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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Nature. Author manuscript; available in PMC 2009 August 3.
Published in final edited form as:
PMCID: PMC2719906

Structural Mechanism of WASP Activation by the Enterohaemorrhagic E. coli Effector EspFU


During infection enterohaemorrhagic Escherichia coli (EHEC) usurp the actin cytoskeleton of eukaryotic cells by injecting the EspFU protein into the host cytoplasm1, 2. EspFU controls actin by activating members of the Wiskott-Aldrich Syndrome Protein (WASP) family15. Here we show that EspFU binds the autoinhibitory GTPase binding domain (GBD) in WASP proteins and displaces it from the activity-bearing VCA domain. This interaction potently activates WASP and N-WASP in vitro and induces localized actin assembly in cells. In the solution structure of the GBD-EspFU complex, EspFU forms an amphipathic helix that binds the GBD, mimicking interactions of the VCA in autoinhibited WASP. Thus, EspFU activates by competing directly for the VCA binding site on the GBD. This mechanism is distinct from that used by the eukaryotic activators Cdc42 and SH2 domains, which globally destabilize the GBD fold to release the VCA68. Such diversity of mechanism in WASP proteins is distinct from other multi-modular systems, and may result from the intrinsically unstructured nature of the isolated GBD and VCA elements. The structural incompatibility of the GBD complexes with EspFU and Cdc42/SH2, plus high affinity EspFU binding, enable EHEC to potently hijack the eukaryotic cytoskeletal machinery.

Pathogenic bacteria often target the host actin cytoskeleton during infection to assist entry, cell-to-cell spreading, and intimate attachment to host membranes913. EHEC (serotype O157:H7) is a food-born human pathogen that causes severe diarrhea and hemolytic uremic syndrome14. During infection, EHEC binds eukaryotic host intestinal epithelial cells and secretes Tir (Translocated Intimin Receptor) and EspFU (also known as TccP), into the host cell1, 2. The secreted Tir localizes in the host plasma membrane, and upon clustering by binding to the bacterial outer surface protein intimin, recruits EspFU via its C-terminal cytoplasmic domain3. EspFU in turn hijacks members of the Wiskott-Aldrich Syndrome Protein (WASP) family, which locally stimulate the actin nucleation factor, Arp2/3 complex15. Together, these interactions induce the formation of an actin-rich membrane protrusion beneath the site of EHEC attachment, termed an actin pedestal1, 2, 15.

WASP proteins activate Arp2/3 complex through a C-terminal VCA domain16. In free WASP, this activity is blocked through intramolecular interactions with a central GTPase binding domain (GBD). Structural and biochemical studies have shown that the GBD sequesters an amphipathic helix in the C region of the VCA that is necessary for activation of Arp2/3 complex, resulting in autoinhibition7, 17, 18. WASP can be allosterically activated by a number of intracellular ligands, including Cdc42 and (for phosphorylated WASP) SH2 domains8, 19. These ligands bind to WASP and N-WASP in a manner that is structurally incompatible with the autoinhibited fold of the GBD. Thus, they activate by globally destabilizing the GBD, consequently releasing the VCA6, 8, 20, 21.

EspFU is composed of an N-terminal signal sequence necessary for translocation into the eukaryotic host, followed by 2–7 nearly identical 47 residue repeats (Fig. 1A)22. Each full repeat consists of an N-terminal 27 residue hydrophobic segment and a C-terminal 20 residue proline-rich segment. Previous studies showed that EspFU can bind the N-WASP GBD and activate N-WASP toward Arp2/3 complex in actin assembly assays in vitro1, 2, 4. A similar sequence is found in the related bacterial effector EspF, which also can activate N-WASP (Fig. S1)23.

