LptB‐LptF coupling mediates the closure of the substrate‐binding cavity in the LptB₂FGC transporter through a rigid‐body mechanism to extract LPS

Abstract

Lipopolysaccharides (LPS) are essential envelope components in many Gram‐negative bacteria and provide intrinsic resistance to antibiotics. LPS molecules are synthesized in the inner membrane and then transported to the cell surface by the LPS transport (Lpt) machinery. In this system, the ATP‐binding cassette (ABC) transporter LptB2FGC extracts LPS from the inner membrane and places it onto a periplasmic protein bridge through a poorly understood mechanism. Here, we show that residue E86 of LptB is essential for coupling the function of this ATPase to that of its partners LptFG, specifically at the step where ATP binding drives the closure of the LptB dimer and the collapse of the LPS‐binding cavity in LptFG that moves LPS to the Lpt periplasmic bridge. We also show that defects caused by changing residue E86 are suppressed by mutations altering either LPS structure or transmembrane helices in LptG. Furthermore, these suppressors also fix defects in the coupling helix of LptF, but not of LptG. Together, these results support a transport mechanism in which the ATP‐driven movements of LptB and those of the substrate‐binding cavity in LptFG are bi‐directionally coordinated through the rigid‐body coupling, with LptF’s coupling helix being important in coordinating cavity collapse with LptB dimerization.

Assembly and Maintenance of Lipids at the Bacterial Outer Membrane

“…More recently, genetics have provided additional mechanistic insight into the closure of the LptFG cavity that LptB triggers when binding ATP. Changes to the essential groove region (residue E86 of LptB), like the R144H change, can reduce the ability of the LptB dimer to bind ATP and attain the closed-dimer conformation that triggers cavity collapse . Both of these defects in LptB can be suppressed by changing either the structure of LPS via Δ lpxM or the TM helices in LptG.…”

“…These studies also revealed that, although they are structurally similar, the coupling helices of LptFG play distinct roles in the transport cycle, as changing equivalent residues in each coupling helix does not confer the same phenotypes . Indeed, as we describe below, recent studies have proposed that the conserved glutamate in the coupling helix of LptF, but not the structurally equivalent residue in that of LptG, is critical in mediating the closure of the LptFG cavity …”

“…Further insight into the NBD–TMD coupling that is mediated by the interactions between the LptFG coupling helices and the groove region in LptB surprisingly came from the antibiotic novobiocin . This antibiotic targets DNA gyrase and is often used in assessing the effect of Lpt defects on outer-membrane permeability because it is hydrophobic. ,,, Serendipitously, novobiocin was found to suppress defects in LPS transport caused by specific mutations that we now know affect NBD–TMD coupling. , Structural and biochemical studies have demonstrated that, in addition to binding to its canonical target, DNA gyrase, novobiocin also binds to the groove region of LptB, where it forms contacts with residues that were previously identified as being important for LptB function. ,,, As demonstrated by an in vitro reconstitution LPS release assay, through this binding, novobiocin increases LPS transport, which leads to suppression of coupling defects . The mechanism for how novobiocin increases LPS transport remains to be elucidated, but these findings open the door for the development of small molecules that might interfere with LPS transport.…”

Cardiolipin aids in lipopolysaccharide transport to the gram-negative outer membrane

“…6). Therefore, MsbA cannot transport penta-acylated lipid A as efficiently as hexa-acylated lipid A. Penta-acylated lipid A produced by lpxM mutants was recently shown to have altered binding to the hydrophobic pockets in the Lpt system, which transports LPS from the periplasmic leaflet of the IM to the OM, further supporting that the acylation state of the substrate affects LPS transport systems (48).…”

Resensitizing resistant Escherichia Coli ST131 to Macrolide using Fluoroquinolones ‎

“…The malEFG, metNIQ, and potABCD operons are responsible for the biosynthesis of maltose, L/D methionine, and spermidine ABC transporters, respectively [28][29][30][31]. The lptFG operon alongside with lptCAB operon involves in the biosynthesis of the lipopolysaccharide ABC transporter system [32,33]. The genes enriched in the lipoprotein-macrolide ABC transporter network are all downregulated (Figure 4b).…”