Phosphorylation-independent interaction between 14-3-3 and exoenzyme S: from structure to pathogenesis.
14-3-3 proteins are phosphoserine/phosphothreonine-recognizing adapter proteins that regulate the activity of a vast array of targets. There are also examples of 14-3-3 proteins binding their targets via unphosphorylated motifs. Here we present a structural and biological investigation of the phosphorylation-independent interaction between 14-3-3 and exoenzyme S (ExoS), an ADP-ribosyltransferase toxin of Pseudomonas aeruginosa. ExoS binds to 14-3-3 in a novel binding mode mostly relying on hydrophobic contacts. The 1.5 A crystal structure is supported by cytotoxicity analysis, which reveals that substitution of the corresponding hydrophobic residues significantly weakens the ability of ExoS to modify the endogenous targets RAS/RAP1 and to induce cell death. Furthermore, mutation of key residues within the ExoS binding site for 14-3-3 impairs virulence in a mouse pneumonia model. In conclusion, we show that ExoS binds 14-3-3 in a novel reversed orientation that is primarily dependent on hydrophobic residues. This interaction is phosphorylation independent and is required for the function of ExoS.
Ottmann
Christian
C
Department of Structural Biology, Max-Planck-Institute for Molecular Physiology, Dortmund, Germany.
Yasmin
Lubna
L
Weyand
Michael
M
Veesenmeyer
Jeffrey L
JL
Diaz
Maureen H
MH
Palmer
Ruth H
RH
Francis
Matthew S
MS
Hauser
Alan R
AR
Wittinghofer
Alfred
A
Hallberg
Bengt
B
D048948:14-3-3 Proteins / Q000235:genetics* / Q000378:metabolism*; D036002:ADP Ribose Transferases / Q000378:metabolism*; D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism*; D015153:Blotting, Western; D003461:Crystallography; D017931:DNA Primers; D005260:Female; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008968:Molecular Conformation; D009154:Mutation / Q000235:genetics; D011014:Pneumonia / Q000382:microbiology*; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000472:pathogenicity*; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism
D048948:14-3-3 Proteins; D001427:Bacterial Toxins; D017931:DNA Primers; D036002:ADP Ribose Transferases; C444820:exoenzyme S; D020827:rap1 GTP-Binding Proteins