14-3-3 protein C-terminal stretch occupies ligand binding groove and is displaced by phosphopeptide binding

14-3-3 proteins are important regulators of numerous cellular signaling circuits. They bind to phosphorylated protein ligands and regulate their functions by a number of different mechanisms. The C-terminal part of the 14-3-3 protein is known to be involved in the regulation of 14-3-3 binding properties. The structure of this region is unknown; however, a possible location of the C-terminal stretch within the ligand binding groove of the 14-3-3 protein has been suggested. To fully understand the role of the C-terminal stretch in the regulation of the 14-3-3 protein binding properties, we investigated the physical location of the C-terminal stretch and its changes upon the ligand binding. For this purpose, we have used Forster resonance energy transfer ( FRET) measurements and molecular dynamics simulation. FRET measurements between Trp(242) located at the end of the C-terminal stretch and a dansyl group attached at two different cysteine residues (Cys(25) or Cys(189)) indicated that in the absence of the ligand, the C-terminal stretch occupies the ligand binding groove of 14-3-3 protein. Our data also showed that phosphopeptide binding displaces the C-terminal stretch from the ligand binding groove. Intramolecular distances calculated from FRET measurements fit well with distances obtained from molecular dynamics simulation of full-length 14-3-3 zeta protein.