Energetics of lipid transport by the ABC transporter MsbA is lipid dependent

Abstract

The ABC multidrug exporter MsbA mediates the translocation of lipopolysaccharides and phospholipids across the plasma membrane in Gram-negative bacteria. Although MsbA is structurally well characterised, the energetic requirements of lipid transport remain unknown. Here, we report that, similar to the transport of small-molecule antibiotics and cytotoxic agents, the flopping of physiologically relevant long-acyl-chain 1,2-dioleoyl (C18)-phosphatidylethanolamine in proteoliposomes requires the simultaneous input of ATP binding and hydrolysis and the chemical proton gradient as sources of metabolic energy. In contrast, the flopping of the large hexa-acylated (C12-C14) Lipid-A anchor of lipopolysaccharides is only ATP dependent. This study demonstrates that the energetics of lipid transport by MsbA is lipid dependent. As our mutational analyses indicate lipid and drug transport via the central binding chamber in MsbA, the lipid availability in the membrane can affect the drug transport activity and vice versa.

Biosensor for Multimodal Characterization of an Essential ABC Transporter for Next-Generation Antibiotic Research

“…The substratebinding chamber of this mutant lacks three arginine residues (R78, R148, and R296) that are critical for Lipid-A and PE transport. 9 However, consistent with previous comparisons of ethidium transport by MsbA-WT and MsbA-TripRA in cells, the ethidium + transport activity in proteoliposomes near Vmax conditions (Figure S8), as well as the ATPase activity of the TripRA mutant (117 ± 12 nmol Pi/min/mg), were not significantly (P = 0.5509) different from the observations for WT protein (100 ± 13 nmol Pi/min/mg). Therefore, the EIS measurements, which are nonselective for K + , Na + , and perhaps other cations, appear to be associated with ethidium transport rather than lipid flipping by MsbA.…”

“…To confirm that the observed response in MsbA-containing SLBs is linked to ATP binding and hydrolysis by MsbA, 100 nM ATP hydrolysis and transport inhibitor G907 , was introduced into the system. By wedging into a conserved transmembrane pocket, G907 and related quinoline compounds trap MsbA in an inward-facing conformation.…”

“…Using AFM images to estimate the amount of protein in the SLBs, this equates to 100 ± 13 nmol Pi/min/mg equivalent to the ATPase rates obtained for suspended proteoliposomes of the same lipid composition. 9 In the case of the MsbA-ΔK382 SLBs, an ATPase activity of 10 ± 4 nmol Pi/min/mg was obtained, which is ∼10% of the MsbA-WT. From these data, we conclude that the ATP-dependent resistance changes in the MsbA-containing SLBs require ATP binding and subsequent hydrolysis by MsbA.…”

“…They provide important drug targets in our quest to develop next-generation antibiotics . One of these targets is the ATP-binding cassette (ABC) transporter MsbA in the plasma membrane of Gram-negative ESKAPE bacteria and in Escherichia coli, Salmonella typhimurium, Vibrio cholerae, and others. − MsbA plays an essential role in the viability and survival of these bacteria by mediating the translocation of core-Lipid-A and glycerophospholipids across the plasma membrane. − In further steps at the outer leaflet of the plasma membrane, a polysaccharide moiety (O-antigen) is ligated to core Lipid-A to form full-length lipopolysaccharides (LPS) which, together with phospholipids, are essential for forming the protective and rigid outer membrane that make Gram-negative bacteria so impervious to antibiotics. , Furthermore, as MsbA has been experimentally more accessible than some of its mammalian homologues over the past two decades, it has also been studied as a model system, enabling advancements in our understanding of protein structures and transport mechanisms in the ABC superfamily.…”

Drug‐dependent inhibition of nucleotide hydrolysis in the heterodimeric ABC multidrug transporter PatAB from Streptococcus pneumoniae

“…The transport of daunomycin or ethidium was followed in real‐time in L . lactis cells [63]. 50 mL cultures containing cells with expressed PatAB proteins were harvested by centrifugation at 16 °C (6500× g , 10 min) and washed once with wash buffer (50 m m KPi, 5 m m MgSO 4 , pH 7.0).…”