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Characterization of Interactions and Phospholipid Transfer between Substrate Binding Proteins of the OmpC-Mla System

  • Bilge Ercan
    Bilge Ercan
    Department of Chemistry, National University of Singapore, Singapore 117543
    More by Bilge Ercan
  • Wen-Yi Low
    Wen-Yi Low
    Department of Chemistry, National University of Singapore, Singapore 117543
    More by Wen-Yi Low
  • Xuejun Liu
    Xuejun Liu
    School of Pharmacy, University College London, London WC1N 1AX, United Kingdom
    More by Xuejun Liu
  • , and 
  • Shu-Sin Chng*
    Shu-Sin Chng
    Department of Chemistry, National University of Singapore, Singapore 117543
    Singapore Center for Environmental Life Sciences Engineering (SCELSE-NUS), Singapore 117456
    *E-mail: [email protected]
    More by Shu-Sin Chng
Cite this: Biochemistry 2019, 58, 2, 114–119
Publication Date (Web):October 4, 2018
Copyright © 2018 American Chemical Society

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    Abstract Image

    The outer membrane (OM) of Gram-negative bacteria is a permeability barrier that impedes the entry of external insults, such as antibiotics and bile salts. This barrier function depends critically on the asymmetric lipid distribution across the bilayer, with lipopolysaccharides (LPS) facing outside and phospholipids (PLs) facing inside. In Escherichia coli, the OmpC-Mla system is believed to maintain OM lipid asymmetry by removing surface exposed PLs and shuttling them back to the inner membrane (IM). How proteins in the pathway interact to mediate PL transport across the periplasm is not known. Evidence for direct transfer of PLs between these proteins is also lacking. In this study, we mapped the interaction surfaces between the two PL-binding proteins, MlaC and MlaD, using site-specific in vivo photo-cross-linking, and obtained a physical picture for how these proteins may transfer PLs. Furthermore, we demonstrated using purified proteins that MlaD spontaneously transfers PLs to MlaC, suggesting that the latter has a higher affinity for PLs. Our work provides insights into the mechanism of bacterial intermembrane lipid transport important for the maintenance of OM lipid asymmetry.

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    Supporting Information

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.8b00897.

    • Materials and methods, bacteria strains, plasmids, and primers used in this study, MlaCV171pBpa-His interactions with membrane proteins, analysis of SDS/EDTA sensitivity, MlaC-His copurification with PLs and interaction with the periplasmic soluble domain of MlaD, TLC analyses, and SEC profiles (PDF)

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    24. Nicolas Coudray, Georgia L Isom, Mark R MacRae, Mariyah N Saiduddin, Gira Bhabha, Damian C Ekiert. Structure of bacterial phospholipid transporter MlaFEDB with substrate bound. eLife 2020, 9
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    33. Paola Sperandeo, Alessandra M. Martorana, Alessandra Polissi. Lipopolysaccharide Biosynthesis and Transport to the Outer Membrane of Gram-Negative Bacteria. 2019, 9-37.

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