Residues of 14-3-3ζ Required for Activation of Exoenzyme S of Pseudomonas aeruginosa

• Published in: Biochemistry (Easton) 1999-09, Vol.38 (37), p.12159-12164
• Main Authors: Zhang, Lixin, Wang, Haining, Masters, Shane C, Wang, Bingcheng, Barbieri, Joseph T, Fu, Haian
• Format: Article
• Language: English
• Subjects: 14-3-3 protein; 14-3-3 Proteins; ADP Ribose Transferases - metabolism; Bacterial Toxins; Binding Sites - genetics; Conserved Sequence; Enzyme Activation - genetics; exoenzyme S; Mutagenesis, Site-Directed; Poly(ADP-ribose) Polymerases - metabolism; Protein Isoforms - chemistry; Protein Isoforms - genetics; Protein Isoforms - metabolism; Protein Isoforms - physiology; Proteins - chemistry; Proteins - genetics; Proteins - metabolism; Proteins - physiology; Pseudomonas aeruginosa; Pseudomonas aeruginosa - enzymology; Pseudomonas aeruginosa - genetics; Structure-Activity Relationship; Tyrosine 3-Monooxygenase; Valine - genetics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi991019l

Exoenzyme S (ExoS) is a mono-ADP-ribosyltransferase secreted by the opportunistic pathogen Pseudomonas aeruginosa. ExoS requires a eukaryotic factor, the 14-3-3 protein, for enzymatic activity. Here, two aspects of the activation 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 β, ζ, η, σ, and τ, activated ExoS with similar efficiency. This implicates a conserved structure in 14-3-3 that contributes to the interaction between 14-3-3 and ExoS. One candidate structure is the conserved amphipathic groove that mediates the 14-3-3/Raf-1 interaction. The next series of experiments examined the role of individual amino acids of the amphipathic groove of 14-3-3ζ in ExoS activation and showed that ExoS activation required the basic residues lining the amphipathic groove of 14-3-3ζ without extensive involvement of the hydrophobic residues. Strikingly, mutations of Val-176 of 14-3-3ζ that disrupted its interaction with Raf-1 did not affect the binding and activation of ExoS by 14-3-3. Thus, ExoS selectively employs residues in the Raf-binding groove for its association with 14-3-3 proteins.