Figure 1
A single repeat of EspFU activates WASP/N-WASP with high potency

To understand EspFU-mediated activation, we examined fragments containing the full 47-residue fifth repeat (R47, Fig. 1, Fig. S1) and an N-terminal 33-residue fragment lacking most of the proline-rich sequence (R33). In Arp2/3-mediated pyrene-actin assembly assays, R47 and R33 significantly and similarly enhanced the activities of autoinhibited N-WASP and WASP proteins (N-WASPC, and GBD-VCA, respectively; Fig. 1B, Fig. S2). Titration of R47 into N-WASPC produced a monotonic increase in actin assembly rate (Fig. 1C and Fig. S3), which reached a plateau value similar to that of the NWASP VCA. Thus, the endpoint of the titration produces a state which biochemically resembles free N-WASP VCA. Fitting the data to a single-site binding isotherm yields a KAct of 24 nM, approximating the KD of R47 for N-WASPC, 35 nM (Fig. S4). EspFU binds N-WASP > 100-fold more strongly than does Cdc4221, resulting in much greater potency in actin assembly assays (Fig. 1C). This much higher affinity should enable EspFU to hijack N-WASP away from its endogenous regulators in vivo21.

Cdc42 and SH2 domains activate WASP by physically displacing the VCA from the GBD7, 8. EspFU R33 disrupts GBD-VCA interactions similarly (Fig. S5), indicating that it also binds the GBD in a manner that is incompatible with the autoinhibitory interactions. The combined data show that EspFU R33 is functionally equivalent to Cdc42 and SH2 domains: all three ligands activate WASP/N-WASP by releasing the VCA from the GBD.

The WASP GBD is largely unstructured on its own, but folds to a well-defined conformation upon binding VCA7. 1H/15N and 1H/13C amide and methyl TROSY NMR spectra showed that EspFU R33 or a five-repeat fragment, R5, also induced folding of the GBD (Fig. S6). Reciprocally, the GBD induces folding of the unstructured R33 (Fig. S6B, Fig. S7). Titrations of R33 and R5 into GBD saturated at 1:1 and 0.2:1 EspFU:GBD stoichiometry, respectively, showing that each repeat can bind N-WASP. The endpoint spectra are strikingly similar, indicating that the GBD folds to the same structure on each repeat, and that the molecular nature of allosteric activation can be understood through analysis of the R33 complex.

We determined the solution structure of the WASP GBD in complex with EspFU R33 using NMR spectroscopy (Fig. 2, Fig. S8; Table S1). The complex can be considered in three stacked structural layers7. The first layer is composed of a short N-terminal i+4 β hairpin (β1, 252–253; β2, 257–258) and α1 helix (265–274) from the GBD. The second layer lies behind the first and consists of GBD helices α2–α4 (α2, 278–281; α3, 284–296; α4, 300–306), which have a roughly planar C-shaped arrangement. The third layer is an amphipathic α5 helix (3–14) and an extended arm (16–20) from EspFU. These elements contact the back of the GBD and are roughly antiparallel to α3 and α2, respectively (Fig. 2A, structural details in Fig. S9). WASP residues 242–249 and EspFU residues 21–33 appear to be largely disordered in solution, based on the absence of long range 1H-1H NOEs and low heteronuclear {1H}-15N NOEs (Fig. S10). The EspFU relative, EspF, is expected to bind the GBD through an analogous amphipathic α5 helix, but may not use an extended arm, as these C-terminal residues are not conserved (Fig. S1).

Figure 2
Structures of the WASP GBD in complex with different ligands

To better understand the physical basis of EspFU activity, we examined a series of single repeat mutants based on the structure. First, we fused these proteins C-terminally to the WASP GBD through a short linker. Since the GBD and R33 are only folded when bound to each other, the thermodynamic stability of the fusions reports on the relative strengths of interaction between the two elements. We mutated three conserved hydrophobic residues, V4, L8 and L12, which lie at the center of the interface between α5 and the GBD (Fig. S9). The L12A (R33VL*) and V4A/L8A (R33**L) mutants have substantially reduced stability relative to wild type in the GBD-R33 fusions, demonstrating the importance of α5 in the interaction of N-WASP with EspFU. Truncations from R33 to R30 or R27 decrease stability by ~2 kcal/mol. Thus, residues beyond Ala30 contribute thermodynamically to GBD-R33 interaction, despite the absence of discrete structure in these regions. Similar contributions to affinity through disordered regions have been observed in other systems as well24. Truncation of all (R14) or part (R18) of the EspFU C-terminal arm decreases the stability further, indicating that the arm contributes significantly to binding of the GBD to EspFU. These unfolding studies mirror direct binding measurements. R33 and R18 bind N-WASPC with KD values of 35 and 4000 nM, respectively (Fig. 1D), corresponding to a difference in free energy of 2.9 kcal/mol. This value agrees closely with the stability difference between the two GBD-fusions (2.6 kcal/mol, Fig. 1D). Finally, these differences in binding correspond well to the activities of the EspFU mutants in N-WASP mediated actin assembly assays, where activity decreases in concert with strength of interaction (Figs. 1B, D).

