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