N terminus (1:1:3 Ac2O/pyridine/ ments, FC-A was dissolved in ethanol, which was evaporated before addition of

E1160 | www.pnas.org/cgi/doi/10.1073/pnas.1516631113 Stevers et al. Downloaded by guest on September 26, 2021 the protein. The final concentrations in these experiments were 0.1 mM 14-3-3β antibody anti-mouse IgG conjugated with FITC (Sigma; 1:100 dilution in PBS) was PNAS PLUS with 0.2 mM FC-A in the cell and 0.5 mM peptide in the syringe. incubated for 1 h, and the cells were washed three times with PBS and analyzed on the plate reader again. Background fluorescence was subtracted from the Crystallography. See SI Materials and Methods and Table S1 for details. signal, after which the signal was normalized to the DMSO control.

Cell Experiments. This experiment was performed as previously described by ACKNOWLEDGMENTS. We thank Rowin J. P. M. de Visser, Annelies C. Wauters, Carlile et al. (27). In brief, 3HA-tagged F508del-CFTR–expressing baby hamster and Niels M. Leijten for help with peptide synthesis and purification; Dr. Steven Weeks and the K. U. Leuven Laboratory for Biocrystallography for sharing the kidney cells were seeded in 96-well plates (Corning; half-area, black-sided, clear- PKA coexpression plasmid; and Dr. Tom F. A. de Greef for stimulating and bottom) at 15,000 cells per well. After a 24-h incubation at 37 °C, the cells were helpful discussions. We thank the team from the Max Planck Institute treated with different concentrations of FC-A or VX-809 for 24 h [final DMSO Dortmund and the staff of beamline X10SA (PXII) of the Swiss Light Source concentration 1% (vol/vol)]. The cells were fixed with 4% (vol/vol) para- Villigen for data collection. Funding was granted by the Netherlands Organi- formaldehyde, washed with PBS, and then blocked with FBS [5% (vol/vol) in PBS]. zation for Scientific Research via Gravity Program 024.001.035 and Graduate Program 2012 022.004.027, Marie Curie Action PIAPP-GA-2011-286418 14-3- Mouse monoclonal anti-HA antibody (Sigma; 1:150 dilution in PBS) was incubated 3Stabs, and the Collaborative Research Center SFB1093 of the Deutsche overnight, and after three washes with PBS the background fluorescence was Forschungsgemeinschaft (DFG). D.Y.T. is a Canada Research Chair, and his work measured on a plate reader (excitation 488 nm, emission 510 nm). The secondary was supported by the Canadian Institutes of Health Research.

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