Structural mechanism of WASP activation by the enterohaemorrhagic E-coli effector EspF(U)

Department of Biochemistry and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
Nature (Impact Factor: 42.35). 08/2008; 454(7207):1009-13. DOI: 10.1038/nature07160
Source: PubMed

ABSTRACT During infection, enterohaemorrhagic Escherichia coli (EHEC) takes over the actin cytoskeleton of eukaryotic cells by injecting the EspF(U) protein into the host cytoplasm. EspF(U) controls actin by activating members of the Wiskott-Aldrich syndrome protein (WASP) family. Here we show that EspF(U) binds to the autoinhibitory GTPase binding domain (GBD) in WASP proteins and displaces it from the activity-bearing VCA domain (for verprolin homology, central hydrophobic and acidic regions). This interaction potently activates WASP and neural (N)-WASP in vitro and induces localized actin assembly in cells. In the solution structure of the GBD-EspF(U) complex, EspF(U) forms an amphipathic helix that binds the GBD, mimicking interactions of the VCA domain in autoinhibited WASP. Thus, EspF(U) activates WASP 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 VCA. Such diversity of mechanism in WASP proteins is distinct from other multimodular systems, and may result from the intrinsically unstructured nature of the isolated GBD and VCA elements. The structural incompatibility of the GBD complexes with EspF(U) and Cdc42/SH2, plus high-affinity EspF(U) binding, enable EHEC to hijack the eukaryotic cytoskeletal machinery effectively.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The human pathogen Listeria monocytogenes is able to directly spread to neighboring cells of host tissues, a process recently linked to the virulence factor InlC. InlC targets the sixth SH3 domain (SH3-6) of human Tuba, disrupting its physiological interaction with the cytoskeletal protein N-WASP. The resulting loss of cortical actin tension may slacken the junctional membrane, allowing protrusion formation by motile Listeria. Complexes of Tuba SH3-6 with physiological partners N-WASP and Mena reveal equivalent binding modes but distinct affinities. The interaction surface of the infection complex InlC/Tuba SH3-6 is centered on phenylalanine 146 of InlC stacking upon asparagine 1569 of Tuba. Replacing Phe146 by alanine largely abrogates molecular affinity and in vivo mimics deletion of inlC. Collectively, our findings indicate that InlC hijacks Tuba through its LRR domain, blocking the peptide binding groove to prevent recruitment of its physiological partners.
    Structure 12/2013; DOI:10.1016/j.str.2013.10.017 · 6.79 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Many actin-binding proteins (ABPs) use complex multi-domain architectures to integrate and coordinate multiple signals and interactions with the dynamic remodeling of actin cytoskeleton. In these proteins, small segments that are intrinsically disordered in their unbound native state can be functionally as important as identifiable folded units. These functional intrinsically disordered regions (IDRs) are however difficult to identify and characterize in vitro. Here, we try to summarize the state of the art in understanding the structural features and interfacial properties of IDRs involved in actin self-assembly dynamics. Recent structural and functional insights into the regulation of widespread, multi-functional WH2/β-thymosin domains, and of other IDRs such as those associated with WASP/WAVE, formin or capping proteins are examined. Understanding the functional versatility of IDRs in actin assembly requires apprehending by multiple structural and functional approaches their large conformational plasticity and dynamics in their interactions. In many modular ABPs, IDRs relay labile interactions with multiple partners and act as interaction hubs in interdomain and protein-protein interfaces. They thus control multiple conformational transitions between the inactive and active states or between various active states of multi-domain ABPs, and play an important role to coordinate the high turnover of interactions in actin self-assembly dynamics. © 2013 Wiley Periodicals, Inc.
    Cytoskeleton 11/2013; 70(11). DOI:10.1002/cm.21140 · 3.01 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The GTP-ase binding domain (GBD) of the signaling protein Wiskott-Aldrich Syndrome Protein (WASP) is intrinsically disordered and mutations in it have been linked with Wiskott-Aldrich Syndrome (WAS), an X-linked disorder characterized by thrombocytopenia, eczema and recurrent infections. Here, we use molecular dynamics simulations and the semi-empirical GROMOS 45A3 force field to study interaction of the GBD domain of WASP with a fragment of the protein EspF(U) as well as with the VCA domain of WASP (auto-inhibited state). EspF(U) is secreted and used by enterohaemorrhagic Escherichia colt to hijack eukaryotic cytoskeletal machinery, and it does so by competitively disrupting the auto-inhibitory interaction between GBD and VCA domains of WASP. In addition, naturally occurring mutations in the VCA domain cause different variants of WAS. Our simulations confirm that the EspF(U) domain binds the GBD domain similarly to the VCA domain, which explains why these two binding partners are competitive binders of the GBD domain. Furthermore, we propose a possible mechanism to explain the higher affinity of EspF(U) for the GBD domain. Finally, we show that the mutations in the VCA domain responsible for Wiskott-Aldrich syndrome can cause formation of beta-sheets in the VCA domain. This effect, combined with the mutation-induced rearrangement of the salt bridge network, consequently disables tight binding between GBD and VCA domains. Overall, our results provide a microscopic, dynamic picture behind the two main ways through which the interactions involving the GBD domain of WASP participate in different disease processes.(doi: 10.5562/cca1806)
    Croatica Chemica Acta 09/2011; 84(2):211-220. DOI:10.5562/cca1806 · 0.56 Impact Factor