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Structural mechanism of WASP activation by the enterohaemorrhagic E. coli effector EspFU

Abstract

During infection, enterohaemorrhagic Escherichia coli (EHEC) takes over 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) family1,2,3,4,5. Here we show that EspFU 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–EspFU complex, EspFU forms an amphipathic helix that binds the GBD, mimicking interactions of the VCA domain in autoinhibited WASP. Thus, EspFU 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 VCA6,7,8. 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 EspFU and Cdc42/SH2, plus high-affinity EspFU binding, enable EHEC to hijack the eukaryotic cytoskeletal machinery effectively.

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Figure 1: A single repeat of EspF U activates WASP/N-WASP with high potency.
Figure 2: Structures of the WASP GBD in complex with different ligands.
Figure 3: EspF U induces actin pedestal formation.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates of the 20 final lowest energy conformers have been deposited in the Protein Data Bank under accession number 2K42.

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Acknowledgements

We thank T. Otomo for discussion and assistance on biochemical assays and NMR spectroscopy; P. Li, I. Martins, C. A. Amezcua, K. H. Gardner and Q. Wu for assistance with NMR spectroscopy and structure calculations; G. K. Amarasinghe and D. W. Leung for sharing reagents; D. Trobaugh, J. Rennie and D. Robbins for technical assistance; S. B. Padrick for help with Mathematica and X. Yao and S. B. Padrick for assistance in writing and for critical reading of the manuscript. This work was supported by grants from the National Institute of Health (NIH-R01-GM56322 to M.K.R.; NIH-R01-AI46454 to J.M.L.), Welch Foundation (I–1544 to M.K.R.) and a Chilton Foundation Fellowship to H.-C.C.

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Cheng, HC., Skehan, B., Campellone, K. et al. Structural mechanism of WASP activation by the enterohaemorrhagic E. coli effector EspFU. Nature 454, 1009–1013 (2008). https://doi.org/10.1038/nature07160

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