To test how the biophysical properties of EspFU-N-WASP interaction relate to biological function, we examined the ability of EspFU derivatives to induce localized actin assembly in mammalian cells. To uncouple Tir-mediated recruitment of EspFU from EspFU function in pedestal generation after recruitment, we replaced the C-terminal cytoplasmic domain of Tir with two repeats of EspFU (giving TirΔC-R2). We expressed TirΔC-R2 in mouse fibroblast-like cells, where it inserted into the plasma membrane, and then clustered it by adding E. coli expressing intimin. Immunostaining revealed foci of clustered Tir and N-WASP coincident with bound bacteria (Fig. 3A). Similarly, F-actin strongly colocalized with Tir. In contrast, a control Tir derivative (TirΔC) lacking cytoplasmic elements did not recruit N-WASP or produce actin pedestals (Fig. 3). Mutation of Leu12 to Ala in both EspFU repeats (giving TirΔC-R2 VL*) also abolished both actin assembly and N-WASP recruitment (Fig. 3A), consistent with the decreased binding of this mutant to N-WASP in vitro (Fig. 1). To examine the activity of EspFU truncation mutants, we modified this assay to analyze derivatives of one EspFU repeat unit. As predicted by in vitro data (Fig. 1), the triple V4A/L8A/L12A mutant, TirΔCR47***, was also unable to assemble actin (Fig. S12). Truncation of R47 to R33 decreased activity, likely due to the loss of most of the proline-rich region, which in EspF is known to bind SH3-containing proteins to promote actin assembly23 (Fig. 3B). R30 and R27 showed even lower actin assembly levels, consistent with the diminished N-WASPC binding by these proteins compared to R33 (Fig. 1). Finally, R14, which bound N-WASPC only very weakly (Fig. 1), did not promote actin assembly above background levels. Together, these data show that the ability of EspFU derivatives to promote actin assembly in vivo reflects the physical interactions between EspFU and WASP in vitro.

Figure 3
EspFU induces actin pedestal formation

The GBD-R33 and GBD-C complexes are remarkably similar (Fig. 2). In both cases, ligand binding causes the GBD to fold into a β-hairpin and four helices. The backbone r. m. s. d. between the average GBD coordinates in the two structures is only 0.95 Å (residues 247–310). This structural similarity is reflected in high chemical shift similarity: the average GBD chemical-shift differences are 0.015 ppm for HN, 0.062 ppm for Hα, 0.043 ppm for Hβ, 0.21 ppm for Cα, and 0.30 ppm for Cβ. The most significant chemical shift differences between the two complexes occur in α2, which contacts the extended arm of EspFU in the GBD-R33 complex, but is solvent exposed in the GBD-C complex. The WASP C helix and EspFU helix bind the same site on the GBD in the same orientation through analogous hydrophobic contacts involving similar sequences (Fig. 1A, Fig. S9). EspFU makes additional contacts to the GBD through its C-terminal arm, which are not seen in the bound C region helix in autoinhibited WASP. These contacts contribute to the much higher WASP affinity of EspFU compared to its eukaryotic counterparts21. The combined structural and biochemical data lead to a clear and simple mechanism for WASP/N-WASP activation by a single repeat element of EspFU: displacement of the VCA through competitive binding to a common site on the folded GBD.

