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

Proc Natl Acad Sci U S A. 2016 Mar 1;113(9):E1152-61. doi: 10.1073/pnas.1516631113. Epub 2016 Feb 17.

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.

Keywords: disordered protein; multivalency; protein–protein interaction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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

Substances

  • 14-3-3 Proteins
  • Cystic Fibrosis Transmembrane Conductance Regulator

Associated data

  • PDB/5D2D
  • PDB/5D3E
  • PDB/5D3F