Residues of 14-3-3 zeta required for activation of exoenzyme S of Pseudomonas aeruginosa

Exoenzyme S (ExoS) is a mono-ADP-ribosyltransferase secreted by the opportu nistic pathogen Pseudomonas aeruginosa. ExoS requires a eukaryotic factor, the 14-3-3 protein, for enzymatic activity. Here, two aspects of the activa tion of the ADP-ribosyltransferase activity of ExoS by 14-3-3 proteins are examined. Initial studies showed that several isoforms of 14-3-3, including beta, zeta, eta, sigma, and tau, activated ExoS with similar efficiency. T his implicates a conserved structure in 14-3-3 that contributes to the inte raction between 14-3-3 and ExoS. One candidate structure is the conserved a mphipathic groove that mediates the 14-3-3/Raf-1 interaction. The next seri es of experiments examined the role of individual amino acids of the amphip athic groove of 14-3-3 zeta in ExoS activation and showed that ExoS activat ion required the basic residues lining the amphipathic groove of 14-3-3 zet a without extensive involvement of the hydrophobic residues. Strikingly, mu tations of Val-176 of 14-3-3 zeta that disrupted its interaction with Raf-1 did not affect the binding and activation of ExoS by 14-3-3. Thus, ExoS se lectively employs residues in the Raf-binding groove for its association wi th 14-3-3 proteins.