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 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 beta, zeta, eta, sigma, and tau, 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 14-3-3 zeta in ExoS activation and showed that ExoS activation required the basic residues lining the amphipathic groove of 14-3-3 zeta without extensive involvement of the hydrophobic residues. Strikingly, mutations 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 selectively employs residues in the Raf-binding groove for its association with 14-3-3 proteins.
Zhang
L
L
Department of Pharmacology, Graduate Program in Cell and Developmental Biology, Emory University, Atlanta, Georgia 30322, USA.
Wang
H
H
Masters
S C
SC
Wang
B
B
Barbieri
J T
JT
Fu
H
H
D048948:14-3-3 Proteins; D036002:ADP Ribose Transferases / Q000378:metabolism*; D001427:Bacterial Toxins; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D004789:Enzyme Activation / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D013329:Structure-Activity Relationship; D014446:Tyrosine 3-Monooxygenase; D014633:Valine / Q000235:genetics
D048948:14-3-3 Proteins; D001427:Bacterial Toxins; D020033:Protein Isoforms; D011506:Proteins; D014446:Tyrosine 3-Monooxygenase; D036002:ADP Ribose Transferases; D011065:Poly(ADP-ribose) Polymerases; C444820:exoenzyme S; D014633:Valine