Lipopolysaccharide Transport Involves Long-Range Coupling between Cytoplasmic and Periplasmic Domains of the LptB ₂ FGC Extractor

Abstract

The cell surface of the Gram-negative cell envelope contains lipopolysaccharide (LPS) molecules, which form a permeability barrier against hydrophobic antibiotics. The LPS transport (Lpt) machine composed of LptB2FGCADE forms a proteinaceous trans-envelope bridge that allows for the rapid and specific transport of newly synthesized LPS from the inner membrane (IM) to the outer membrane (OM). This transport is powered from the IM by the ATP-binding cassette transporter LptB2FGC. The ATP-driven cycling between closed- and open-dimer states of the ATPase LptB2 is coupled to the extraction of LPS by the transmembrane domains LptFG. However, the mechanism by which LPS moves from a substrate-binding cavity formed by LptFG at the IM to the first component of the periplasmic bridge, the periplasmic β-jellyroll domain of LptF, is poorly understood. To better understand how LptB2FGC functions in Escherichia coli, we searched for suppressors of a defective LptB variant. We found that defects in LptB2 can be suppressed by both structural modifications to the core oligosaccharide of LPS and changes in various regions of LptFG, including a periplasmic loop in LptF that connects the substrate-binding cavity in LptFG to the periplasmic β-jellyroll domain of LptF. These novel suppressors suggest that interactions between the core oligosaccharide of LPS and periplasmic regions in the transporter influence the rate of LPS extraction by LptB2FGC. Together, our genetic data reveal a path for the bi-directional coupling between LptB2 and LptFG that extends from the cytoplasm to the entrance to the periplasmic bridge of the transporter. IMPORTANCE Gram-negative bacteria are intrinsically resistant to many antibiotics due to the presence of lipopolysaccharide (LPS) at their cell surface. LPS is transported from its site of synthesis at the inner membrane to the outer membrane by the Lpt machine. Lpt proteins form a transporter that spans the entire envelope and is thought to function similarly to a PEZ candy dispenser. This trans-envelope machine is powered by the cytoplasmic LptB ATPase through a poorly understood mechanism. Using genetic analyses in Escherichia coli, we found that LPS transport involves long-ranging bi-directional coupling across cellular compartments between cytoplasmic LptB and periplasmic regions of the Lpt transporter. This knowledge could be exploited in developing antimicrobials that overcome the permeability barrier imposed by LPS.

The transmembrane α‐helix of LptC participates in LPS extraction by the LptB2FGC transporter

“…As stated earlier, the Q‐loop motif in the NBDs (i.e., ATPase LptB) and the coupling helices in the TMDs (i.e., LptFG) are thought to function in NBD–TMD coupling in ABC transporters (Figure 6b) (Davidson et al, 2008; Luo et al, 2017; Simpson et al, 2016). Accordingly, residue E86 in the Q‐loop of LptB, and residues E84 and E88 in the coupling helices of LptF and LptG, respectively, had been identified as important in coupling the closure of the LptB dimer with that of the LPS‐binding cavity in LptFG (Lundstedt et al, 2020; Lundstedt, Simpson, et al, 2021; Simpson et al, 2016). However, it is not clear whether these residues affect the partial cavity closure caused by the removal of LptC's TM helix (transition from state II to III, Figure 6a) and/or the total closure of the cavity (transition from state III to IV, Figure 6a).…”

“…We therefore suggest that the intercalation of LptC's TM helix into the substrate-binding cavity allows LptB (through its residue E86) to properly coordinate the total collapse of the cavity and release of LPS into the periplasmic bridge. Since LptB's activity has been previously shown to be influenced by residues in the TM helices of LptFG and the periplasmic region of LptF (Baeta et al, 2021;Benedet et al, 2016;Lundstedt et al, 2020), and the physical structure of the Lipid A and core regions of LPS (Lundstedt, Simpson, et al, 2021)…”

“…We therefore suggest that the intercalation of LptC’s TM helix into the substrate‐binding cavity allows LptB (through its residue E86) to properly coordinate the total collapse of the cavity and release of LPS into the periplasmic bridge. Since LptB's activity has been previously shown to be influenced by residues in the TM helices of LptFG and the periplasmic region of LptF (Baeta et al, 2021; Benedet et al, 2016; Lundstedt et al, 2020), and the physical structure of the Lipid A and core regions of LPS (Lundstedt, Simpson, et al, 2021), it remains unknown whether LptC's TM helix prevents premature closing of the substrate‐binding cavity to ensure proper binding of LPS and ATP and/or coordination with the placement of LPS onto the β‐jellyroll of LptF. Moreover, if the Lpt system functions akin to a PEZ candy dispenser and LPS travels as a stream, this slow step might be necessary to maintain a certain flow rate along the Lpt bridge.…”

“…Our data have also shown a functional link between LptC's TM helix and NBD–TMD coupling since lptC ∆TM suppresses lptB ( E86Q ) and lptF ( E84A ) /lptG ( E88A ). We previously had concluded that these lptB/F/G mutants are defective in the closure of the LPS‐binding cavity (Lundstedt et al, 2020; Lundstedt, Simpson, et al, 2021; Simpson et al, 2016). Combining our previous and present data, we propose that, in wild‐type complexes, the removal of LptC's TM helix involves NBD–TMD coupling and specifically residue E86 in LptB and the coupling helices of LptFG.…”

“…Furthermore, that lptB ( E86Q ) is completely suppressed by lptC ∆TM , while the more defective lptB ( E86A ) allele is not, and that lptC ∆TM can only partially suppress lptF ( E84A ) /lptG ( E88A ) suggest that residue E86 of LptB and residues E84 and E88 in the coupling helices of LptF and LptG, respectively, play additional roles in LPS transport. Indeed, earlier work implicated these residues in the transition to state IV that occurs when the LptB dimer closes around two ATP molecules to trigger the total collapse of the LPS‐binding cavity and movement of LPS to the Lpt bridge (Lundstedt et al, 2020; Lundstedt, Simpson, et al, 2021; Simpson et al, 2016).…”