The Gram-negative bacterial outer membrane (OM) is a unique bilayer that forms an efficient permeation barrier to protect the cell from noxious compounds1 , 2. The defining characteristic of the OM is lipid asymmetry, with phospholipids comprising the inner leaflet and lipopolysaccharides comprising the outer leaflet1,2,3. This asymmetry is maintained by the Mla pathway, a six-component system that is widespread in Gram-negative bacteria and is thought to mediate retrograde transport of misplaced phospholipids from the outer leaflet of the OM to the cytoplasmic membrane4. The OM lipoprotein MlaA performs the first step in this process via an unknown mechanism that does not require external energy input. Here we show, using X-ray crystallography, molecular dynamics simulations and in vitro and in vivo functional assays, that MlaA is a monomeric α-helical OM protein that functions as a phospholipid translocation channel, forming a ~20-Å-thick doughnut embedded in the inner leaflet of the OM with a central, amphipathic pore. This architecture prevents access of inner leaflet phospholipids to the pore, but allows outer leaflet phospholipids to bind to a pronounced ridge surrounding the channel, followed by diffusion towards the periplasmic space. Enterobacterial MlaA proteins form stable complexes with OmpF/C5,6, but the porins do not appear to play an active role in phospholipid transport. MlaA represents a lipid transport protein that selectively removes outer leaflet phospholipids to help maintain the essential barrier function of the bacterial OM.

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  1. 1.

    Henderson, J. C. et al. The power of asymmetry: architecture and assembly of the Gram-negative outer membrane lipid bilayer. Annu. Rev. Microbiol. 70, 255–278 (2016).

  2. 2.

    May, K. L. & Silhavy, T. J. Making a membrane on the other side of the wall. Biochim. Biophys. Acta. 1862, 1386-1393 (2017).

  3. 3.

    Nikaido, H. Molecular basis of bacterial outer membrane permeability revisited. Microbiol. Mol. Biol. Rev. 67, 593–656 (2003).

  4. 4.

    Malinverni, J. C. & Silhavy, T. J. An ABC transport system that maintains lipid asymmetry in the Gram-negative outer membrane. Proc. Natl Acad. Sci. USA 106, 8009–8014 (2009).

  5. 5.

    Ekiert, D. C. et al. Architectures of lipid transport systems for the bacterial outer membrane. Cell 169, 273–285 (2017).

  6. 6.

    Chong, Z.-S., Woo, W.-F. & Chng, S.-S. Osmoporin OmpC forms a complex with MlaA to maintain outer membrane lipid asymmetry in Escherichia coli. Mol. Microbiol. 98, 1133–1146 (2015).

  7. 7.

    Snijder, H. J. et al. Structural evidence for dimerization-regulated activation of an integral membrane phospholipase. Nature 401, 717–721 (1999).

  8. 8.

    Bishop, R. E. et al. Transfer of palmitate from phospholipids to lipid A in outer membranes of Gram-negative bacteria. EMBO J. 19, 5071–5080 (2000).

  9. 9.

    Casali, N. & Riley, L. W. A phylogenomic analysis of the Actinomycetales mce operons. BMC Genomics 8, 60 (2007).

  10. 10.

    Awai, K., Xu, C., Tamot, B. & Benning, C. A phosphatidic acid-binding protein of the chloroplast inner envelope membrane involved in lipid trafficking. Proc. Natl Acad. Sci. USA 103, 10817–10822 (2006).

  11. 11.

    Thong, S. et al. Defining key roles for auxiliary proteins in an ABC transporter that maintains bacterial outer membrane lipid asymmetry. eLife 5, e19042 (2016).

  12. 12.

    Zhao, L. et al. Deletion of the vacJ gene affects the biology and virulence in Haemophilus parasuis serovar 5. Gene 603, 42–53 (2017).

  13. 13.

    Carpenter, C. D. et al. The Vps/VacJ ABC transporter is required for intercellular spread of Shigella flexneri. Infect. Immun. 82, 660–669 (2014).

  14. 14.

    Suzuki, T. et al. Identification and characterization of a chromosomal virulence gene, vacJ, required for intercellular spreading of Shigella flexneri. Mol. Microbiol. 11, 31–41 (1994).

  15. 15.

    Shen, L. et al. PA2800 plays an important role in both antibiotic susceptibility and virulence in Pseudomonas aeruginosa. Curr. Microbiol. 65,601–609 (2012).

  16. 16.

    Roier, S. et al. A novel mechanism for the biogenesis of outer membrane vesicles in Gram-negative bacteria. Nat. Commun. 7, 10515 (2016).

  17. 17.

    Nikaido, H. Restoring permeability barrier function to outer membrane. Chem. Biol. 12, 507–509 (2005).

  18. 18.

    Li, G.-W., Burkhardt, D., Gross, C. & Weissman, J. S. Quantifying absolute protein synthesis rates reveals principles underlying allocation of cellular resources. Cell 157, 624–635 (2014).

  19. 19.

    Holm, L. & Rosenström, P. Dali server: conservation mapping in 3D. Nucleic Acids Res. 38, W545–W549 (2010).

  20. 20.

    Arunmanee, W. et al. Gram-negative trimeric porins have specific LPS binding sites that are essential for porin biogenesis. Proc. Natl Acad. Sci. USA 113, E5034–E5043 (2016).

  21. 21.

    Takeuchi, Y. & Nikaido, H. Persistence of segregated phospholipid domains in phospholipid-lipopolysaccharide mixed bilayers: studies with spin-labeled phospholipids. Biochemistry 20, 523–529 (1981).

  22. 22.

    Ashkenazy, H., Erez, E., Martz, E., Pupko, T. & Ben-Tal, N. ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids. Nucleic Acids Res. 38, W529–W533 (2010).

  23. 23.

    Nichols, R. J. et al. Phenotypic landscape of a bacterial cell. Cell 144,143–156 (2011).

  24. 24.

    Sutterlin, H. A. et al. Disruption of lipid homeostasis in the Gram-negative cell envelope activates a novel cell death pathway. Proc. Natl Acad. Sci. USA 113, E1565–E1574 (2016).

  25. 25.

    Filip, C., Fletcher, G., Wulff, J. L. & Earhart, C. F. Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate. J. Bacteriol. 115, 717–722 (1973).

  26. 26.

    Steeb, B. et al. Parallel exploitation of diverse host nutrients enhances Salmonella virulence. PLoS Pathog. 9, e1003301 (2013).

  27. 27.

    Dong, C. et al. Wza the translocon for E. coli capsular polysaccharides defines a new class of membrane protein. Nature 444, 226–229 (2006).

  28. 28.

    Chandran, V. et al. Structure of the outer membrane complex of a type IV secretion system. Nature 462, 1011–1015 (2009).

  29. 29.

    Hankins, H. M., Baldridge, R. D., Xu, P. & Graham, T. R. Role of flippases, scramblases and transfer proteins in phosphatidylserine subcellular distribution. Traffic 16, 35–47 (2015).

  30. 30.

    Daleke, D. L. Regulation of transbilayer plasma membrane phospholipid asymmetry. J. Lipid Res. 44, 233–42 (2003).

  31. 31.

    Guzman, L. M., Belin, D., Carson, M. J. & Beckwith, J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177, 4121–4130 (1995).

  32. 32.

    Miroux, B. & Walker, J. E. Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260, 289–298 (1996).

  33. 33.

    Cronan, J. E. A family of arabinose-inducible Escherichia coli expression vectors having pBR322 copy control. Plasmid 55, 152–157 (2006).

  34. 34.

    Prilipov, A., Phale, P. S., Van Gelder, P., Rosenbusch, J. P. & Koebnik, R. Coupling site-directed mutagenesis with high-level expression: large scale production of mutant porins from E. coli. FEMS Microbiol. Lett. 163,65–72 (1998).

  35. 35.

    Kabsch, W. Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr. D Biol. Crystallogr. 66, 133–144 (2010).

  36. 36.

    Waterman, D. G. et al. Diffraction-geometry refinement in the DIALS framework. Acta Crystallogr. D Struct. Biol. 72, 558–575 (2016).

  37. 37.

    Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution? Acta Crystallogr. D Biol. Crystallogr. 69, 1204–1214 (2013).

  38. 38.

    Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D Biol. Crystallogr. 62, 72–82 (2006).

  39. 39.

    McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

  40. 40.

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

  41. 41.

    Terwilliger, T. C. et al. Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard. Acta Crystallogr. D Biol. Crystallogr. 64, 61–69 (2008).

  42. 42.

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

  43. 43.

    Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 68, 352–367 (2012).

  44. 44.

    Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

  45. 45.

    The PyMOL molecular graphics system, Version 1.8 Schrödinger, LLC.

  46. 46.

    Bond, C. S. & Schüttelkopf, A. W. ALINE: aWYSIWYG protein-sequence alignment editor for publication-quality alignments. Acta Crystallogr. D Biol. Crystallogr. 65, 510–512 (2009).

  47. 47.

    Ho, B. K. & Gruswitz, F. HOLLOW: generating accurate representations of channel and interior surfaces in molecular structures. BMC Struct. Biol. 8,49 (2008).

  48. 48.

    Moriarty, N. W., Grosse-Kunstleve, R. W. & Adams, P. D. Electronic Ligand Builder and Optimization Workbench (eLBOW): a tool for ligand coordinate and restraint generation. Acta Crystallogr. D Biol. Crystallogr. 65,1074–1080 (2009).

  49. 49.

    Gotfredsen, M. & Gerdes, K. The Escherichia coli relBE genes belong to a new toxin-antitoxin gene family. Mol. Microbiol. 29, 1065–1076 (1998).

  50. 50.

    Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006.0008 (2006).

  51. 51.

    Cherepanov, P. P. & Wackernagel, W. Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene 158, 9–14 (1995).

  52. 52.

    Hoiseth, S. K. & Stocker, B. A. D. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature 291, 238–239 (1981).

  53. 53.

    Abraham, M. J. et al. GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1–2, 19–25 (2015).

  54. 54.

    Klauda, J. B. et al. Update of the CHARMM all-atom additive force field for lipids: validation on six lipid types. J. Phys. Chem. B 114, 7830–7843 (2010).

  55. 55.

    Herzog, F. A., Braun, L., Schoen, I. & Vogel, V. Improved side chain dynamics in MARTINI simulations of protein–lipid interfaces. J. Chem. Theory Comput. 12, 2446–2458 (2016).

  56. 56.

    Yesylevskyy, S. O., Schäfer, L. V., Sengupta, D. & Marrink, S. J. Polarizable water model for the coarse-grained MARTINI force field. PLoS Comput. Biol. 6, e1000810 (2010).

  57. 57.

    Periole, X., Cavalli, M., Marrink, S.-J. & Ceruso, M. A. Combining an elastic network with a coarse-grained molecular force field: structure, dynamics, and intermolecular recognition. J. Chem. Theory Comput. 5, 2531–2543 (2009).

  58. 58.

    Hsu, P.-C., Jefferies, D. & Khalid, S. Molecular dynamics simulations predict the pathways via which pristine fullerenes penetrate bacterial membranes. J. Phys. Chem. B 120, 11170–11179 (2016).

  59. 59.

    Bennett, W. F. D. & Tieleman, D. P. Water defect and pore formation in atomistic and coarse-grained lipid membranes: pushing the limits of coarse graining. J. Chem. Theory Comput. 7, 2981–2988 (2011).

  60. 60.

    Hoover, W. G. Canonical dynamics: equilibrium phase-space distributions. Phys. Rev. A 31, 1695–1697 (1985).

  61. 61.

    Andersen, H. C. & C., H. Molecular dynamics simulations at constant pressure and/or temperature. J. Chem. Phys. 72, 2384–2393 (1980).

  62. 62.

    Parrinello, M. & Rahman, A. Polymorphic transitions in single crystals: a new molecular dynamics method. J. Appl. Phys. 52, 7182–7190 (1981).

  63. 63.

    Darden, T., York, D. & Pedersen, L. Particle mesh Ewald: An Nlog(N) method for Ewald sums in large systems. J. Chem. Phys. 98, 10089–10092 (1993).

  64. 64.

    Vermeer, L. S., de Groot, B. L., Réat, V., Milon, A. & Czaplicki, J. Acyl chain order parameter profiles in phospholipid bilayers: computation from molecular dynamics simulations and comparison with 2H NMR experiments. Eur. Biophys. J. 36, 919–931 (2007).

  65. 65.

    Bussi, G., Donadio, D. & Parrinello, M. Canonical sampling through velocity rescaling. J. Chem. Phys. 126, 14101–14107 (2007).

  66. 66.

    Berendsen, H. J. C., Postma, J. P. M., van Gunsteren, W. F., DiNola, A. & Haak, J. R. Molecular dynamics with coupling to an external bath. J. Chem. Phys. 81, 3684–3690 (1984).

  67. 67.

    Jo, S., Vargyas, M., Vasko-Szedlar, J., Roux, B. & Im, W. PBEQ-Solver for online visualization of electrostatic potential of biomolecules. Nucleic Acids Res. 36, W270–W275 (2008).

  68. 68.

    Smart, O. S., Neduvelil, J. G., Wang, X., Wallace, B. & Sansom, M. S. HOLE: a program for the analysis of the pore dimensions of ion channel structural models. J. Mol. Graph. 14, 354–360 (1996).

  69. 69.

    Humphrey, W. F., Dalke, A. & Schulten, K. VMD – visual molecular dynamics. J. Mol. Graph. 14, 33–38 (1996).

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We would like to thank the staff at beam lines I24, I04 and I04-1 of the Diamond Light Source UK for beam time (proposal mx13587) and assistance with data collection. The research of S.S.K., U.K., D.B. and B.v.d.B. has received support from the Innovative Medicines Initiatives Joint Undertaking under Grant Agreement No. 115525, resources that are composed of financial contributions from the European Union’s seventh framework programme (FP7/2007–2013) and European Federation of Pharmaceutical Industries and Associations companies in-kind contribution.

Author information


  1. Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK

    • Javier Abellón-Ruiz
    • , Arnaud Baslé
    •  & Bert van den Berg
  2. Department of Physics & Earth Sciences, Jacobs University Bremen, 28759, Bremen, Germany

    • Shreyas S. Kaptan
    •  & Ulrich Kleinekathöfer
  3. Focal Area Infection Biology, University of Basel, CH-4056, Basel, Switzerland

    • Beatrice Claudi
    •  & Dirk Bumann


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B.v.d.B and J.A-R. purified MlaA proteins, crystallized MlaA–OmpF complexes, and determined crystal structures. J.A-R. constructed MlaA variant proteins and conducted in vitro functional assays. S.S.K carried out and analysed molecular dynamics simulations. U.K. supervised the computational studies. A.B. collected X-ray diffraction data and maintained the Newcastle Structural Biology Laboratory. B.C. carried out competitive fitness experiments, supervised by D.B. B.v.d.B and J.A-R designed experiments and B.v.d.B wrote the paper, with input from all co-authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Bert van den Berg.

Electronic supplementary material

  1. Supplementary Information

    Supplementary Figures 1–9, Supplementary Tables 1–3, Supplementary References.

  2. Life Sciences Reporting Summary

  3. Supplementary Video 1

    Atomistic molecular dynamics simulation (200 ns) of the KpMlaA–OmpF complex, showing the movement of the POPE molecule interacting with MlaA (oxygens, red; nitrogens, blue). MlaA is shown as a cartoon and coloured light blue (OmpF; grey). Phosphate head groups are shown as olive and green balls for the outer and inner leaflets respectively, water molecules as small blue spheres. The centre of the channel is indicated by the orange line. Residue Asp152, interacting with the POPE head group is shown.

  4. Supplementary Video 2

    Coarse-grained molecular dynamics simulation (2 μs) of the KpMlaA–OmpF complex.

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