Electrostatic interactions play a minor role in the binding of ExoS to 14-3-3 proteins.
14-3-3 proteins belong to a family of conserved molecules expressed in all eukaryotic cells that play an important role in a multitude of signalling pathways. 14-3-3 proteins bind either to phosphoserine/phosphothreonine residues or to sequence-specific non-phosphorylated motifs in more than 200 interaction partners [Pozuelo Rubio, Geraghty, Wong, Wood, Campbell, Morrice and Mackintosh (2004) Biochem. J. 379, 395-408]. These interactions result in cell-cycle regulation, apoptosis, stress responses, cell metabolism and malignant transformation. One example of a phosphorylation-independent interaction is the binding of 14-3-3 to ExoS (exoenzyme S), a bacterial ADP-ribosyltransferase toxin of Pseudomonas aeruginosa. In the present study, we have utilized additional biochemical and infection analyses to define further the structural basis of the interaction between ExoS and 14-3-3. An ExoS leucine-substitution mutant dramatically reduced the interaction potential with 14-3-3 suggesting that Leu422, Leu423, Leu426 and Leu428 of ExoS are important for its interaction with 14-3-3, its enzymatic activity and cytotoxicity. However, ExoS substitution mutants of residues that interact with 14-3-3 through an electrostatic interaction, such as Ser416, His418, Asp424 and Asp427, showed no reduction in their interaction potential with 14-3-3. These ExoS substitution mutants were also as aggressive as wild-type ExoS at inducing cell death and to modify endogenous ExoS target within the cell. In conclusion, electrostatic interaction between ExoS and 14-3-3 via polar residues (Ser416, His418, Asp424 and Asp427) appears to be of secondary importance. Thus the interaction between the 'roof' of the groove of 14-3-3 and ExoS relies more on hydrophobic interaction forces, which probably contributes to induce cell death after ExoS infection and activation.
Yasmin
Lubna
L
Department of Molecular Biology, Umeå University, Sweden.
Veesenmeyer
Jeffrey L
JL
Diaz
Maureen H
MH
Francis
Matthew S
MS
Ottmann
Christian
C
Palmer
Ruth H
RH
Hauser
Alan R
AR
Hallberg
Bengt
B
D048948:14-3-3 Proteins / Q000737:chemistry / Q000378:metabolism*; D036002:ADP Ribose Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016923:Cell Death; D005260:Female; D006367:HeLa Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D051379:Mice; D008807:Mice, Inbred BALB C; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000737:chemistry*; D055672:Static Electricity
D048948:14-3-3 Proteins; D001427:Bacterial Toxins; D036002:ADP Ribose Transferases; C444820:exoenzyme S