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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.


SEC-MALS analysis of the MlaF(His-E)DB complex.Molecular mass: 256 kDa (predicted, MlaF2E2D6B2), 285 ( ± 0.5%) kDa (observed). Molecular masses of DDM fraction in complex and free micelles are 35 ( ± 4.4%) kDa and 65 ( ± 2.1%) kDa, respectively. Numbers stated after ± show statistical consistency of analysis.DOI:http://dx.doi.org/10.7554/eLife.19042.007
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fig1s4: SEC-MALS analysis of the MlaF(His-E)DB complex.Molecular mass: 256 kDa (predicted, MlaF2E2D6B2), 285 ( ± 0.5%) kDa (observed). Molecular masses of DDM fraction in complex and free micelles are 35 ( ± 4.4%) kDa and 65 ( ± 2.1%) kDa, respectively. Numbers stated after ± show statistical consistency of analysis.DOI:http://dx.doi.org/10.7554/eLife.19042.007

Mentions: MlaD appears to form an oligomeric structure stable to SDS when purified as part of the IM complex. Full length MlaD comprises an N-terminal transmembrane helix and a large C-terminal periplasmic substrate-binding domain (Figure 2A) (UniProtKB P64604) (Magrane and the UniProt consortium, 2011). To determine whether this oligomeric state is dependent on the transmembrane helix or its association with the complex, we over-expressed and purified the substrate-binding domain (or soluble domain) of MlaD (sMlaD-His) alone for characterization. We found that sMlaD-His does not exist in the monomeric state, as judged by its SEC profile (Figure 2B). Multi-angle light scattering (MALS) analysis revealed that the absolute molar mass of purified sMlaD-His is ~110.7 kDa, establishing that these are in fact hexamers (Figure 2C). Furthermore, the experimental molar mass of the MlaFEDB complex (~285 kDa by SEC-MALS) is consistent with the presence of six copies of MlaD (Figure 1—figure supplement 4). Remarkably, hexamers formed by sMlaD-His alone are also resistant to SDS denaturation (Figure 2B). These results indicate that oligomerization and extreme stability are unique properties of the soluble domain.10.7554/eLife.19042.008Figure 2.MlaD forms SDS-resistant hexamers via its soluble domain.


Defining key roles for auxiliary proteins in an ABC transporter that maintains bacterial outer membrane lipid asymmetry
SEC-MALS analysis of the MlaF(His-E)DB complex.Molecular mass: 256 kDa (predicted, MlaF2E2D6B2), 285 ( ± 0.5%) kDa (observed). Molecular masses of DDM fraction in complex and free micelles are 35 ( ± 4.4%) kDa and 65 ( ± 2.1%) kDa, respectively. Numbers stated after ± show statistical consistency of analysis.DOI:http://dx.doi.org/10.7554/eLife.19042.007
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5016091&req=5

fig1s4: SEC-MALS analysis of the MlaF(His-E)DB complex.Molecular mass: 256 kDa (predicted, MlaF2E2D6B2), 285 ( ± 0.5%) kDa (observed). Molecular masses of DDM fraction in complex and free micelles are 35 ( ± 4.4%) kDa and 65 ( ± 2.1%) kDa, respectively. Numbers stated after ± show statistical consistency of analysis.DOI:http://dx.doi.org/10.7554/eLife.19042.007
Mentions: MlaD appears to form an oligomeric structure stable to SDS when purified as part of the IM complex. Full length MlaD comprises an N-terminal transmembrane helix and a large C-terminal periplasmic substrate-binding domain (Figure 2A) (UniProtKB P64604) (Magrane and the UniProt consortium, 2011). To determine whether this oligomeric state is dependent on the transmembrane helix or its association with the complex, we over-expressed and purified the substrate-binding domain (or soluble domain) of MlaD (sMlaD-His) alone for characterization. We found that sMlaD-His does not exist in the monomeric state, as judged by its SEC profile (Figure 2B). Multi-angle light scattering (MALS) analysis revealed that the absolute molar mass of purified sMlaD-His is ~110.7 kDa, establishing that these are in fact hexamers (Figure 2C). Furthermore, the experimental molar mass of the MlaFEDB complex (~285 kDa by SEC-MALS) is consistent with the presence of six copies of MlaD (Figure 1—figure supplement 4). Remarkably, hexamers formed by sMlaD-His alone are also resistant to SDS denaturation (Figure 2B). These results indicate that oligomerization and extreme stability are unique properties of the soluble domain.10.7554/eLife.19042.008Figure 2.MlaD forms SDS-resistant hexamers via its soluble domain.

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.