Structure of 14-3-3ζ complexed with ExoS peptides. (A) Structure of the 14-3-3ζ dimer in complex with a 15-amino-acid (in 14-3-3ζ A, residues 416–430) or a 10-amino-acid (in 14-3-3ζ B, residues 421–430) peptide of ExoS. The 14-3-3 dimer is shown as a blue ribbon and the ExoS peptides are shown as stick models, with carbon, oxygen and nitrogen atoms coloured in yellow, red and blue, respectively. (B) Stereo view of monomer A of 14-3-3ζ complexed to 15 amino acids of ExoS (416–430). Colour coding is as in panel A. An omit electron density map of the ExoS peptide contoured at 3σ is shown in red.

Residues 416–430 of ExoS binding to 14-3-3ζ. (A) Binding of ExoS (416–430) to 14-3-3ζ. The 14-3-3 protein is shown as blue surface with residues contacting ExoS leucines 422, 423, 426 and 428 coloured in green. ExoS (416–430) is shown as sticks with carbon, nitrogen and oxygen atoms coloured in yellow, blue and red, respectively. Important residues of 14-3-3ζ contacting ExoS are labelled white and the corresponding residues in ExoS are labelled yellow. Leucines 422, 423, 426 and 428 of ExoS constitute an extensive hydrophobic interaction surface with the nonpolar ‘roof' of the 14-3-3 binding groove consisting of residues Pro165, Ile166, Leu172, Leu216, Ile217 and Leu220. (B) Schematic representation of the interface between 14-3-3ζ and ExoS (418–429). Residues from ExoS are shown as sticks coloured as in panel A. Residues from 14-3-3 are represented as blue-filled squares. Arrows indicate polar interactions.

Role of the phosphopeptide coordinating basic pocket of 14-3-3 in ExoS binding. (A) 2F_o−F_c electron density map contoured at 2σ showing the basic pocket of 14-3-3ζ and neighbouring 14-3-3 residues as well as the ExoS peptides and several coordinated water molecules. (B) Superimposition of the mode I consensus 14-3-3 binding peptide (magenta carbons) (PDB code 1QJB) and ExoS (yellow carbons) and selected residues from 14-3-3ζ, in complex with either mode 1 (green carbons) or ExoS (blue carbons). Electrostatic interactions of less than 3.5 Å are shown as dotted lines. The positions of Asp424 and Asp427 are too remote from the basic pocket of 14-3-3ζ (Arg56, Arg127, Lys49 and Tyr128) to mimic the binding of the phosphorylated amino acid of the mode I consensus peptide. In addition, the triangular cluster of Lys49, Arg56 and Arg127 is disrupted by a significant side-chain movement of Lys49 in the ExoS complex structure.

Comparison of binding of ExoS and serotonin-N-acetyl transferase (AANAT) to 14-3-3. (A) The ExoS peptide is coordinated by 14-3-3ζ in an opposite orientation relative to AANAT. AANAT and also all other structurally known 14-3-3 interactors are coordinated in an N- to C-terminal direction starting from the phosphoresidue binding basic pocket to the exit point of the groove. In contrast, ExoS is coordinated from the C- to N-terminus starting from the equivalent position in the basic pocket. Both peptides are coloured in rainbow (blue, N-terminus; red, C-terminus). For clarity, only the main-chain atoms are shown. (B) Comparison of the exit/entry points of ExoS and the AANAT peptide. Both peptides are bound in elongated conformation. With AANAT, the proline at position +2 after the phosphorylated threonine serves the exit of the peptide from the 14-3-3 groove. In contrast, the ExoS peptide maintains intimate contact to the 14-3-3 binding groove before exiting the 14-3-3 dimer.

Cytotoxicity and host cell modification following infection of mammalian cells with ExoS variants. (A) Viability of HeLa cells 24 h after intoxication with ExoS variants. HeLa cells were infected for 2 h in the presence of 0.1% arabinose with Yersinia (YPIII) carrying plasmids expressing different variants of ExoS. Lanes 1 and 2, uninfected cells; lane 3, YPIII(pMF384) expressing wild-type ExoS(wt); lane 4, YPIII(pMF516) expressing ExoS(LDL426-428AAA); lane 5, YPIII(pMF697) expressing ExoS(L422A); lane 6, YPIII(pMF662) expressing ExoS(L423A); lane 7, YPIII(pMF582) expressing ExoS(L426A); lane 8, YPIII(pMF583) expressing ExoS(L428A), lane 9, YPIII(pMF523) expressing ExoS(D424A:D427A). Both loose and semi-detached cytotoxic cells were washed free from bacteria and transferred to a new Petri dish and incubated overnight with medium containing gentamicin. A Trypan blue exclusion assay was performed 24 h after infection to quantitate the percentage of dead cells. Error bars represent s.e.m.±. (B) Viability of J774 cells 24 h after intoxication with ExoS variants. J774 cells were infected with Yersinia (YPIII) expressing variants of ExoS as described in panel A. (C) ExoS secretion and (D) expression after each Yersinia strain was induced in the presence of arabinose and analysed using anti-ExoS antibodies. (E–I) ExoS-mediated ADP-ribosylation of HeLa cell proteins following infection and, before harvest, stimulation with EGF (50 ng/ml) for 5 min. HeLa cells were lysed and Western blot analysis was performed on immunoblotted filters with anti-RAS (D), anti-RAP1 (F), anti-phospho-ERK1/2 (G), anti-phospho-PKB/Akt (H) and anti-pan-PKB/Akt (I) antibodies.

Virulence of ExoS containing substitutions predicted to affect binding to 14-3-3, as measured in a mouse model of acute pneumonia. (A) Bacterial load in the lungs of mice infected with P. aeruginosa secreting ExoS variants. Each symbol represents the bacterial load in the lung of an individual animal at 18 h post-infection. Bars represent the median values. Bacterial loads associated with infection with PA99null+S(LDL426-428AAA) differed significantly from those associated with PA99null+S (P<0.05, Student's t-test). (B) Survival of mice infected with P. aeruginosa secreting ExoS variants. Data points represent the number of animals surviving in each experimental group over time (n=10 mice per strain, pooled from two separate experiments). Survival of mice infected with PA99null+S(LDL426-428AAA) differed significantly from that of mice infected with PA99null+S (P<0.0001 log-rank test for equality of survivor functions).