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Substrate-engaged type III secretion system structures reveal gating mechanism for unfolded protein translocation

View ORCID ProfileSean Miletic, View ORCID ProfileDirk Fahrenkamp, View ORCID ProfileNikolaus Goessweiner-Mohr, View ORCID ProfileJiri Wald, View ORCID ProfileMaurice Pantel, View ORCID ProfileOliver Vesper, Vadim Kotov, View ORCID ProfileThomas C. Marlovits
doi: https://doi.org/10.1101/2020.12.17.423328
Sean Miletic
1University Medical Center Hamburg-Eppendorf (UKE), Martinistrasse 52, D-20246 Hamburg, Germany.
2Centre for Structural Systems Biology (CSSB), Notkestrasse 85, D-22607 Hamburg, Germany.
3Deutsches Elektronen-Synchrotron Zentrum (DESY), Notkestrasse 85, D-22607 Hamburg, Germany.
4Institute of Molecular Biotechnology GmbH (IMBA), Austrian Academy of Sciences, Dr. Bohr-Gasse 5, A-1030 Vienna, Austria.
5Research Institute of Molecular Pathology (IMP), Campus-Vienna-Biocenter 1, A-1030 Vienna, Austria.
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Dirk Fahrenkamp
1University Medical Center Hamburg-Eppendorf (UKE), Martinistrasse 52, D-20246 Hamburg, Germany.
2Centre for Structural Systems Biology (CSSB), Notkestrasse 85, D-22607 Hamburg, Germany.
3Deutsches Elektronen-Synchrotron Zentrum (DESY), Notkestrasse 85, D-22607 Hamburg, Germany.
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Nikolaus Goessweiner-Mohr
1University Medical Center Hamburg-Eppendorf (UKE), Martinistrasse 52, D-20246 Hamburg, Germany.
3Deutsches Elektronen-Synchrotron Zentrum (DESY), Notkestrasse 85, D-22607 Hamburg, Germany.
4Institute of Molecular Biotechnology GmbH (IMBA), Austrian Academy of Sciences, Dr. Bohr-Gasse 5, A-1030 Vienna, Austria.
5Research Institute of Molecular Pathology (IMP), Campus-Vienna-Biocenter 1, A-1030 Vienna, Austria.
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  • ORCID record for Nikolaus Goessweiner-Mohr
Jiri Wald
1University Medical Center Hamburg-Eppendorf (UKE), Martinistrasse 52, D-20246 Hamburg, Germany.
2Centre for Structural Systems Biology (CSSB), Notkestrasse 85, D-22607 Hamburg, Germany.
3Deutsches Elektronen-Synchrotron Zentrum (DESY), Notkestrasse 85, D-22607 Hamburg, Germany.
4Institute of Molecular Biotechnology GmbH (IMBA), Austrian Academy of Sciences, Dr. Bohr-Gasse 5, A-1030 Vienna, Austria.
5Research Institute of Molecular Pathology (IMP), Campus-Vienna-Biocenter 1, A-1030 Vienna, Austria.
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Maurice Pantel
1University Medical Center Hamburg-Eppendorf (UKE), Martinistrasse 52, D-20246 Hamburg, Germany.
2Centre for Structural Systems Biology (CSSB), Notkestrasse 85, D-22607 Hamburg, Germany.
3Deutsches Elektronen-Synchrotron Zentrum (DESY), Notkestrasse 85, D-22607 Hamburg, Germany.
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Oliver Vesper
1University Medical Center Hamburg-Eppendorf (UKE), Martinistrasse 52, D-20246 Hamburg, Germany.
2Centre for Structural Systems Biology (CSSB), Notkestrasse 85, D-22607 Hamburg, Germany.
3Deutsches Elektronen-Synchrotron Zentrum (DESY), Notkestrasse 85, D-22607 Hamburg, Germany.
4Institute of Molecular Biotechnology GmbH (IMBA), Austrian Academy of Sciences, Dr. Bohr-Gasse 5, A-1030 Vienna, Austria.
5Research Institute of Molecular Pathology (IMP), Campus-Vienna-Biocenter 1, A-1030 Vienna, Austria.
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Vadim Kotov
1University Medical Center Hamburg-Eppendorf (UKE), Martinistrasse 52, D-20246 Hamburg, Germany.
2Centre for Structural Systems Biology (CSSB), Notkestrasse 85, D-22607 Hamburg, Germany.
3Deutsches Elektronen-Synchrotron Zentrum (DESY), Notkestrasse 85, D-22607 Hamburg, Germany.
4Institute of Molecular Biotechnology GmbH (IMBA), Austrian Academy of Sciences, Dr. Bohr-Gasse 5, A-1030 Vienna, Austria.
5Research Institute of Molecular Pathology (IMP), Campus-Vienna-Biocenter 1, A-1030 Vienna, Austria.
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Thomas C. Marlovits
1University Medical Center Hamburg-Eppendorf (UKE), Martinistrasse 52, D-20246 Hamburg, Germany.
2Centre for Structural Systems Biology (CSSB), Notkestrasse 85, D-22607 Hamburg, Germany.
3Deutsches Elektronen-Synchrotron Zentrum (DESY), Notkestrasse 85, D-22607 Hamburg, Germany.
4Institute of Molecular Biotechnology GmbH (IMBA), Austrian Academy of Sciences, Dr. Bohr-Gasse 5, A-1030 Vienna, Austria.
5Research Institute of Molecular Pathology (IMP), Campus-Vienna-Biocenter 1, A-1030 Vienna, Austria.
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  • For correspondence: marlovits@marlovitslab.org
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Abstract

