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Insight into Lipopolysaccharide Translocation by Cryo-EM structures of a LptDE Transporter in Complex with Pro-Macrobodies

View ORCID ProfileMathieu Botte, View ORCID ProfileDongchun Ni, View ORCID ProfileStephan Schenck, View ORCID ProfileIwan Zimmermann, View ORCID ProfileMohamed Chami, View ORCID ProfileNicolas Bocquet, View ORCID ProfilePascal Egloff, View ORCID ProfileDenis Bucher, View ORCID ProfileMatilde Trabuco, View ORCID ProfileRobert K.Y. Cheng, View ORCID ProfileJanine D. Brunner, View ORCID ProfileMarkus A. Seeger, View ORCID ProfileHenning Stahlberg, View ORCID ProfileMichael Hennig
doi: https://doi.org/10.1101/2021.03.23.436624
Mathieu Botte
1leadXpro AG, Park Innovaare, 5234 Villigen, Switzerland
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Dongchun Ni
2C-CINA, Biozentrum, University of Basel, Mattenstr. 24, 4058 Basel, Switzerland
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  • ORCID record for Dongchun Ni
Stephan Schenck
1leadXpro AG, Park Innovaare, 5234 Villigen, Switzerland
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Iwan Zimmermann
3Institute of Medical Microbiology, University of Zürich, Gloriastasse 28/30, 8006 Zürich
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Mohamed Chami
2C-CINA, Biozentrum, University of Basel, Mattenstr. 24, 4058 Basel, Switzerland
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Nicolas Bocquet
1leadXpro AG, Park Innovaare, 5234 Villigen, Switzerland
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Pascal Egloff
3Institute of Medical Microbiology, University of Zürich, Gloriastasse 28/30, 8006 Zürich
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Denis Bucher
1leadXpro AG, Park Innovaare, 5234 Villigen, Switzerland
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  • ORCID record for Denis Bucher
Matilde Trabuco
1leadXpro AG, Park Innovaare, 5234 Villigen, Switzerland
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Robert K.Y. Cheng
1leadXpro AG, Park Innovaare, 5234 Villigen, Switzerland
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Janine D. Brunner
4Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institute (PSI), 5232 Villigen, Switzerland
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Markus A. Seeger
3Institute of Medical Microbiology, University of Zürich, Gloriastasse 28/30, 8006 Zürich
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Henning Stahlberg
2C-CINA, Biozentrum, University of Basel, Mattenstr. 24, 4058 Basel, Switzerland
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Michael Hennig
1leadXpro AG, Park Innovaare, 5234 Villigen, Switzerland
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  • For correspondence: michael.hennig@leadxpro.com
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Abstract

Lipopolysaccharides (LPS) are major constituents of the extracellular leaflet in the bacterial outer membrane and form an effective physical barrier for environmental threats and for antibiotics in Gram-negative bacteria 1. The last step of LPS insertion via the Lpt pathway is mediated by the LptD/E protein complex 2. Despite detailed insights from X-ray crystallography into the architecture of LptDE transporter complexes 3–5, no structure of a laterally open LptD transporter has been described, a transient state that occurs during LPS release 6. To facilitate the acquisition of hitherto unknown conformations we subjected LptDE of N. gonorrhoeae to cryo-EM analyses. In complex with newly designed rigid chaperones derived from nanobodies (Pro-Macrobodies, PMbs) we obtained a map of a partially opened LptDE transporter at 3.4 Å resolution and in addition we captured a laterally fully opened LptDE complex from a subset of particles. Our work offers new insights into the mechanism of LPS insertion, provides a structural framework for the development of antibiotics targeting LptD and describes a novel, highly rigid and widely applicable chaperone scaffold to enable structural biology of challenging protein targets.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted March 23, 2021.
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Insight into Lipopolysaccharide Translocation by Cryo-EM structures of a LptDE Transporter in Complex with Pro-Macrobodies
Mathieu Botte, Dongchun Ni, Stephan Schenck, Iwan Zimmermann, Mohamed Chami, Nicolas Bocquet, Pascal Egloff, Denis Bucher, Matilde Trabuco, Robert K.Y. Cheng, Janine D. Brunner, Markus A. Seeger, Henning Stahlberg, Michael Hennig
bioRxiv 2021.03.23.436624; doi: https://doi.org/10.1101/2021.03.23.436624
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Insight into Lipopolysaccharide Translocation by Cryo-EM structures of a LptDE Transporter in Complex with Pro-Macrobodies
Mathieu Botte, Dongchun Ni, Stephan Schenck, Iwan Zimmermann, Mohamed Chami, Nicolas Bocquet, Pascal Egloff, Denis Bucher, Matilde Trabuco, Robert K.Y. Cheng, Janine D. Brunner, Markus A. Seeger, Henning Stahlberg, Michael Hennig
bioRxiv 2021.03.23.436624; doi: https://doi.org/10.1101/2021.03.23.436624

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