Abstract
Autotransporters are virulence factors produced by Gram-negative bacteria. They consist of two domains, an N-terminal 'passenger' domain and a C-terminal β-domain. β-domains form β-barrel structures in the outer membrane while passenger domains are translocated into the extracellular space. In some autotransporters, the two domains are separated by proteolytic cleavage. Using X-ray crystallography, we solved the 2.7-Å structure of the post-cleavage state of the β-domain of EspP, an autotransporter produced by Escherichia coli strain O157:H7. The structure consists of a 12-stranded β-barrel with the passenger domain–β-domain cleavage junction located inside the barrel pore, approximately midway between the extracellular and periplasmic surfaces of the outer membrane. The structure reveals an unprecedented intra-barrel cleavage mechanism and suggests that two conformational changes occur in the β-domain after cleavage, one conferring increased stability on the β-domain and another restricting access to the barrel pore.
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Acknowledgements
We thank J. Peterson for technical support, E. Udho and A. Finkelstein for discussions, J. Shiloach and L. Trinh for help with fermentation, and A. Hickman for critically reading the manuscript. This work was supported by the Intramural Research Program of the US National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases. Diffraction data were collected at the SER-CAT 22-ID and 22-BM beamlines at the Advanced Photon Source, Argonne National Laboratory. Supporting institutions may be found at http://www.ser-cat.org/members.html.
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T.J.B. purified, crystallized and determined the crystal structure of EspP. N.D. and H.D.B. designed and performed mutagenesis and cell biological assays. P.L. and S.K.B. were involved in crystallization, data collection, structure analysis and discussions. T.J.B., H.D.B. and S.K.B. wrote the manuscript.
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Supplementary Figs. 1–6, Supplementary Tables 1–2 and Supplementary Methods (PDF 972 kb)
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Barnard, T., Dautin, N., Lukacik, P. et al. Autotransporter structure reveals intra-barrel cleavage followed by conformational changes. Nat Struct Mol Biol 14, 1214–1220 (2007). https://doi.org/10.1038/nsmb1322
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DOI: https://doi.org/10.1038/nsmb1322
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