# Regulated Assembly of LPS, Its Structural Alterations and Cellular Response to LPS Defects
## 7 Transport of LPS
### LPS Translocation and Assembly in the Outer Membrane  
After the flipping of LPS by MsbA to the periplasmic side of IM, LPS is transported for its final localization in the OM which requires a complex of seven essential conserved proteins LptA-LptG, whose components reside in every cell compartment and form a single transenvelope complex spanning from the IM to the OM. Here, we briefly summarise key elements, as this topic has been aptly reviewed recently [44; 45; 46; 47]. The Lpt system is organized into the IM complex comprised of LptB2 CFG and the OM component of 1:1 complex of LptDE, which are bridged by a periplasmic protein component LptA. This transenvelope complex acts as a single unit to transport LPS, since depletion of any component leads to LPS accumulation at the IM [44; 45; 46; 47]. Mechanistically, the ABC transporter LptB2 FG extracts LPS from the IM in an ATP-dependent manner to deliver to LptC, and LPS transfer from LptC to LptA requires additional ATP hydrolysis [44; 45; 46; 47], allowing LPS transit across the aqueous periplasm for its final delivery to LptD. The evidence from co-sedimentation, pull-down experiments, photo-cross-linking, mutational and structural analyses, assembly of liposomes from IM components and OM components in the presence or absence of LptA, shows that, while the N-terminus of LptA interacts with LptC at the IM, the C-terminus of LptA interacts with the periplasmic domain of the OM LptD, creating a continuous bridge of antiparallel \(\beta\)-strands between the IM and the OM [44; 45; 46; 47]. The presence of \(\beta\)-jellyroll domains in LptG, LptF, LptC, LptA and LptD can sequester the acyl chains of LPS from the aqueous periplasm, using ATPase activity of LptB to transfer LPS over transenvelope bridge [48]. In support of the single stable bridge model of LPS transport, it was shown that LptA can promote association of liposomes containing the IM complex of LptBCFG with OM liposomes containing the OM LptDE [49]. Furthermore, ATP-dependent transfer of LPS from LptBFG to LptC and LptA has been demonstrated in liposomes and transfer to LptA was shown to be increased when LptC was present [49]. Structural studies of LptD and LptE revealed that LptD folds into two domains: a \(\beta\)-jellyroll and a \(\beta\)-barrel [50; 51]. The \(\beta\)-jellyroll extends away from the OM and interacts with the \(\beta\)-jellyroll domain of LptA suitable for binding to lipid A, while leaving the LPS oligosaccharidescharide exposed. LPS could be delivered directly into the cavity of the larger domain of LptD \(\beta\)-barrel, which is composed of 26 membrane-spanning \(\beta\)-strands. LptE adopts a roll-like structure located inside the barrel of LptD to form a unique two-protein 'barrel and plug' architecture [50; 51]. The plugging of LptE inside of the LptD barrel causes a diminished lumen size to 45A \(\times\) 35A on the periplasmic side. However, such a space is sufficient to accommodate LPS. In these structures, \(\beta\)-strands 1 and 2 are distorted and weak hydrogen bonding between \(\beta\)-strands 1 and 26 could support a lateral opening for LPS migration. In this process, positioning of the lipid A part could be assisted by LptE, as it is known to bind LPS and oligosaccharidescharide could be placed in the hydrophilic cavity of LptD chamber. Finally, LptD folding into the functional state can be a rate-limiting step. Several periplasmic folding catalysts like DsbA, DsbC, SurA, FkpA are known to play important roles in the folding of OMPs and periplasmic proteins [52]. Folding of LptD requires the SurA periplasmic folding catalyst and other periplasmic folding factors, like Skp and KkpA, could further modulate efficient folding of LptD [22]. LptD has two disulfide bridges between four non-consecutive cysteine residues, which require the periplasmic DsbA disulfide oxidoreductase and interaction of LptD with LptE to achieve correct folding with native disulfide bridges [53].