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

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 t...

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Published in:	Proceedings of the National Academy of Sciences - PNAS Vol. 113; no. 9; p. E1152
Main Authors:	Stevers, Loes M, Lam, Chan V, Leysen, Seppe F R, Meijer, Femke A, van Scheppingen, Daphne S, de Vries, Rens M J M, Carlile, Graeme W, Milroy, Lech G, Thomas, David Y, Brunsveld, Luc, Ottmann, Christian
Format:	Journal Article
Language:	English
Published:	United States 01.03.2016
Subjects:	14-3-3 Proteins - chemistry 14-3-3 Proteins - metabolism Amino Acid Sequence Binding Sites Calorimetry Cystic Fibrosis Transmembrane Conductance Regulator - chemistry Cystic Fibrosis Transmembrane Conductance Regulator - metabolism Models, Molecular Molecular Sequence Data protein–protein interaction multivalency disordered protein
ISSN:	1091-6490, 1091-6490
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Summary:	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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1091-6490 1091-6490
DOI:	10.1073/pnas.1516631113