title: 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.
authors.affiliation: Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands; Institute for Complex Molecular Systems, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands;
authors.forename: Loes M
authors.initials: LM
authors.lastname: Stevers
authors.affiliation: Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands; Institute for Complex Molecular Systems, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands;
authors.forename: Chan V
authors.initials: CV
authors.lastname: Lam
authors.affiliation: Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands; Institute for Complex Molecular Systems, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands;
authors.forename: Seppe F R
authors.initials: SF
authors.lastname: Leysen
authors.affiliation: Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands; Institute for Complex Molecular Systems, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands;
authors.forename: Femke A
authors.initials: FA
authors.lastname: Meijer
authors.affiliation: Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands; Institute for Complex Molecular Systems, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands;
authors.forename: Daphne S
authors.initials: DS
authors.lastname: van Scheppingen
authors.affiliation: Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands; Institute for Complex Molecular Systems, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands;
authors.forename: Rens M J M
authors.initials: RM
authors.lastname: de Vries
authors.affiliation: Cystic Fibrosis Translational Research Centre, Department of Biochemistry, McGill University, Montreal, QC, Canada, H3G 1Y6;
authors.forename: Graeme W
authors.initials: GW
authors.lastname: Carlile
authors.affiliation: Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands; Institute for Complex Molecular Systems, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands;
authors.forename: Lech G
authors.initials: LG
authors.lastname: Milroy
authors.affiliation: Cystic Fibrosis Translational Research Centre, Department of Biochemistry, McGill University, Montreal, QC, Canada, H3G 1Y6;
authors.forename: David Y
authors.initials: DY
authors.lastname: Thomas
authors.affiliation: Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands; Institute for Complex Molecular Systems, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands;
authors.forename: Luc
authors.initials: L
authors.lastname: Brunsveld
authors.affiliation: Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands; Institute for Complex Molecular Systems, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands; Department of Chemistry, University of Duisburg-Essen, 45141 Essen, Germany c.ottmann@tue.nl.
authors.forename: Christian
authors.initials: C
authors.lastname: Ottmann
mesh_terms: D048948:14-3-3 Proteins / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002151:Calorimetry; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data
chemical_list: D048948:14-3-3 Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator
keywords: disordered protein; multivalency; protein–protein interaction