Enterohaemorrhagic Escherichia coli exploits a tryptophan switch to hijack host f-actin assembly

Abstract

Intrinsically disordered protein (IDP)-mediated interactions are often characterized by low affinity but high specificity. These traits are essential in signaling and regulation that require reversibility. Enterohaemorrhagic Escherichia coli (EHEC) exploit this situation by commandeering host cytoskeletal signaling to stimulate actin assembly beneath bound bacteria, generating "pedestals" that promote intestinal colonization. EHEC translocates two proteins, EspF(U) and Tir, which form a complex with the host protein IRTKS. The interaction of this complex with N-WASP triggers localized actin polymerization. We show that EspF(U) is an IDP that contains a transiently α-helical N-terminus and dynamic C-terminus. Our structure shows that single EspF(U) repeat forms a high-affinity trimolecular complex with N-WASP and IRTKS. We demonstrate that bacterial and cellular ligands interact with IRTKS SH3 in a similar fashion, but the bacterial protein has evolved to outcompete cellular targets by utilizing a tryptophan switch that offers superior binding affinity enabling EHEC-induced pedestal formation.

Figure 1. Structural characterization of free EspF_U

A) Amino acid sequence of EspF_U fifth repeat (R47₅) along with N-WASP GBD and IRTKS/IRSp53 SH3 binding epitopes. B) Structural disorder prediction for R47₅ based on IUPred and Disprot algorithms. C) ¹⁵N-¹H correlation (HSQC) spectrum of ¹⁵N,¹³C labeled EspF_U R47₅, recorded at 800 MHz ¹H frequency. Narrow range of ¹H chemical shifts is a signature of disordered nature of EspF_U R47₅. D) Analysis of ¹³Cα and secondary chemical shifts in unbound EspF_U R47₅. Deviations from residue specific random coil chemical shifts are shown, which take into account the nearest neighbor effects and temperature (Kjaergaard & Poulsen, 2011). E) Values of reduced spectral density functions at three frequencies 0.87ω_H, ω_N and 0 against the primary sequence of EspF_U R47₅. Transiently populated α-helix as well as XPxXP motifs are shown above histograms. See also Figures S1 and S2.

Figure 2. Structure and dynamics ternary complex

Structure of trimolecular complex between N-WASP GBD, EspF_U R47₅ and IRTKS SH3. A) Ribbon presentation of the lowest energy conformation of the ternary complex. N-WASP GBD orange, EspF_U R47₅ green and IRTKS SH3 magenta. B) Superimposition of 20 lowest energy conformers of N-WASP GBD 212–270: EspF_U 502–521(left) and IRTKS SH3 343–400: EspF_U 527–540 (right). Same coloring as in A). C) Superimposition of GBD domains from N-WASP:EspF_U (red) and the WASP:EspF_U complexes (Cheng et al., 2008) (blue) for residues 216–262. M238, C239 from N-WASP GBD and the corresponding R35 and A36 from WASP are shown in stick presentation. D) Chemical shift perturbations observed ¹H-¹⁵N HSQC spectra between unbound EspF_U R47₅ (blue contours), in complex with IRTKS SH3 (red contours) and in complex with N-WASP GBD and IRTKS SH3 (green contours). E) Comparison of steady state heteronuclear {¹H}-¹⁵N NOEs for unbound EspF_U R47₅ (blue bars) and associated to trimolecular complex with N-WASP GBD and IRTKS SH3 (red bars). F) Color coding of observed {¹H}-¹⁵N NOEs in the complex on the structure of EspF_U R47₅ reflects the increased rigidity on ps-ns timescales for N-WASP binding residues 3–21 and IRTKS SH3 binding epitope H26-V40 (blue coloring). Sustained flexibility on ps-ns timescales (colored green and yellow) is observed for the linker residues 22–24 that connect GBD and SH3 binding domains. The C-terminal end of EspF_U R47₅ remains highly disordered also in the complex. See also Figure S3.

Figure 3. Chemical shift perturbation mapping of IRTKS SH3 upon addition of peptides from EspF_U and Eps8

Full assignment of resonances is given for IRTKS SH3:EspF_U R47₅ complex (upper left panel). Selected boxed resonances with assignments capture the embedded binding affinity and show that all the peptides bind to the same binding site on IRTKS SH3. The assignments corresponds to the saturated state with IRTKS SH3:peptide shown in blue. Saturated state for EspF_U R47₅ and Eps8^A33W correspond to SH3:peptide molar ratio 1:1 and for EspF_U^W33A and Eps8 to SH3:peptide molar ratio 1:3.25. Free IRTKS resonances are shown in red and other colors correspond to intermediate states between free and saturated states.

Figure 4. Structural and functional role of the W-switch

A) Alignment of tandem PxxP containing ligands. Delphilin is a protein that we predicted as a potential novel IRTKS ligand, but this has not been experimentally tested. B) EspF_U W33 establishes a T-shaped edge-to-face arrangement with IRTKS W378. C) Schematic showing a single repeat of EspF_UC. The N-WASP binding helix “H” (Cheng et al., 2008) and IRTKS binding “P” (Weiss et al., 2009; Vingadassolom et al., 2009) domains are indicated. Asterisk indicates the site of the W33A mutation. D) Plasmids encoding the LexA DNA binding domain-IRTKS_SH3 and the indicated GAL4 AD-EspF_U fusions were co-transformed into a yeast two-hybrid reporter strain L40. “PW33A” indicates the mutant with the alanine substitution of residue W33. β-galactosidase activity was assessed as an average of three co-transformants in Miller Units (MU) with error bars indicating the standard deviation. Results are representative of at least three experiments. D) FLCs expressing myc-tagged GFP-EspF_U fusions were infected with EPEC KC12, which requires ectopic expression of EspF_U for pedestal formation. Red asterisks (and corresponding red stripes in schematic) indicate W33A mutations. Monolayers were stained with DAPI (blue), anti-myc antibody (green), and Alexa568-phalloidin (red). E) FLCs expressing HA-tagged Tir-EspF_U fusion protein carrying W33A mutations were infected with intimin-expressing E. coli K12. Monolayers were stained with DAPI (blue), anti-HA antibody (green), and Alexa568-phalloidin (red). F) An engineered tryptophan mutation in Eps8 dramatically enforces its intracellular association with IRTKS. Human 293T cells were transfected with an expression vector for IRTKS tagged with a biotin acceptor domain together with a vector for a Myc-tagged wild-type Eps8 (wt) or a mutated derivative (mut) containing an EspF_U-like tryptophan-containing linker between the PxxP motifs in the IRTKS SH3 domain binding region. IRTKS from lysates of these cells was precipitated using streptavidin-coated beads. Proteins precipitated from these lysates with streptavidin-coated beads were examined by Western blotting using anti-Myc antibodies and labeled streptavidin to detect Eps8 and IRTKS proteins, respectively. Part of the total lysates was similarly analyzed for Eps8 and IRTKS expression without prior affinity selection, as indicated.