title: The Shigella Virulence Factor IcsA Relieves N-WASP Autoinhibition by Displacing the Verprolin Homology/Cofilin/Acidic (VCA) Domain.
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.
authors.affiliation: From the Cambridge Institute for Medical Research, Department of Pathology, University of Cambridge, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, United Kingdom and.
authors.forename: Rui P M
authors.initials: RP
authors.lastname: Mauricio
authors.affiliation: the European Molecular Biology Laboratory (EMBL), Hamburg Outstation, c/o DESY, Hamburg 22067, Germany.
authors.forename: Cy M
authors.initials: CM
authors.lastname: Jeffries
authors.affiliation: the European Molecular Biology Laboratory (EMBL), Hamburg Outstation, c/o DESY, Hamburg 22067, Germany.
authors.forename: Dmitri I
authors.initials: DI
authors.lastname: Svergun
authors.affiliation: From the Cambridge Institute for Medical Research, Department of Pathology, University of Cambridge, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, United Kingdom and jed55@cam.ac.uk.
authors.forename: Janet E
authors.initials: JE
authors.lastname: Deane
mesh_terms: D051339:Actin Depolymerizing Factors / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D000072417:Protein Domains; D017386:Sequence Homology, Amino Acid; D012763:Shigella flexneri / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D051300:Wiskott-Aldrich Syndrome Protein Family / Q000235:genetics / Q000378:metabolism*; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D051339:Actin Depolymerizing Factors; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D014157:Transcription Factors; C495771:WASL protein, human; D051300:Wiskott-Aldrich Syndrome Protein Family; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; C058483:virG protein, Shigella flexneri
keywords: actin polymerization; bacterial pathogenesis; fluorescence anisotropy; host-pathogen interaction; small-angle X-ray scattering (SAXS); virulence factor