14-3-3-ZETA BINDS A PHOSPHORYLATED RAF PEPTIDE AND AN UNPHOSPHORYLATED PEPTIDE VIA ITS CONSERVED AMPHIPATHIC GROOVE

14-3-3 proteins bind a variety of molecules involved in signal transdu ction, cell cycle regulation and apoptosis. 14-3-3 binds ligands such as Raf-1 kinase and Bad by recognizing the phosphorylated consensus mo tif, RSX-pSXP, but must bind unphosphorylated ligands, such as glycopr otein Ib and Pseudomonas aeruginosa exoenzyme S, via a different motif . Here we report the crystal structures of the zeta isoform of 14-3-3 in complex with two peptide ligands: a Raf-derived phosphopeptide (pS- Raf-259, LSQRQRSTpSTPNVHMV) and an unphosphorylated peptide derived fr om phage display (R18, PH-CVPRDLSWLDLEANMCLP) that inhibits binding of exoenzyme S and Raf-1. The two peptides bind within a conserved amphi pathic groove on the surface of 14-3-3 at overlapping but distinct sit es. The phosphoserine of pS-Raf-259 engages a cluster of basic residue s (Lys(49) Arg(56), Arg(60), and Arf(127)), whereas R18 binds via the amphipathic sequence, WLDLE, with its two acidic groups coordinating t he same basic cluster. 14-3-3 is dimeric, and its two peptide-binding grooves are arranged in an antiparallel fashion, 30 Angstrom apart. Th e ability of each groove to bind different peptide motifs suggests how 14-3-3 can act in signal transduction by inducing either homodimer or heterodimer formation in its target proteins.