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Defining key roles for auxiliary proteins in an ABC transporter that maintains bacterial outer membrane lipid asymmetry

View Article: PubMed Central - PubMed

ABSTRACT

In Gram-negative bacteria, lipid asymmetry is critical for the function of the outer membrane (OM) as a selective permeability barrier, but how it is established and maintained is poorly understood. Here, we characterize a non-canonical ATP-binding cassette (ABC) transporter in Escherichia coli that provides energy for maintaining OM lipid asymmetry via the transport of aberrantly localized phospholipids (PLs) from the OM to the inner membrane (IM). We establish that the transporter comprises canonical components, MlaF and MlaE, and auxiliary proteins, MlaD and MlaB, of previously unknown functions. We further demonstrate that MlaD forms extremely stable hexamers within the complex, functions in substrate binding with strong affinity for PLs, and modulates ATP hydrolytic activity. In addition, MlaB plays critical roles in both the assembly and activity of the transporter. Our work provides mechanistic insights into how the MlaFEDB complex participates in ensuring active retrograde PL transport to maintain OM lipid asymmetry.

Doi:: http://dx.doi.org/10.7554/eLife.19042.001

No MeSH data available.


Related in: MedlinePlus

MS analyses of sMlaD-His.(A) Positive-mode ESI-MS spectrum of sMlaD-His under denaturing conditions (50% acetonitrile 0.2% formic acid), revealing monomeric sMlaD-His at various charge states. Inset: Deconvoluted spectrum of monomeric sMlaD-His(experimental 17828.32 Da, theoretical 17827.77 Da). (B) Positive mode ESI-MS spectrum of sMlaD-His under non-denaturing conditions (20 mM ammonium acetate, pH 6.9) with increasing collision energies (0 V (black), 80 V (green), 100 V (pink) and 150 V (blue)), progressively revealing hexameric sMlaD-His with four to zero bound PL molecules.DOI:http://dx.doi.org/10.7554/eLife.19042.011
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fig3s2: MS analyses of sMlaD-His.(A) Positive-mode ESI-MS spectrum of sMlaD-His under denaturing conditions (50% acetonitrile 0.2% formic acid), revealing monomeric sMlaD-His at various charge states. Inset: Deconvoluted spectrum of monomeric sMlaD-His(experimental 17828.32 Da, theoretical 17827.77 Da). (B) Positive mode ESI-MS spectrum of sMlaD-His under non-denaturing conditions (20 mM ammonium acetate, pH 6.9) with increasing collision energies (0 V (black), 80 V (green), 100 V (pink) and 150 V (blue)), progressively revealing hexameric sMlaD-His with four to zero bound PL molecules.DOI:http://dx.doi.org/10.7554/eLife.19042.011

Mentions: To further characterize the interaction between MlaD and PLs, we analyzed purified sMlaD-His using MS. In denaturing MS, the monomeric form of sMlaD-His (deconvoluted mass 17,828 Da) is the only molecular species observed (Figure 3—figure supplement 2A). In native MS, we detected the hexameric form of sMlaD-His with bound ligands at various charge states (Figure 3C). Deconvolution of the native MS spectrum indicates the molecular weight of the native hexamers to be ~110 kDa, suggesting the presence of at least four bound PL molecules (~750 Da each, in agreement with the average mass of PE and PG) and therefore, strong protein-PL interactions. By increasing the collision energy in the mass spectrometer collision cell, we are then able to destabilize the native structure, revealing the presence of hexamers binding three, two, one or zero PL molecules (Figure 3—figure supplement 2B). This suggests that the presence of ligands is not strictly required for the formation of sMlaD hexamers. Taken together, our data establish that hexameric sMlaD is a PL-binding complex with at least four binding sites.


Defining key roles for auxiliary proteins in an ABC transporter that maintains bacterial outer membrane lipid asymmetry
MS analyses of sMlaD-His.(A) Positive-mode ESI-MS spectrum of sMlaD-His under denaturing conditions (50% acetonitrile 0.2% formic acid), revealing monomeric sMlaD-His at various charge states. Inset: Deconvoluted spectrum of monomeric sMlaD-His(experimental 17828.32 Da, theoretical 17827.77 Da). (B) Positive mode ESI-MS spectrum of sMlaD-His under non-denaturing conditions (20 mM ammonium acetate, pH 6.9) with increasing collision energies (0 V (black), 80 V (green), 100 V (pink) and 150 V (blue)), progressively revealing hexameric sMlaD-His with four to zero bound PL molecules.DOI:http://dx.doi.org/10.7554/eLife.19042.011
© Copyright Policy
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC5016091&req=5

fig3s2: MS analyses of sMlaD-His.(A) Positive-mode ESI-MS spectrum of sMlaD-His under denaturing conditions (50% acetonitrile 0.2% formic acid), revealing monomeric sMlaD-His at various charge states. Inset: Deconvoluted spectrum of monomeric sMlaD-His(experimental 17828.32 Da, theoretical 17827.77 Da). (B) Positive mode ESI-MS spectrum of sMlaD-His under non-denaturing conditions (20 mM ammonium acetate, pH 6.9) with increasing collision energies (0 V (black), 80 V (green), 100 V (pink) and 150 V (blue)), progressively revealing hexameric sMlaD-His with four to zero bound PL molecules.DOI:http://dx.doi.org/10.7554/eLife.19042.011
Mentions: To further characterize the interaction between MlaD and PLs, we analyzed purified sMlaD-His using MS. In denaturing MS, the monomeric form of sMlaD-His (deconvoluted mass 17,828 Da) is the only molecular species observed (Figure 3—figure supplement 2A). In native MS, we detected the hexameric form of sMlaD-His with bound ligands at various charge states (Figure 3C). Deconvolution of the native MS spectrum indicates the molecular weight of the native hexamers to be ~110 kDa, suggesting the presence of at least four bound PL molecules (~750 Da each, in agreement with the average mass of PE and PG) and therefore, strong protein-PL interactions. By increasing the collision energy in the mass spectrometer collision cell, we are then able to destabilize the native structure, revealing the presence of hexamers binding three, two, one or zero PL molecules (Figure 3—figure supplement 2B). This suggests that the presence of ligands is not strictly required for the formation of sMlaD hexamers. Taken together, our data establish that hexameric sMlaD is a PL-binding complex with at least four binding sites.

View Article: PubMed Central - PubMed

ABSTRACT

In Gram-negative bacteria, lipid asymmetry is critical for the function of the outer membrane (OM) as a selective permeability barrier, but how it is established and maintained is poorly understood. Here, we characterize a non-canonical ATP-binding cassette (ABC) transporter in Escherichia coli that provides energy for maintaining OM lipid asymmetry via the transport of aberrantly localized phospholipids (PLs) from the OM to the inner membrane (IM). We establish that the transporter comprises canonical components, MlaF and MlaE, and auxiliary proteins, MlaD and MlaB, of previously unknown functions. We further demonstrate that MlaD forms extremely stable hexamers within the complex, functions in substrate binding with strong affinity for PLs, and modulates ATP hydrolytic activity. In addition, MlaB plays critical roles in both the assembly and activity of the transporter. Our work provides mechanistic insights into how the MlaFEDB complex participates in ensuring active retrograde PL transport to maintain OM lipid asymmetry.

Doi:: http://dx.doi.org/10.7554/eLife.19042.001

No MeSH data available.


Related in: MedlinePlus