R18 peptide is a non-phosphorylated ligand of 14-3-3 that was originally isolated from a phage display screen, and the peptide sequences revealed a tetrapeptide motif, WLDL, located in the middle of the R18 peptide. This peptide exhibits a high affinity for many isoforms of 14-3-3 with estimated Kd values of 70-90 nM, similar to those of the 14-3-3 binding phosphopeptides. Recognition of multiple isoforms of 14-3-3 suggests the targeting of R18 to a structure that is common among 14-3-3 proteins, such as the conserved ligand-binding groove.
R18 is a peptide selected from a phage display library for its high affinity for 14-3-3 proteins. It effectively blocked the binding of 14-3-3 to the kinase Raf-1, a physiological ligand of 14-3-3, and effectively abolished the protective role of 14-3-3 against phosphatase-induced inactivation of Raf-1. In the 14-3-3ζ - R18 complex, R18 assumes an extended conformation in the amphipathic groove, similar to phosphorylated peptides. Its core pentapeptide, WLDLE sequence is located in the phosphoserine binding site with its two acidic residues, Asp and Glu, next to the basic cluster of 14-3-3. Two Leu of R18 interacts with amino acids on the hydrophobic side of the 14-3-3 groove, including L172 and L220. Thus, R18 assumes a true amphipathic structure.
The binding of R18 to 14-3-3 appears to be specific because R18 recognizes only 14-3-3 proteins in total cell lysates despite the presence of many other proteins. Functionally, the R18 peptide can abolish the association of 14-3-3 with both phosphorylated and unphosphorylated protein ligands, including Raf-1, ASK1, and ExoS. The potent inhibitory effect of R18 on 14-3-3-ligand interactions can be explained by the localization of R18 in the conserved amphipathic groove of 14-3-3. Thus, R18 is likely able to block the interaction of 14-3-3 with most or all of its ligands and may serve as a general antagonist of 14-3-3 proteins. R18 represents a potentially useful tool for the dissection of the biological function of 14-3-3.
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