We have described the autoinhibited GBD-VCA structure as composed of three layers, analogous to the three layers of the GBD-R33 structure, with the C region of the VCA substituting for R337. Three structurally distinct mechanisms for relieving autoinhibition of WASP/N-WASP are now known that involve attacks on distinct regions of this fold (Fig. S13). Cdc42 globally destabilizes the GBD by binding to the first layer of structure. Similarly, following phosphorylation of the conserved Tyr291/256 in WASP/N-WASP, SH2 domains can also destabilize the GBD by binding to this site in the second layer. EHEC have evolved the simplest and structurally most direct means of releasing the VCA: EspFU binds to the GBD-VCA interface, displacing the VCA from the folded GBD by structural competition. Of the three known allosteric mechanisms, only this latter one is akin to those found in most other autoinhibited multi-domain systems, where activation is driven by rearrangements of intact functional modules. The diversity of WASP regulation likely originates from the intrinsically unstructured nature of the GBD, which enables its broad binding specificity. Since the folded GBD is structurally incompatible with Cdc42/SH2 binding, EspFU can displace the VCA while simultaneously preventing interactions with eukaryotic activators, thus sequestering WASP proteins away from their normal signaling pathways. This, coupled with very high affinity binding, allows EHEC to subvert the host actin cytoskeleton to its own ends.

Methods Summary

Protein Expression and Purification

EspFU and WASP proteins were expressed in bacteria as either His6- or GST-fusions, and purified by affinity and conventional chromatographies. Bovine Arp2/3 complex and rabbit skeletal muscle actin were purified as described25, 26.

Biochemical Assays

All biochemical assays were performed in KMEI buffer (10 mM imidazole pH 7.0, 50 mM KCl, 1 mM MgCl2, and 1 mM EGTA), or KMEI plus 5 mM β-mercaptoethanol at 25 °C. Actin Assembly. Actin assembly assays were performed as described25, with 4 µM actin (5% pyrene labeled) and 10 nM Arp2/3 complex. Filament barbed end concentrations were calculated as described27. Chemical Denaturation. GBD-EspFU fusion proteins (10 µM) were denatured with guanidine hydrochloride. Tryptophan fluorescence emission (λex = 295 nm, λem = 320 nm, 355 nm) was fit to a six parameter equation28 to yield ΔGunfolding. Isothermal Titration Calorimetry. EspFU proteins were titrated into N-WASPC in a Microcal VP-ITC microcalorimeter. Baseline was corrected for EspFU addition to buffer. Data were fit to a single site binding model.

Mammalian Cell Culture

Mouse fibroblast-like cells were transfected with HA-Tir-EspFU fusions as described1. Transfected cells were treated with E. coli expressing intimin for 3.5 hours (for Tir-R2 fusions) or antibodies recognizing the extracellular domain of EHEC Tir (gift from A. Donohue-Rolfe, Tufts University School of Veterinary Medicine), followed by S. aureus particles (Pansorbin; Calbiochem) (for Tir fusions with a single EspFU repeat).

Immunofluorescence Microscopy

Monolayers were fixed and permeabilized as described29. Cells were treated with HA.11 or α-N-WASP (gift from S. Snapper), washed and treated with Alexa488 goat anti-mouse antibody (1:200; Molecular Probes) or Alexa568 goat anti-rabbit antibody (1:150; Molecular Probes) respectively. F-actin was stained with Alexa568-phalloidin (1:100; Molecular Probes).

NMR Spectroscopy and Structure Calculation

The structure of the complex of GBD (residues 242–310) and R33 (residues 1–33 in Fig. 1, corresponding to residues 268–300 in EspFU (Fig. S1)) was determined in iterative fashion based on NOE, dihedral and hydrogen bond restraints using ARIA 2.130.

