Combining Mutations That Inhibit Two Distinct Steps of the ATP Hydrolysis Cycle Restores Wild-Type Function in the Lipopolysaccharide Transporter and Shows that ATP Binding Triggers Transport

Abstract

ATP-binding cassette (ABC) transporters constitute a large family of proteins present in all domains of life. They are powered by dynamic ATPases that harness energy from binding and hydrolyzing ATP through a cycle that involves the closing and reopening of their two ATP-binding domains. The LptB2FGC exporter is an essential ABC transporter that assembles lipopolysaccharides (LPS) on the surface of Gram-negative bacteria to form a permeability barrier against many antibiotics. LptB2FGC extracts newly synthesized LPS molecules from the inner membrane and powers their transport across the periplasm and through the outer membrane. How LptB2FGC functions remains poorly understood. Here, we show that the C-terminal domain of the dimeric LptB ATPase is essential for LPS transport in Escherichia coli. Specific changes in the C-terminal domain of LptB cause LPS transport defects that can be repaired by intragenic suppressors altering the ATP-binding domains. Surprisingly, we found that each of two lethal changes in the ATP-binding and C-terminal domains of LptB, when present in combined form, suppressed the defects associated with the other to restore LPS transport to wild-type levels both in vivo and in vitro. We present biochemical evidence explaining the effect that each of these mutations has on LptB function and how the observed cosuppression results from the opposing lethal effects these changes have on the dimerization state of the LptB ATPase. We therefore propose that these sites modulate the closing and reopening of the LptB dimer, providing insight into how the LptB2FGC transporter cycles to export LPS to the cell surface and how to inhibit this essential envelope biogenesis process. IMPORTANCE Gram-negative bacteria are naturally resistant to many antibiotics because their surface is covered by the glycolipid LPS. Newly synthesized LPS is transported across the cell envelope by the multiprotein Lpt machinery, which includes LptB2FGC, an unusual ABC transporter that extracts LPS from the inner membrane. Like in other ABC transporters, the LptB2FGC transport cycle is driven by the cyclical conformational changes that a cytoplasmic, dimeric ATPase, LptB, undergoes when binding and hydrolyzing ATP. How these conformational changes are controlled in ABC transporters is poorly understood. Here, we identified two lethal changes in LptB that, when combined, remarkably restore wild-type transport function. Biochemical studies revealed that the two changes affect different steps in the transport cycle, having opposing, lethal effects on LptB’s dimerization cycle. Our work provides mechanistic details about the LptB2FGC extractor that could be used to develop Lpt inhibitors that would overcome the innate antibiotic resistance of Gram-negative bacteria.

Cryo-EM structure of the full-length WzmWzt ABC transporter required for lipid-linked O antigen transport

“…In the case of the LPS ABC transporter LptB 2 FGC, replacing Tyr234 (equivalent to Wzt Tyr232) with alanine (LptB Y234A) abolished LPS transport, underscoring its importance for function. Notably, substituting LptB Tyr234 with phenylalanine (LptB Y234F) had only a minimal effect on LPS transport (42). Thus, the C-terminal helix of the NBD with its conserved aromatic residue may function as a general modulator of ATPase activity responding to different stimuli, such as O antigen binding to WzmWzt.…”

LptB2FG is an ABC transporter with Adenylate Kinase activity regulated by LptC/A recruitment

“…Number of mutations in LptB 2 FG were studied for their effect on the complex activity, either biochemically or for their in vivo functionality (23, 24). Two SMA-LptB 2 FG complexes carrying already characterized mutations were purified, one on LptF periplasmic side, and one in the ATPase domain(LptB).…”

Assembly and Maintenance of Lipids at the Bacterial Outer Membrane

“…The Lpt system poses the additional challenge of requiring two membranes and components in every compartment of the cell (Figures and ). Nevertheless, biochemical studies have overcome these challenges and have been invaluable for characterizing Lpt factors, characterizing if and how they interact with one another and LPS, determining the role of ATP in LPS transport, and ultimately providing the full in vitro reconstitution of the Lpt machine. ,,− These accomplishments have involved a variety of methodologies that we highlight below.…”

“…More recently, this reconstitution assay was modified to quantifiably measure LPS incorporation into the outer membrane by preloading outer-membrane-like proteoliposomes with dansylated polymyxin B nonapeptide, which binds to LPS and increases in fluorescence when it is incorporated into the membrane . Additional modifications to these reconstitutions and implementation of ATPase assays have been used to examine the effect of small molecules on the transporter, as well as to provide mechanistic insight into transport. ,, …”

“…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.…”