Structural analysis of 14-3-3 phosphopeptide complexes identifies a dual role for the nuclear export signal of 14-3-3 in ligand binding

We have solved the high-resolution X-ray structure of 14-3-3 bound to two d ifferent phosphoserine peptides, representing alternative substrate-binding motifs. These structures reveal an evolutionarily conserved network of pep tide-protein interactions within all 14-3-3 isotypes, explain both binding motifs, and identify a novel intrachain phosphorylation-mediated loop struc ture in one of the peptides. A 14-3-3 mutation disrupting Raf signaling alt ers the ligand-binding cleft, selecting a different phosphopeptide-binding motif and different substrates than the wild-type protein. Many 14-3-3: pep tide contacts involve a C-terminal amphipathic alpha helix containing a put ative nuclear export signal, implicating this segment in both ligand and Cr m1 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 t he Crm1 nuclear export machinery.