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

Botte, M.; Ni, D.; Schenck, S.; Zimmermann, I.; Chami, M.; Bocquet, N.; Egloff, P.; Bucher, D.; Trabuco, M.; Cheng, R. K. Y.; Brunner, J. D.; Seeger, M. A.; Stahlberg, H.; Hennig, M.

doi:10.1101/2021.03.23.436624

bioRxiv · 10.1101/2021.03.23.436624

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

Botte, M.·Ni, D.·Schenck, S.·Zimmermann, I.·Chami, M.·Bocquet, N.·Egloff, P.·Bucher, D.·Trabuco, M.·Cheng, R. K. Y.·Brunner, J. D.·Seeger, M. A.·Stahlberg, H.·Hennig, M.

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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 [A] 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.

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Botte, M., Ni, D., Schenck, S., Zimmermann, I., Chami, M., Bocquet, N., Egloff, P., Bucher, D., Trabuco, M., Cheng, R. K. Y., Brunner, J. D., Seeger, M. A., Stahlberg, H., Hennig, M.. 2021-03-23. Insight into Lipopolysaccharide Translocation by Cryo-EM structures of a LptDE Transporter in Complex with Pro-Macrobodies. https://doi.org/10.1101/2021.03.23.436624

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