Structural Basis of Lipopolysaccharide Assembly by the Outer Membrane Translocon Holo-Complex

Chen, H.; Siroy, A.; Morales, V.; Gurvic, D.; Quentin, Y.; Balor, S.; Abuta'a, Y. A.; Marteau, M.; Froment, C.; Caumont-Sarcos, A.; Marcoux, J.; Stansfeld, P. J.; Fronzes, R.; Ieva, R.

doi:10.1101/2025.04.10.648201

bioRxiv · 10.1101/2025.04.10.648201

Structural Basis of Lipopolysaccharide Assembly by the Outer Membrane Translocon Holo-Complex

Chen, H.·Siroy, A.·Morales, V.·Gurvic, D.·Quentin, Y.·Balor, S.·Abuta'a, Y. A.·Marteau, M.·Froment, C.·Caumont-Sarcos, A.·Marcoux, J.·Stansfeld, P. J.·Fronzes, R.·Ieva, R.

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Abstract

Lipopolysaccharide (LPS) assembly at the surfaces-exposed leaflet of the bacterial outer membrane (OM) is mediated by the OM LPS translocon. An essential transmembrane {beta}-barrel protein, LptD, and a cognate lipoprotein, LptE, translocate LPS selectively into the OM external leaflet via a poorly understood mechanism. Here, we characterize two additional translocon subunits, the lipoproteins LptM and LptY (formerly YedD). We use single-particle cryo-EM analysis, functional assays and molecular dynamics simulations to visualize the roles of LptM and LptY at the translocon holo-complex LptDEMY, uncovering their impact on LptD conformational dynamics. Whereas LptY binds and stabilizes the periplasmic LptD {beta}-taco domain that functions as LPS receptor, LptM intercalates the lateral gate of the {beta}-barrel domain, promoting its opening and access by LPS. Remarkably, we demonstrate a conformational switch of the LptD {beta}-taco/{beta}-barrel interface alternating between contracted and extended states. The LptD {beta}-strand 1, which defines the mobile side of the lateral gate, binds LPS and performs a stroke movement toward the external leaflet during the contracted-to-extended state transition. Our findings establish a detailed mechanistic framework explaining the selective transport of LPS to the membrane external leaflet.

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Chen, H., Siroy, A., Morales, V., Gurvic, D., Quentin, Y., Balor, S., Abuta'a, Y. A., Marteau, M., Froment, C., Caumont-Sarcos, A., Marcoux, J., Stansfeld, P. J., Fronzes, R., Ieva, R.. 2025-04-10. Structural Basis of Lipopolysaccharide Assembly by the Outer Membrane Translocon Holo-Complex. https://doi.org/10.1101/2025.04.10.648201

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