Abstract
Shigella flexneri is a bacterial pathogen that invades cells of the gastrointestinal tract, causing severe dysentery. Shigella mediates intracellular motility and spreading via actin comet tail formation. This process is dependent on the surface-exposed, membrane-embedded virulence factor IcsA, which recruits the host actin regulator N-WASP. Although it is clear that Shigella requires N-WASP for this process, the molecular details of this interaction and the mechanism of N-WASP activation remain poorly understood. Here, we show that co-expression of full-length IcsA and the Shigella membrane protease IcsP yields highly pure IcsA passenger domain (residues 53–758). We show that IcsA is monomeric and describe the solution structure of the passenger domain obtained by small-angle X-ray scattering (SAXS) analysis. The SAXS-derived models suggest that IcsA has an elongated shape but, unlike most other autotransporter proteins, possesses a central kink revealing a distinctly curved structure. Pull-down experiments show direct binding of the IcsA passenger domain to both the WASP homology 1 (WH1) domain and the GTPase binding domain (GBD) of N-WASP and no binding to the verprolin homology/cofilin/acidic (VCA) region. Using fluorescence polarization experiments, we demonstrate that IcsA binding to the GBD region displaces the VCA peptide and that this effect is synergistically enhanced upon IcsA binding to the WH1 region. Additionally, domain mapping of the IcsA interaction interface reveals that different regions of IcsA bind to the WH1 and GBD domains of N-WASP. Taken together, our data support a model where IcsA and N-WASP form a tight complex releasing the N-WASP VCA domain to recruit the host cell machinery for actin tail formation.
Highlights
Shigella flexneri is a bacterial pathogen that invades cells of the gastrointestinal tract, causing severe dysentery
We demonstrate that IcsA binding to the GTPase binding domain (GBD) region displaces the verprolin homology/cofilin/acidic (VCA) peptide and that this effect is synergistically enhanced upon IcsA binding to the WASP homology 1 (WH1) region
We have shown that this protein is correctly folded, monodisperse, and competent to bind the relevant host factor N-WASP
Summary
The Passenger Domain of IcsA Is a Monodisperse Elongated Protein—Expression in Escherichia coli of full-length IcsA including the N-terminal secretion signal and the C-terminal transporter domain followed by immunoblotting reveals a protein of ϳ120 kDa present in the cell pellet consistent with membrane-bound IcsA (Fig. 1B). The early volume at which IcsA elutes off of the size exclusion column (13.3 ml) suggests that the protein may adopt an elongated structure in solution (compared with BSA at 66 kDa, which elutes at 14.2 ml) This observation is consistent with structural predictions that IcsA will possess a -helix fold similar to that of other autotransporters. The individual models representing the low resolution structure of IcsA[53–758] fit the SAXS data (2 ϭ 1.9) with no systematic deviations observed, as assessed using Correlation Map (CorMap p ϭ 0.84; Fig. 3 (D and E)) (30 –32) Both the GASBOR and DAMMIN models show a characteristic ϳ90° “bend” in the IcsA[53–758] monomer that occurs approximately halfway along the length of the protein so that the overall structure of the protein adopts an L-shaped conformation. The dummy atom model volume/2 provides an empirical estimate of molecular mass
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