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

Lubna Yasmin , Jeffrey L Veesenmeyer , Maureen H Diaz , Matthew S Francis , Christian Ottmann , Ruth H Palmer , Alan R Hauser , Bengt Hallberg (1)

1 Department of Molecular Biology,

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 signaling pathways. 14-3-3 proteins bind to either phosphoserine/phosphothreonine residues or to sequence-specific non-phosphorylated motifs in more than 200 interaction partners (Pozuelo Rubio, Geraghty, Wong, Wood, Campbell, Morrice, Mackintosh. 2004, Biochemical Journal, 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 exoenzyme S (ExoS), a bacterial ADP-ribosyltransferase toxin of Pseudomonas (P) aeruginosa. Here we have utilized additional biochemical and infection analyses to further define 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 ExoS Leucine residues-422, 423, 426, 428 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 Ser-416, His-418, and Asp-424 and Asp-427 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 (S416, H418, D424 and D427) 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.

Mots clés: Life Sciences

Lubna Yasmin, Jeffrey L Veesenmeyer, Maureen H Diaz, Matthew S Francis, Christian Ottmann, et al.. Electrostatic interactions play a minor role in the binding of ExoS to 14-3-3.. Biochemical Journal, 2010, 427 (2), pp.217-224. ⟨10.1042/BJ20100043⟩. ⟨hal-00479290⟩