Structural Analysis of 14-3-3 Phosphopeptide Complexes Identifies a Dual Role for the Nuclear Export Signal of 14-3-3 in Ligand Binding

Abstract

We have solved the high-resolution X-ray structure of 14-3-3 bound to two different phosphoserine peptides, representing alternative substrate-binding motifs. These structures reveal an evolutionarily conserved network of peptide-protein interactions within all 14-3-3 isotypes, explain both binding motifs, and identify a novel intrachain phosphorylation-mediated loop structure in one of the peptides. A 14-3-3 mutation disrupting Raf signaling alters the ligand-binding cleft, selecting a different phosphopeptide-binding motif and different substrates than the wild-type protein. Many 14-3-3: peptide contacts involve a C-terminal amphipathic alpha helix containing a putative nuclear export signal, implicating this segment in both ligand and Crm1 binding. Structural homology between the 14-3-3 NES structure and those within I kappa B alpha and p53 reveals a conserved topology recognized by the Crm1 nuclear export machinery.

Fluorescent Amino Acids: Modular Building Blocks for the Assembly of New Tools for Chemical Biology

“…The residue at the (-)2 position was shown to be buried inside a hydrophobic pocket on binding the target protein by X-ray crystallography. 32 peptide-bound HLA-DR protein was also determined, confirming the occupation of the 4-DAPA side chain in the expected hydrophobic pocket. 38 These phthalimide and naphthalimide-based FlAAs have also been incorporated into peptides recognizing PDZ and SH2 domains revealing their versatility in diverse applications.…”

Significance of 14-3-3 Self-Dimerization for Phosphorylation-dependent Target Binding

“…The dimeric nature of the 14-3-3 proteins was established soon with their discovery through biochemical means (Boston et al, 1982) and confirmed by crystallographic analysis (Liu et al, 1995;Xiao et al, 1995). The structure of the 14-3-3 protein crystal showed the existence of a binding groove in each 14-3-3 half-dimer and cocrystallization of 14-3-3 with short synthetic phosphopeptides showed the ability of each part of the dimer to bind a phosphopeptide independently (Yaffe et al, 1997;Rittinger et al, 1999). These findings suggested that the ability of 14-3-3 to bind its target proteins should be independent of 14-3-3 dimerization.…”

“…Crystal structure analyses of 14-3-3, either alone or with bound peptides, indicate that each 14-3-3 half dimer can bind a target peptide independently (Yaffe et al, 1997;Rittinger et al, 1999). Combined, these findings suggest that dimerization may not be necessary for 14-3-3 to bind its phosphorylated targets.…”

The crystal structure of the non-liganded 14-3-3σ protein: insights into determinants of isoform specific ligand binding and dimerization

“…The structure was determined by molecular replacement using the Molrep program from the CCP4 suite [25]. The structure of the 14-3-3ζ isoform taken from the PDB entry 1QJB [26] was used as a probe structure after removing the phosphopeptide part. The initial R-factor of the model was 0.46.…”