Abstract
Lipopolysaccharide (LPS) is a main component of the outer membrane of Gram-negative bacteria, which is essential for the vitality of most Gram-negative bacteria and plays a critical role for drug resistance. LptD/E complex forms a N-terminal LPS transport slide, a hydrophobic intramembrane hole and the hydrophilic channel of the barrel, for LPS transport, lipid A insertion and core oligosaccharide and O-antigen polysaccharide translocation, respectively. However, there is no direct evidence to confirm that LptD/E transports LPS from the periplasm to the external leaflet of the outer membrane. By replacing LptD residues with an unnatural amino acid p-benzoyl-L-phenyalanine (pBPA) and UV-photo-cross-linking in E.coli, the translocon and LPS intermediates were obtained at the N-terminal domain, the intramembrane hole, the lumenal gate, the lumen of LptD channel, and the extracellular loop 1 and 4, providing the first direct evidence and “snapshots” to reveal LPS translocation steps across the outer membrane.
Highlights
The N-terminal domain of LptD directly delivers lipid A into the hydrophobic intramembrane hole of LptD, where it is inserted into the hydrophobic bilayer of the outer membrane, while the hydrophilic core oligosaccharide and O-antigen polysaccharide are translocated through the channel formed by LptD and LptE18
We selected 9 residues at the N-terminal domain for incorporation of pBPA. All these residues are located along the hydrophobic groove of the N-terminal domain with their side chains pointing to the core of the jellyroll structure
Molecular dynamics simulations and functional assays have identified the intramembrane hole within LptD, which we suggest is where the lipid A of lipopolysaccharide is directly inserted into the hydrophobic bilayer of the outer membrane[18]
Summary
The N-terminal domain of LptD directly delivers lipid A into the hydrophobic intramembrane hole of LptD, where it is inserted into the hydrophobic bilayer of the outer membrane, while the hydrophilic core oligosaccharide and O-antigen polysaccharide are translocated through the channel formed by LptD and LptE18. To confirm whether the O-antigen and core oligosaccharide are translocated through the LptD channel, 11 residues located in the lumen of the channel were selected for the incorporation of pBPA and UV-crosslinking (Fig. 1b and text below).
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have