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ATP disrupts lipid-binding equilibrium to drive retrograde transport critical for bacterial outer membrane asymmetry.

ABSTRACT: The hallmark of the gram-negative bacterial envelope is the presence of the outer membrane (OM). The OM is asymmetric, comprising lipopolysaccharides (LPS) in the outer leaflet and phospholipids (PLs) in the inner leaflet; this critical feature confers permeability barrier function against external insults, including antibiotics. To maintain OM lipid asymmetry, the OmpC-Mla system is believed to remove aberrantly localized PLs from the OM and transport them to the inner membrane (IM). Key to the system in driving lipid trafficking is the MlaFEDB ATP-binding cassette transporter complex in the IM, but mechanistic details, including transport directionality, remain enigmatic. Here, we develop a sensitive point-to-point in vitro lipid transfer assay that allows direct tracking of [14C]-labeled PLs between the periplasmic chaperone MlaC and MlaFEDB reconstituted into nanodiscs. We reveal that MlaC spontaneously transfers PLs to the IM transporter in an MlaD-dependent manner that can be further enhanced by coupled ATP hydrolysis. In addition, we show that MlaD is important for modulating productive coupling between ATP hydrolysis and such retrograde PL transfer. We further demonstrate that spontaneous PL transfer also occurs from MlaFEDB to MlaC, but such anterograde movement is instead abolished by ATP hydrolysis. Our work uncovers a model where PLs reversibly partition between two lipid-binding sites in MlaC and MlaFEDB, and ATP binding and/or hydrolysis shift this equilibrium to ultimately drive retrograde PL transport by the OmpC-Mla system. These mechanistic insights will inform future efforts toward discovering new antibiotics against gram-negative pathogens.

SUBMITTER: Low WY

PROVIDER: S-EPMC8685716 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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ATP disrupts lipid-binding equilibrium to drive retrograde transport critical for bacterial outer membrane asymmetry.

Low Wen-Yi WY Thong Shuhua S Chng Shu-Sin SS

Proceedings of the National Academy of Sciences of the United States of America 20211201 50

The hallmark of the gram-negative bacterial envelope is the presence of the outer membrane (OM). The OM is asymmetric, comprising lipopolysaccharides (LPS) in the outer leaflet and phospholipids (PLs) in the inner leaflet; this critical feature confers permeability barrier function against external insults, including antibiotics. To maintain OM lipid asymmetry, the OmpC-Mla system is believed to remove aberrantly localized PLs from the OM and transport them to the inner membrane (IM). Key to the ...[more]

PMID: 34873038

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Project description:Voltage-dependent anion channel (VDAC), the major channel of the mitochondrial outer membrane, serves as a principal pathway for ATP, ADP, and other respiratory substrates across this membrane. Using umbrella-sampling simulations, we established the thermodynamic and kinetic components governing ATP transport across the VDAC1 channel. We found that there are several low-affinity binding sites for ATP along the translocation pathway and that the main barrier for ATP transport is located around the center of the channel and is formed predominantly by residues in the N-terminus. The binding affinity of ATP to an open channel was found to be in the millimolar to micromolar range. However, we show that this weak binding increases the ATP translocation probability by about 10-fold compared with the VDAC pore in which attractive interactions were artificially removed. Recently, it was found that free dimeric tubulin induces a highly efficient, reversible blockage of VDAC reconstituted into planar lipid membranes. It was proposed that by blocking VDAC permeability for ATP/ADP and other mitochondrial respiratory substrates tubulin controls mitochondrial respiration. Using the Rosetta protein-protein docking algorithm, we established a tentative structure of the VDAC-tubulin complex. An extensive set of equilibrium and nonequilibrium (under applied electric field) molecular dynamics (MD) simulations was used to establish the conductance of the open and blocked channel. It was found that the presence of the unstructured C-terminal tail of tubulin in the VDAC pore decreases its conductance by more than 40% and switches its selectivity from anionic to cationic. The subsequent 1D potential of mean force (PMF) computations for the VDAC-tubulin complex show that the state renders ATP transport virtually impossible. A number of residues pivotal for tubulin binding to the channel were identified that help to clarify the molecular details of VDAC-tubulin interaction and to provide new insight into the mechanism of the control of mitochondria respiration by VDAC.

Dataset Information

ATP disrupts lipid-binding equilibrium to drive retrograde transport critical for bacterial outer membrane asymmetry.

Publications

ATP disrupts lipid-binding equilibrium to drive retrograde transport critical for bacterial outer membrane asymmetry.

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