ATP-Driven Allosteric Regulation of 14-3-3: Positive Modulation of ATP Hydrolysis and Negative Regulation of Peptide Binding.

Many 14-3-3 paralogs, except sigma, could bind and hydrolyze ATP. However, the catalytic residues and the significance of ATP binding or hydrolysis remain unknown. Here we confirm that there are two binding pockets for ATP, one at the peptide binding amphipathic pocket and the other at the dimer interface. As predicted by a new computational method, CLICK, and by limited proteolysis coupled to mass spectroscopy, we identify E131 and E180 as the catalytic residues. We further confirm that ATP hydrolysis is an inherent property of 14-3-3, and mutations result in either gain or loss of ATPase activity. The dimeric fold of the protein is mandatory for ATP hydrolysis but not for peptide binding. While ATP at the dimer interface acts as an allosteric activator of ATP hydrolysis, it acts as a selective negative regulator of a nonphosphopeptide, originating from ExoS, a pathogenic Pseudomonas protein. This study for the first time, unveils the hidden allosteric properties of the 14-3-3 proteins and its role in excluding specific ligands of disease relevance.