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

L.M. Stevers, Chan V. Lam, S.F.R. Leysen, F.A. Meijer, D.S. van Scheppingen, R.M.J.M. de Vries, G.W. Carlile, L.G. Milroy, D.Y. Thomas, L. Brunsveld, Chr. Ottmann

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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.

Original languageEnglish
Pages (from-to)E1152-E1161
Number of pages10
JournalProceedings of the National Academy of Sciences of the United States of America (PNAS)
Volume113
Issue number9
DOIs
Publication statusPublished - 1 Mar 2016

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Keywords

  • protein-protein interaction
  • disordered protein
  • multivalency

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