Additional Methods

Protein Expression and Purification

To simplify nomenclature, EspFU residues are numbered 1–47 below and throughout the text (cf. Fig. 1), corresponding to residues 268–314 of the full length protein (Fig. S1). WASP GBD (residues 242–310), EspFU R33 (residues 1–33), R33 mutants, GBD-R33 (WASP 242-310 -GGSGGSHM- EspFU 1-33) and other GBD-repeat fusions were cloned into a modified pGEX-2T vector with a Tev protease cleavage site at the C-terminus of GST. EspFU R5 (residues 80–314) was cloned into a pET15b vector. EspFU R47 (residues 1–47) was cloned into a modified pET19b vector with a His10-ubiquitin-Tev protease site at the N-terminus. All proteins were expressed in E. coli strain BL21(DE3). Uniform 15N/13C labeling of proteins was carried out in M9 minimum media supplemented with 15NH4Cl and 13C-gluose as the sole nitrogen and carbon sources. Samples of U-[15N,2H], Ile-[13CδH3], Leu-[13CH3,12CD3], Val-[13CH3,12CD3]) labeled proteins were obtained from bacterial growth in M9 D2O media containing 3 g/l of U-[2H]-glucose, 1 g/l of 15NH4Cl, and 50 mg/l of 2-keto-3-methyl-d3-3-d1-4-13C-butyrate and 50 mg/l 4-[13C,1H]-3,3-2H-α-ketobutyrate (the latter two, added 1 hr before induction)31. The 3,3-[1H] or the 3-[1H] positions of commercially available α-ketobutyrate or 2-keto-3-methyl-butyrate were exchanged to 2H prior to use, according to the procedure of Gardner et al.32. EspFU R14 and R18 peptides were synthesized at the UT Southwestern Protein Chemistry Technology Center and quantified by amino acid analysis.

Cells expressing GBD and GBD-EspFU fusions were lysed by sonication in GST buffer (20 mM Tris pH 8.0, 100 mM NaCl, 1 mM DTT, and 1 mM EDTA) with protease inhibitors. Cleared lysates were applied to glutathione sepharose 4B (GE Healthcare). Following wash, GST-tagged proteins were eluted in GST buffer containing 10 mM reduced glutathione. Proteins were concentrated, cleaved with Tev protease to separate the GST tag, diluted three-fold into QA buffer (20 mM Tris pH 8.0, 1 mM DTT, and 1 mM EDTA) and loaded onto an anion exchange column (Mono Q, GE Healthcare) equilibrated in QA buffer. The column was developed with a linear gradient from 0% to 50% QB buffer (Buffer A plus 1 M NaCl) in 10 bed volumes. Protein-containing fractions were further purified by size exclusion chromatography (Superdex 75 column, GE Healthcare). GST-GBD was purified similarly, except that the Tev protease cleavage was omitted. EspFU R33 and its mutants were purified similarly, except that in the anion-exchange step the protein was collected in the flow through. His10-ubiquitin tagged EspFU R47 was retained from cleared bacterial lysate on a Ni-NTA column (Qiagen), eluted with an imidazole gradient, desalted into QA buffer and cleaved with Tev protease to remove the His10-ubiquitin tag. Subsequent anion exchange and size exclusion chromatographies were the same as described for EspFU R33. His6-EspFU R5 was purified by Ni affinity chromatography and buffer exchanged into SA buffer (50 mM sodium phosphate pH 6.0, 1 mM DTT, and 1 mM EDTA). Thrombin (GE Healthcare) cleaved EspFU R5 was purified by cation exchange (Mono S, GE Healthcare) in a gradient from 300 mM to 600 mM NaCl over 10 column volumes. Fractions containing EspFU R5 were pooled and further purified by gel filtration chromatography (Superdex 200, GE Healthcare). WASP GBD-VCA, N-WASPC (residues 193–501), bovine Arp2/3 complex and rabbit skeletal muscle actin were purified as described previously2527.

Pull-Down Assays

GST-GBD was immobilized on Glutathione-sepharose 4B beads (~75 nmole/ml beads, GE Healthcare). Beads (60 µl) were incubated with 30 µM EspFU R33 and 15 µM WASP VCA separately in a final volume of 230 µl and washed three times. The same volume of beads containing VCA-bound GST-GBD was further incubated with 200 µM R33 and washed four times. Flow through and beads were analyzed by SDS-PAGE stained with Coomassie Blue.

