Abstract
The fidelity and specificity of information flow within a cell is controlled by scaffolding proteins that assemble and link enzymes into signalling circuits1,2. These circuits can be inhibited by bacterial effector proteins that post-translationally modify individual pathway components3,4,5,6. However, there is emerging evidence that pathogens directly organize higher-order signalling networks through enzyme scaffolding7,8, and the identity of the effectors and their mechanisms of action are poorly understood. Here we identify the enterohaemorrhagic Escherichia coli O157:H7 type III effector EspG as a regulator of endomembrane trafficking using a functional screen, and report ADP-ribosylation factor (ARF) GTPases and p21-activated kinases (PAKs) as its relevant host substrates. The 2.5 Å crystal structure of EspG in complex with ARF6 shows how EspG blocks GTPase-activating-protein-assisted GTP hydrolysis, revealing a potent mechanism of GTPase signalling inhibition at organelle membranes. In addition, the 2.8 Å crystal structure of EspG in complex with the autoinhibitory Iα3-helix of PAK2 defines a previously unknown catalytic site in EspG and provides an allosteric mechanism of kinase activation by a bacterial effector. Unexpectedly, ARF and PAKs are organized on adjacent surfaces of EspG, indicating its role as a ‘catalytic scaffold’ that effectively reprograms cellular events through the functional assembly of GTPase-kinase signalling complex.
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Acknowledgements
We would like to thank our colleagues, specifically K. Orth, M. Rosen, M. Cobb, J. Seeman, C. Brautigam and T. Fox, for helpful discussions in preparation of this manuscript, and we are particularly indebted to members of the Structural Biology Lab and mass spectrometry facilities for their efforts on this project. We would also like to thank J. Goldberg and G. Bokoch for providing valuable reagents. We would particularly like to thank J. Cherfils for providing preliminary insights into this work and for key reagents. The structure shown in this report is derived from work performed on beamlines 19-BM and 19-ID at the Structural Biology Center, Advanced Photon Source, Argonne National Laboratory. Argonne National Laboratory is operated by UChicago Argonne, LLC, for the US Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357. R.C.O. was supported by a NIH Molecular Microbiology training grant (5T32AI007520-12). This work was supported by the Welch Foundation (#I-1704) and a grant from the NIH (NIAID; 1RO1AI083359-01) to N.M.A.
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N.M.A. and A.S.S. had the general ideas for this manuscript. A.S.S., N.M.A. and S.M.B. crystallized the protein complexes and D.R.T. solved the complex structures. N.M.A., A.S.S., S.E.S., B.A.W., L.E.R. and R.C.O. planned, performed and interpreted the experiments. N.M.A. and A.S.S. wrote the manuscript and all authors provided editorial input.
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Selyunin, A., Sutton, S., Weigele, B. et al. The assembly of a GTPase–kinase signalling complex by a bacterial catalytic scaffold. Nature 469, 107–111 (2011). https://doi.org/10.1038/nature09593
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DOI: https://doi.org/10.1038/nature09593
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