Structural Analysis of the 14-3-3ζ/Chibby Interaction Involved in Wnt/β-Catenin Signaling

Abstract

The partially disordered Chibby (Cby) is a conserved nuclear protein that antagonizes the Wnt/β-catenin signaling pathway. By competing with the Tcf/Lef family proteins for binding to β-catenin, Cby abrogates the β-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 β-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 14-3-3ζ/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 14-3-3ζ. 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.

Fig 1

(A) Crystal structure of the 14-3-3ζ /Cby 18-mer complex. Each 14-3-3ζ monomer (coloured orange and blue) is bound to one Cby phosphopeptide. (B) Final 2F _o − F _c electron density of 14-3-3ζ (blue mesh) and Cby 18-mer (green mesh) contoured to 1σ. (C) Polar contacts (black hashed lines) between 14-3-3ζ residues (light blue sticks) and the Cby 18-mer (green sticks).

Fig 2. Structural comparison of 14-3-3ζ-bound Cby with other 14-3-3 binding motifs comprising various +2 residues.

Residues K49 and R56 are coloured yellow on the white surface representation of 14-3-3ζ. Structural and sequence alignment of Cby (green sticks) with the binding-motifs of (A). Raf1 (pink sticks, PDB: 3CU8), (B) PKCε (orange sticks, PDB: 2WH0), (C) Histone H3 (white sticks, PDB: 2C1N), (D) β2 integrin (blue sticks, PDB: 2V7D) and (E) α 4 integrin (purple sticks. PDB: 4HKC). The Cα RMSD values were computed by subtracting a Cα distance matrix between the -2 and +1 residues of Cby and each peptide as well as the -2 and most C-terminal residue in the respective alignments.

Fig 3

(A) ¹H-¹⁵N TROSY-HSQC spectrum and backbone resonance assignment of ²H/¹³C/¹⁵N labeled 14-3-3ζΔC12. (B) Secondary structure propensity (SSP) scores for 14-3-3ζΔC12. SSP scores were calculated based upon the ¹³Cα/β chemical shifts.

Fig 4. NMR titration experiments of 14-3-3ζΔC12 with Cby peptides.

(A) ¹H-¹⁵N TROSY-HSQC spectra of 14-3-3ζΔC12 alone (black) and with 3 molar equivalents of the Cby 7-mer (red) and Cby 18-mer (blue). (B) ¹H-¹⁵N TROSY-HSQC spectra of 14-3-3ζΔC12 alone (black) and with 3 molar equivalents of the WT Cby 18-mer (blue) and Cby S22P 18-mer (blue).

Fig 5. The mapping of chemical shifts on the crystal structure of the 14-3-3ζ /Cby complex.

Due to the crowding of some peaks, the chemical shifts of some residues could not be confidently traced and were excluded from the analysis. (A) (Above) A monomer of the 14-3-3ζ /Cby crystal structure interface based on Cby residues (¹⁸SApSLSNLH²⁵). Residues R56, R127 and Y128 which contact the Cby phosphate group are coloured red, residues interfacing the peptide are coloured cyan, and 14-3-3ζ residues coloured magenta are found along the 14-3-3 dimer interface. (Below) The 14-3-3ζ dimer bound to Cby. (B) (Cby 7-mer), (C) (Cby 18-mer) and (D) (Cby S22P 18-mer). Residues with traceable assigned resonances are coloured on a blue—white—yellow gradient (0 ppm to 0.1 ppm) based on their combined chemical shift [Δω = ((Δδ¹H)² +(0.2*Δδ¹⁵N)²)^1/2] at a 3:1 peptide:protein ratio. Residues coloured in orange represent peaks that broadened out to disappearance upon addition of peptide. Residues R56, R127 and Y128 are coloured pink.

Fig 6. The 14-3-3ζ, Cby, β-catenin tripartite complex.

Model of dimeric 14-3-3ζ bound to two molecules of full-length Cby. The models of two full-length Cby proteins have disordered residues 30–73 of the N-terminus and 100–126 of the C-terminus presented in a highly extended fashion, and are shown forming a coiled-coil from residues 73–100. β-catenin (PDB: 2Z6H) is shown with its Cby binding-site (helix C) in red which then binds along the C-terminus (64–126) of Cby.