Mammalian Cell Culture and Immunofluorescence Microscopy

To create transfection plasmids expressing Tir-EspFU fusions (all N-terminally HA-tagged), DNA fragments encoding EspFU R14, R30, R33, R47, R2 (repeats 4–5) and point mutants were cloned into the KpnI and BamHI sites of pHN-TirΔC1. Mouse fibroblast-like cells were cultured in six-well plates in DMEM plus 10% FBS and transfected with 1 µg of plasmid per well for 12–16 h using Lipofectamine Plus reagent (Invitrogen)1. Cells were reseeded onto 12-mm glass coverslips to achieve 50–75% confluency after an additional 24 h of growth. Transfected cells expressing TirΔC-R2 proteins were treated with an E. coli expressing intimin for 3.5 hours. Cells expressing TirΔC fused to a single EspFU repeat were treated with a 1:200 dilution of antibodies raised against the extracellular domain of EHEC Tir (anti-TirM) for 30 min, washed to remove excess antibody, and then treated with S. aureus particles (Pansorbin; Calbiochem) for a further 2.5 h. In all cases, monolayers were fixed in PBS+4% paraformaldehyde for 30 minutes and permeabilized with 0.1% Triton-X-100 as described29. Cells were then treated with HA.11 (diluted 1:500 in PBS+1%BSA) or HA.11 and α-N-WASP (1:500) for 30 minutes prior to washing and addition of Alexa488 goat anti-mouse antibody (1:200; Molecular Probes) or Alexa568 goat anti-rabbit antibody (1:150). F-actin was identified by staining with Alexa568-phalloidin (1:100; Molecular Probes).

NMR Spectroscopy and Structure Calculation

The complex between GBD and R33 was isolated by gel filtration chromatography (in 50 mM sodium phosphate pH 6.8, 100 mM NaCl, 1 mM DTT, and 1 mM EDTA), and concentrated to 1-1.5 mM for all NMR experiments. Backbone and sidechain chemical shift assignments were obtained using standard triple-resonance methods in 15N/13C-labeled samples 33, 34. A total of 1552 distance restraints were obtained from 2D 1H-NOESY, 3D 15N-edited NOESY (τmix = 100 ms), and 4D 13C/13C-edited NOESY (τmix = 75 ms)35 spectra. One hundred intermolecular NOEs were identified in a 13C-filtered NOESY spectrum (τmix = 100 ms) of 13C,15N-GBD in complex with unlabeled R33 recorded in D2O36. A total of 165 unambiguously assigned NOEs define the GBD-R33 interface. Backbone dihedral angles were restrained based on chemical shift analyses in the program TALOS37 (maximum of 30° or the standard deviation observed in the TALOS database matches). Hydrogen bond restraints (HN-O distance, 1.5-2.8 Å; N-O distance, 2.4 – 3.5 Å) were applied to amide groups that were protected for > 1 hour in a sample of a 15N-GBD-R33 fusion protein freshly dissolved in D2O NMR buffer. Structures were calculated and iteratively refined using the program ARIA 2.130. Intermolecular NOEs were assigned manually, in iterative fashion, from the 13C-filtered NOESY spectrum, and assigned distance restraints within ARIA 2.1. Other NOEs were assigned automatically within ARIA 2.1. In the final iteration, structures were refined in explicit water to improve sidechain packing and hydrogen bonding38. All spectra were processed and analyzed using NMRPipe39 and NMRView40. The twenty final lowest energy conformers were analyzed with ProCheckNMR41. Structure figures were generated with Ribbons42 and PyMOL43.

Supplementary Material



We thank Dr. Takanori Otomo for discussion and assistance on biochemical assays and NMR spectroscopy; Pilong Li, Ilidio Martins and Drs. Carlos A. Amezcua, Kevin H. Gardner, and Qiong Wu for assistance with NMR spectroscopy and structure calculations; Drs. Gaya K. Amarasinghe and Daisy W. Leung for sharing reagents; Derek Trobaugh, Jason Rennie and Douglas Robbins for technical assistance; Dr. Shae B. Padrick for help with Mathematica, and Drs. Xiaolan Yao and S.B.P. for assistance in writing and for critical reading of the manuscript. This work was supported by grants from the NIH (NIH-R01-GM56322 to M.K.R.; NIH-R01-AI46454 to J.L.), and Welch Foundation (I–1544 to M.K.R.), and a Chilton Foundation Fellowship to H.-C. C.


Accession Number. Atomic coordinates of the 20 final lowest energy conformers have been deposited in the Protein Data Bank under accession code 2K42.


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