Structural basis for regulating lipopolysaccharide transmembrane transport

Gram-negative bacteria are surrounded by a multilayered cell envelope with a mostly impermeable outer membrane that provides intrinsic resistance to many antibiotics1–5. The outer membrane is an asymmetrical bilayer with phospholipids in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet6,7. An LPS transport (Lpt) machine called LptB2FGCADE moves LPS across a protein bridge from the inner membrane to the outer membrane8,9. LPS biosynthesis is regulated to prevent toxic accumulation of LPS molecules in the inner membrane during growth10–12. Whether LPS transport across the Lpt bridge is also regulated has been unclear. Here we present three structures of the trans-envelope Lpt complex in LPS-free, LPS-bound and ATP-bound states, along with a structure of a partial bridge. These structures, combined with biochemical experiments, show that Lpt bridge assembly triggers movement of the transmembrane helix of LptC (TM-LptC) in an LPS-dependent manner, resulting in increased ATP binding and hydrolysis. We also show that LPS transport in vivo requires bridge formation and movement of the TM-LptC. Our data support a model in which assembled Lpt bridges respond to the presence of LPS in the inner membrane to turn on transport by moving the TM-LptC, thus coordinating LPS transport activity with bridge assembly and the presence of LPS at the inner membrane. Structures of the protein machinery in Gram-negative bacteria that transports lipopolysaccharide (LPS) from the cytoplasmic membrane to the outer leaflet of the outer membrane reveal how conformational changes in response to the presence of LPS promote its transport.