© 2017 by The American Society for Biochemistry and Molecular Biology, Inc. 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 smallangle 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 theGBDregion 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.
Mauricio, R. P. M., Jeffries, C. M., Svergun, D. I., & Deane, J. E. (2017). The Shigella virulence factor IcsA relieves N-WASP autoinhibition by displacing the verprolin homology/cofilin/acidic (VCA) domain. Journal of Biological Chemistry, 292(1), 134–145. https://doi.org/10.1074/jbc.M116.758003