Electrostatic interactions play a minor role in the binding of ExoS to 14-3-3 proteins.

Abstract

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.

• Authors: Lubna Yasmin, Jeffrey L Veesenmeyer, Maureen H Diaz, Matthew S Francis, Christian Ottmann, Ruth H Palmer, Alan R Hauser, Bengt Hallberg
• Journal: The Biochemical journal (Biochem J) Vol. 427 Issue 2 Pg. 217-24 (Mar 29 2010) ISSN: 1470-8728 [Electronic] England
• PMID: 20144150 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• 14-3-3 Proteins
• Bacterial Toxins
• ADP Ribose Transferases
• exoenzyme S

Topics

• 14-3-3 Proteins (chemistry, metabolism)
• ADP Ribose Transferases (chemistry, genetics, metabolism)
• Animals
• Bacterial Toxins (chemistry, genetics, metabolism)
• Cell Death
• Female
• HeLa Cells
• Humans
• Hydrophobic and Hydrophilic Interactions
• Mice
• Mice, Inbred BALB C
• Mutagenesis, Site-Directed
• Phosphorylation
• Protein Binding
• Pseudomonas aeruginosa (chemistry)
• Static Electricity