Structural Analysis of the 14-3-3?/Chibby Interaction Involved in Wnt/?-Catenin Signaling.

ABSTRACT: The partially disordered Chibby (Cby) is a conserved nuclear protein that antagonizes the Wnt/?-catenin signaling pathway. By competing with the Tcf/Lef family proteins for binding to ?-catenin, Cby abrogates the ?-catenin-mediated transcription of Wnt signaling genes. Additionally, upon phosphorylation on S20 by the kinase Akt, Cby forms a complex with 14-3-3 to facilitate the nuclear export of ?-catenin, which represents another crucial mechanism for the regulation of Wnt signaling. To obtain a mechanistic understanding of the 14-3-3/Cby interaction, we have extensively characterized the complex using X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and isothermal titration calorimetry (ITC). The crystal structure of the human 14-3-3?/Cby protein-peptide complex reveals a canonical binding mode; however the residue at the +2 position from the phosphorylated serine is shown to be uniquely oriented relative to other solved structures of 14-3-3 complexes. Our ITC results illustrate that although the phosphorylation of S20 is essential for Cby to recognize 14-3-3, residues flanking the phosphorylation site also contribute to the binding affinity. However, as is commonly observed in other 14-3-3/phosphopeptide crystal structures, residues of Cby flanking the 14-3-3 binding motif lack observable electron density. To obtain a more detailed binding interface, we have completed the backbone NMR resonance assignment of 14-3-3?. NMR titration experiments reveal that residues outside of the 14-3-3 conserved binding cleft, namely a flexible loop consisting of residues 203-210, are also involved in binding Cby. By using a combined X-ray and NMR approach, we have dissected the molecular basis of the 14-3-3/Cby interaction.

SUBMITTER: Killoran RC 

PROVIDER: S-EPMC4409382 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature