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Evidence for phospholipid export from the bacterial inner membrane by the Mla ABC transport system

Abstract

The Mla pathway is believed to be involved in maintaining the asymmetrical Gram-negative outer membrane via retrograde phospholipid transport. The pathway is composed of three components: the outer membrane MlaA–OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here, we solve the crystal structure of MlaC in its phospholipid-free closed apo conformation, revealing a pivoting β-sheet mechanism that functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC, we provide evidence that the inner-membrane MlaFEDB machinery exports phospholipids to MlaC in the periplasm. Furthermore, we confirm that the phospholipid export process occurs through the MlaD component of the MlaFEDB complex and that this process is independent of ATP. Our data provide evidence of an apparatus for lipid export away from the inner membrane and suggest that the Mla pathway may have a role in anterograde phospholipid transport.

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Fig. 1: The MlaC-apo structure.
Fig. 2: MlaC requires MlaD for PL loading.
Fig. 3: MlaFEDB is active and exports IM PLs to MlaC.
Fig. 4: QCM–D and ATR-FTIR further suggest MlaFEDB exports IM PLs to MlaC.
Fig. 5: MlaFEDB-driven PL export occurs in an ATPase-independent manner but ATP hydrolysis is linked to MlaC-apo binding.
Fig. 6: Schematic summarizing the questions for the mechanisms of the Mla system arising from this study.

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Data availability

The MlaC-apo X-ray structure has been deposited in the PDB with the accession number 6GKI. In addition, the data that support the findings of this study are available from the corresponding author on request.

References

  1. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Ruiz, N., Kahne, D. & Silhavy, T. J. Advances in understanding bacterial outer-membrane biogenesis. Nat. Rev. Microbiol. 4, 57–66 (2006).

    Article  PubMed  CAS  Google Scholar 

  3. Kamio, Y. & Nikaido, H. Outer membrane of Salmonella typhimurium: accessibility of phospholipid head groups to phospholipase C and cyanogen bromide activated dextran in the external medium. Biochemistry 15, 2561–2570 (1976).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  5. Wu, T. et al. Identification of a multicomponent complex required for outer membrane biogenesis in Escherichia coli. Cell 121, 235–245 (2005).

    Article  CAS  PubMed  Google Scholar 

  6. Malinverni, J. C. et al. YfiO stabilizes the YaeT complex and is essential for outer membrane protein assembly in Escherichia coli. Mol. Microbiol. 61, 151–164 (2006).

    Article  CAS  PubMed  Google Scholar 

  7. Sklar, J. G. et al. Lipoprotein SmpA is a component of the YaeT complex that assembles outer membrane proteins in Escherichia coli. Proc. Natl Acad. Sci. USA 104, 6400–6405 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Knowles, T. J., Scott-Tucker, A., Overduin, M. & Henderson, I. R. Membrane protein architects: the role of the BAM complex in outer membrane protein assembly. Nat. Rev. Microbiol. 7, 206–214 (2009).

    Article  CAS  PubMed  Google Scholar 

  9. Ruiz, N., Gronenberg, L. S., Kahne, D. & Silhavy, T. J. Identification of two inner-membrane proteins required for the transport of lipopolysaccharide to the outer membrane of Escherichia coli. Proc. Natl Acad. Sci. USA 105, 5537–5542 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Narita, S. & Tokuda, H. Biochemical characterization of an ABC transporter LptBFGC complex required for the outer membrane sorting of lipopolysaccharides. FEBS Lett. 583, 2160–2164 (2009).

    Article  CAS  PubMed  Google Scholar 

  11. Chng, S. S., Gronenberg, L. S. & Kahne, D. Proteins required for lipopolysaccharide assembly in Escherichia coli form a transenvelope complex. Biochemistry 49, 4565–4567 (2010).

    Article  CAS  PubMed  Google Scholar 

  12. Tran, A. X., Dong, C. & Whitfield, C. Structure and functional analysis of LptC, a conserved membrane protein involved in the lipopolysaccharide export pathway in Escherichia coli. J. Biol. Chem. 285, 33529–33539 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Okuda, S. & Tokuda, H. Lipoprotein sorting in bacteria. Annu. Rev. Microbiol. 65, 239–259 (2011).

    Article  CAS  PubMed  Google Scholar 

  14. Grabowicz, M. & Silhavy, T. J. Redefining the essential trafficking pathway for outer membrane lipoproteins. Proc. Natl Acad. Sci. USA 114, 4769–4774 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Jones, N. C. & Osborn, M. J. Translocation of phospholipids between the outer and inner membranes of Salmonella typhimurium. J. Biol. Chem. 252, 7405–7412 (1977).

    Article  CAS  PubMed  Google Scholar 

  16. Langley, K. E., Hawrot, E. & Kennedy, E. P. Membrane assembly: movement of phosphatidylserine between the cytoplasmic and outer membranes of Escherichia coli. J. Bacteriol. 152, 1033–1041 (1982).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Donohue-Rolfe, A. M. & Schaechter, M. Translocation of phospholipids from the inner to the outer membrane of Escherichia coli. Proc. Natl Acad. Sci. USA 77, 1867–1871 (1980).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 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).

    Article  CAS  PubMed  Google Scholar 

  19. 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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Abellon-Ruiz, J. et al. Structural basis for maintenance of bacterial outer membrane lipid asymmetry. Nat. Microbiol. 2, 1616–1623 (2017).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Yeow, J. et al. The architecture of the OmpC-MlaA complex sheds light on the maintenance of outer membrane lipid asymmetry in Escherichia coli. J. Biol. Chem. 293, 11325–11340 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Powers, M. J. & Trent, M. S. Phospholipid retention in the absence of asymmetry strengthens the outer membrane permeability barrier to last-resort antibiotics. Proc. Natl Acad. Sci. USA 115, E8518–E8527 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Kamischke, C. et al. The Acinetobacter baumannii Mla system and glycerophospholipid transport to the outer membrane. eLife 8, e40171 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  25. 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).

    Article  CAS  PubMed  Google Scholar 

  26. Hong, M., Gleason, Y., Wyckoff, E. E. & Payne, S. M. Identification of two Shigella flexneri chromosomal loci involved in intercellular spreading. Infect. Immun. 66, 4700–4710 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Cuccui, J. et al. Development of signature-tagged mutagenesis in Burkholderia pseudomallei to identify genes important in survival and pathogenesis. Infect. Immun. 75, 1186–1195 (2007).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  29. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Leney, A. C. & Heck, A. J. Native mass spectrometry: what is in the name? J. Am. Soc. Mass Spectrom. 28, 5–13 (2017).

    Article  CAS  PubMed  Google Scholar 

  31. Ercan, B., Low, W. Y., Liu, X. & Chng, S. S. Characterization of interactions and phospholipid transfer between substrate binding proteins of the OmpC-Mla system. Biochemistry 58, 114–119 (2018).

    Article  PubMed  CAS  Google Scholar 

  32. Giess, F., Friedrich, M. G., Heberle, J., Naumann, R. L. & Knoll, W. The protein-tethered lipid bilayer: a novel mimic of the biological membrane. Biophys. J. 87, 3213–3220 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Shen, H. H. et al. Reconstitution of a nanomachine driving the assembly of proteins into bacterial outer membranes. Nat. Commun. 5, 5078 (2014).

    Article  CAS  PubMed  Google Scholar 

  34. Norby, J. G. Coupled assay of Na+, K+ ATPase activity. Methods Enzym. 156, 116–119 (1988).

    Article  CAS  Google Scholar 

  35. 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).

    Article  CAS  PubMed  Google Scholar 

  36. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Doerrler, W. T. & Raetz, C. R. ATPase activity of the MsbA lipid flippase of Escherichia coli. J. Biol. Chem. 277, 36697–36705 (2002).

    Article  CAS  PubMed  Google Scholar 

  38. Eckford, P. D. & Sharom, F. J. The reconstituted Escherichia coli MsbA protein displays lipid flippase activity. Biochem. J. 429, 195–203 (2010).

    Article  CAS  PubMed  Google Scholar 

  39. Liu, C. E., Liu, P. Q. & Ames, G. F. Characterization of the adenosine triphosphatase activity of the periplasmic histidine permease, a traffic ATPase (ABC transporter). J. Biol. Chem. 272, 21883–21891 (1997).

    Article  CAS  PubMed  Google Scholar 

  40. Chen, J., Sharma, S., Quiocho, F. A. & Davidson, A. L. Trapping the transition state of an ATP-binding cassette transporter: evidence for a concerted mechanism of maltose transport. Proc. Natl Acad. Sci. USA 98, 1525–1530 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Winter, G. xia2: an expert system for macromolecular crystallography data reduction. J. Appl. Crystallogr. 43, 186–190 (2010).

    Article  CAS  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed Central  Google Scholar 

  45. Joosten, R. P. et al. PDB_REDO: automated re-refinement of X-ray structure models in the PDB. J. Appl. Crystallogr. 42, 376–384 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Kiianitsa, K., Solinger, J. A. & Heyer, W. D. NADH-coupled microplate photometric assay for kinetic studies of ATP-hydrolyzing enzymes with low and high specific activities. Anal. Biochem. 321, 266–271 (2003).

    Article  CAS  PubMed  Google Scholar 

  47. Schuck, P. Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys. J. 78, 1606–1619 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Kay, L., Keifer, P. & Saarinen, T. Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity. J. Am. Chem. Soc. 114, 10663–10665 (1992).

    Article  CAS  Google Scholar 

  49. Palmer III, A. G., Cavanagh, J., Byrd, R. A. & Rance, M. Sensitivity improvement in three-dimensional heteronuclear correlation NMR spectroscopy. J. Magn. Reson. 96, 416–424 (1992).

    Google Scholar 

  50. Schleucher, J. et al. A general enhancement scheme in heteronuclear multidimensional NMR employing pulsed field gradients. J. Biomol. NMR 4, 301–306 (1994).

    Article  CAS  PubMed  Google Scholar 

  51. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

    Article  CAS  PubMed  Google Scholar 

  52. Goddard, T. D. & Kneller, D. G. SPARKY 3. University of California, San Francisco (2004).

  53. Fiser, A. & Šali, A. in Methods in Enzymology, Vol. 374 (eds. Charles W. Carter, Jr & Robert, M. S.) 461–491 (Academic Press, 2003).

  54. Li, D.-W. & Brüschweiler, R. NMR-based protein potentials. Angew. Chem. 122, 6930–6932 (2010).

    Article  Google Scholar 

  55. Huang, J. & MacKerell, A. D. CHARMM36 all-atom additive protein force field: validation based on comparison to NMR data. J. Comput. Chem. 34, 2135–2145 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Schmid, N. et al. Definition and testing of the GROMOS force-field versions 54A7 and 54B7. Eur. Biophys. J. 40, 843–856 (2011).

    Article  CAS  PubMed  Google Scholar 

  57. Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W. & Klein, M. L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926–935 (1983).

    Article  CAS  Google Scholar 

  58. Durell, S. R., Brooks, B. R. & Ben-Naim, A. Solvent-induced forces between two hydrophilic groups. J. Phys. Chem. 98, 2198–2202 (1994).

    Article  CAS  Google Scholar 

  59. Neria, E., Fischer, S. & Karplus, M. Simulation of activation free energies in molecular systems. J. Chem. Phys. 105, 1902–1921 (1996).

    Article  CAS  Google Scholar 

  60. Berendsen, H., Postma, J., van Gunsteren, W. & Hermans, J. in Intermolecular Forces (ed. Pullman, B.) 331–342 (D. Reidel Publishing Company, 1981).

  61. Feenstra, K. A., Hess, B. & Berendsen, H. J. C. Improving efficiency of large time-scale molecular dynamics simulations of hydrogen-rich systems. J. Comput. Chem. 20, 786–798 (1999).

    Article  CAS  Google Scholar 

  62. Essmann, U. et al. A smooth particle mesh Ewald method. J. Chem. Phys. 103, 8577–8593 (1995).

    Article  CAS  Google Scholar 

  63. Nosé, S. A molecular dynamics method for simulations in the canonical ensemble. Mol. Phys. 52, 255–268 (1984).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  66. Nosé, S. & Klein, M. L. Constant pressure molecular dynamics for molecular systems. Mol. Phys. 50, 1055–1076 (1983).

    Article  Google Scholar 

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

    Article  Google Scholar 

  68. Sugita, Y. & Okamoto, Y. Replica-exchange molecular dynamics method for protein folding. Chem. Phys. Lett. 314, 141–151 (1999).

    Article  CAS  Google Scholar 

  69. Bussi, G. Hamiltonian replica exchange in GROMACS: a flexible implementation. Mol. Phys. 112, 379–384 (2014).

    Article  CAS  Google Scholar 

  70. Tribello, G. A., Bonomi, M., Branduardi, D., Camilloni, C. & Bussi, G. PLUMED 2: new feathers for an old bird. Comput. Phys. Commun. 185, 604–613 (2014).

    Article  CAS  Google Scholar 

  71. Paramo, T., East, A., Garzón, D., Ulmschneider, M. B. & Bond, P. J. Efficient characterization of protein cavities within molecular simulation trajectories: trj_cavity. J. Chem. Theory Comput. 10, 2151–2164 (2014).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  73. Sauerbrey, G. Z. The use of quartz oscillators for weighing thin layers and for microweighing. Z. für Phys. 155, 206–222 (1959).

    Article  CAS  Google Scholar 

  74. Richter, R., Mukhopadhyay, A. & Brisson, A. Pathways of lipid vesicle deposition on solid surfaces: a combined QCM-D and AFM study. Biophys. J. 85, 3035–3047 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Reviakine, I., Johannsmann, D. & Richter, R. P. Hearing what you cannot see and visualizing what you hear: interpreting quartz crystal microbalance data from solvated interfaces. Anal. Chem. 83, 8838–8848 (2011).

    Article  CAS  PubMed  Google Scholar 

  76. Webster, J., Holt, S. & Dalgliesh, R. INTER the chemical interfaces reflectometer on target station 2 at ISIS. Phys. B 385–386, 1164–1166 (2006).

    Article  CAS  Google Scholar 

  77. Born, M. & Wolf, E. Principles of Optics (Pergamon Press, 1970).

  78. Clifton, L. A., Neylon, C. & Lakey, J. H. Examining protein-lipid complexes using neutron scattering. Methods Mol. Biol. 974, 119–150 (2013).

    Article  CAS  PubMed  Google Scholar 

  79. Clifton, L. A. et al. The effect of lipopolysaccharide core oligosaccharide size on the electrostatic binding of antimicrobial proteins to models of the Gram negative bacterial outer membrane. Langmuir 32, 3485–3494 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Clifton, L. A. et al. An accurate in vitro model of the E. coli envelope. Angew. Chem. Int. Ed. Engl. 54, 11952–11955 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Tamm, L. K. & McConnell, H. M. Supported phospholipid bilayers. Biophys. J. 47, 105–113 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Sivia, D. S. & Webster, J. R. P. The Bayesian approach to reflectivity data. Phys. B 248, 327–337 (1998).

    Article  CAS  Google Scholar 

  83. Sivia, D. S. & Skilling, J. Data Analysis: a Bayesian Tutorial (Oxford Univ. Press, 2006).

  84. Haario, H., Saksman, E. & Tamminen, J. An adaptive Metropolis algorithm. Bernoulli 7, 223–242 (2001).

    Article  Google Scholar 

  85. Haario, H., Laine, M., Mira, A. & Saksman, E. DRAM: efficient adaptive MCMC. Stat. Comput. 16, 339–354 (2006).

    Article  Google Scholar 

  86. Voss, N. R. & Gerstein, M. 3V: cavity, channel and cleft volume calculator and extractor. Nucleic Acids Res. 38, W555–W562 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank D. Ekiert and G. Bhabha for their discussions and for providing the Mla plasmid constructs. We thank the HWB-NMR at the University of Birmingham for providing open access to their Wellcome Trust-funded NMR equipment. This research was supported by the BBSRC grant no. BB/P009840/1 (T.J.K. and G.W.H.) and Wellcome Trust grant no. 208400/Z/17/Z. T.J.P. acknowledges the use of the Iridis high-performance computing resources at the University of Southampton.

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G.W.H., S.C.L.H., C.S.L., P.S., C.H., T.J.P., P.J.W., A.C.L., D.G.W., M.J., V.S., I.T.C., C.H., G.L.I., J.A.B., Y.Y., M.J., D.H., I.R.H., L.A.C., A.L.L. and T.J.K. participated in the conception and design of the work. G.W.H., S.C.L.H., C.S.L., P.S., A.H.M., T.J.P., P.J.W., A.C.L., D.G.W., R.J.P., L.A.C., A.L.L. and T.J.K. participated in the data acquisition, analysis or interpretation of the work. G.W.H., S.C.L.H., C.S.L., P.S., T.J.P., P.J.W., A.C.L., D.G.W., L.A.C., A.L.L. and T.J.K. were involved in writing and editing the manuscript. All authors approved the final version submitted for publication.

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Correspondence to Timothy J. Knowles.

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Hughes, G.W., Hall, S.C.L., Laxton, C.S. et al. Evidence for phospholipid export from the bacterial inner membrane by the Mla ABC transport system. Nat Microbiol 4, 1692–1705 (2019). https://doi.org/10.1038/s41564-019-0481-y

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