Many bacterial pathogens strictly rely on the activity of type III secretion systems (T3SSs) to secrete and translocate effector proteins in order to establish infection. The central component of T3SSs is the needle complex, a supramolecular machine which assembles a continuous conduit crossing the bacterial envelope and the host cell membrane to allow bacterial effectors to gain entry into the host cell cytoplasm to modulate signal transduction processes. Disruption of this process impairs pathogenicity, providing an avenue for antimicrobial design. However, the molecular principles underlying T3 secretion remain elusive. Here, we report the first structure of an active Salmonella enterica sv. Typhimurium needle complex engaged with the late effector protein SptP in two functional states, revealing the complete 800Å-long secretion conduit and unravelling the critical role of the export apparatus (EA) subcomplex in T3 secretion. Unfolded substrates enter the EA through a hydrophilic constriction formed by SpaQ proteins, which enables side chain-independent transport, explaining heterogeneity and structural disorder of signal sequences in T3SS effector proteins. Above, a methionine gasket formed by SpaP proteins functions as a gate that dilates to accommodate substrates but prevents leaky pore formation to maintain the physical boundaries of compartments separated by a biological membrane. Following gate penetration, a moveable SpaR loop first folds up to then act akin to a linear ratchet to steer substrates through the needle complex. Together, these findings establish the molecular basis for substrate translocation through T3SSs, improving our understanding of bacterial pathogenicity and motility of flagellated bacteria, and paves the way for the development of novel concepts combating bacterial infections.

Competing Interest Statement

The authors have declared no competing interest.

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Substrate-engaged type III secretion system structures reveal gating mechanism for unfolded protein translocation
Sean Miletic, Dirk Fahrenkamp, Nikolaus Goessweiner-Mohr, Jiri Wald, Maurice Pantel, Oliver Vesper, Vadim Kotov, Thomas C. Marlovits
bioRxiv 2020.12.17.423328; doi: https://doi.org/10.1101/2020.12.17.423328
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Substrate-engaged type III secretion system structures reveal gating mechanism for unfolded protein translocation
Sean Miletic, Dirk Fahrenkamp, Nikolaus Goessweiner-Mohr, Jiri Wald, Maurice Pantel, Oliver Vesper, Vadim Kotov, Thomas C. Marlovits
bioRxiv 2020.12.17.423328; doi: https://doi.org/10.1101/2020.12.17.423328

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