Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Structural basis for outer membrane lipopolysaccharide insertion


Lipopolysaccharide (LPS) is essential for most Gram-negative bacteria and has crucial roles in protection of the bacteria from harsh environments and toxic compounds, including antibiotics. Seven LPS transport proteins (that is, LptA–LptG) form a trans-envelope protein complex responsible for the transport of LPS from the inner membrane to the outer membrane, the mechanism for which is poorly understood. Here we report the first crystal structure of the unique integral membrane LPS translocon LptD–LptE complex. LptD forms a novel 26-stranded β-barrel, which is to our knowledge the largest β-barrel reported so far. LptE adopts a roll-like structure located inside the barrel of LptD to form an unprecedented two-protein ‘barrel and plug’ architecture. The structure, molecular dynamics simulations and functional assays suggest that the hydrophilic O-antigen and the core oligosaccharide of the LPS may pass through the barrel and the lipid A of the LPS may be inserted into the outer leaflet of the outer membrane through a lateral opening between strands β1 and β26 of LptD. These findings not only help us to understand important aspects of bacterial outer membrane biogenesis, but also have significant potential for the development of novel drugs against multi-drug resistant pathogenic bacteria.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Crystal structure of the LptD–LptE complex.
Figure 2: The LptE and LptD interaction.
Figure 3: The unique barrel and pore of the LptD–LptE complex.
Figure 4: The proposed mechanism of LPS transport.

Accession codes

Primary accessions

Protein Data Bank


  1. 1

    Raetz, C. R. & Whitfield, C. Lipopolysaccharide endotoxins. Annu. Rev. Biochem. 71, 635–700 (2002)

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

    Ruiz, N., Kahne, D. & Silhavy, T. J. Transport of lipopolysaccharide across the cell envelope: the long road of discovery. Nature Rev. Microbiol. 7, 677–683 (2009)

    CAS  Google Scholar 

  3. 3

    Freinkman, E., Okuda, S., Ruiz, N. & Kahne, D. Regulated assembly of the transenvelope protein complex required for lipopolysaccharide export. Biochemistry 51, 4800–4806 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Villa, R. et al. The Escherichia coli Lpt transenvelope protein complex for lipopolysaccharide export is assembled via conserved structurally homologous domains. J. Bacteriol. 195, 1100–1108 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Freinkman, E., Chng, S. S. & Kahne, D. The complex that inserts lipopolysaccharide into the bacterial outer membrane forms a two-protein plug-and-barrel. Proc. Natl Acad. Sci. USA 108, 2486–2491 (2011)

    ADS  CAS  PubMed  Google Scholar 

  6. 6

    Sperandeo, P. et al. Characterization of lptA and lptB, two essential genes implicated in lipopolysaccharide transport to the outer membrane of Escherichia coli. J. Bacteriol. 189, 244–253 (2007)

    CAS  PubMed  Google Scholar 

  7. 7

    Sperandeo, P. et al. Functional analysis of the protein machinery required for transport of lipopolysaccharide to the outer membrane of Escherichia coli. J. Bacteriol. 190, 4460–4469 (2008)

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Wu, T. et al. Identification of a protein complex that assembles lipopolysaccharide in the outer membrane of Escherichia coli. Proc. Natl Acad. Sci. USA 103, 11754–11759 (2006)

    ADS  CAS  PubMed  Google Scholar 

  9. 9

    Chimalakonda, G. et al. Lipoprotein LptE is required for the assembly of LptD by the beta-barrel assembly machine in the outer membrane of Escherichia coli. Proc. Natl Acad. Sci. USA 108, 2492–2497 (2011)

    ADS  CAS  PubMed  Google Scholar 

  10. 10

    Srinivas, N. et al. Peptidomimetic antibiotics target outer-membrane biogenesis in Pseudomonas aeruginosa. Science 327, 1010–1013 (2010)

    ADS  CAS  PubMed  Google Scholar 

  11. 11

    Remaut, H. et al. Fiber formation across the bacterial outer membrane by the chaperone/usher pathway. Cell 133, 640–652 (2008)

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Phan, G. et al. Crystal structure of the FimD usher bound to its cognate FimC–FimH substrate. Nature 474, 49–53 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Fairman, J. W., Noinaj, N. & Buchanan, S. K. The structural biology of beta-barrel membrane proteins: a summary of recent reports. Curr. Opin. Struct. Biol. 21, 523–531 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14

    Chng, S. S., Ruiz, N., Chimalakonda, G., Silhavy, T. J. & Kahne, D. Characterization of the two-protein complex in Escherichia coli responsible for lipopolysaccharide assembly at the outer membrane. Proc. Natl Acad. Sci. USA 107, 5363–5368 (2010)

    ADS  CAS  PubMed  Google Scholar 

  15. 15

    Grabowicz, M., Yeh, J. & Silhavy, T. J. Dominant negative lptE mutation that supports a role for LptE as a plug in the LptD barrel. J. Bacteriol. 195, 1327–1334 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Braun, M. & Silhavy, T. J. Imp/OstA is required for cell envelope biogenesis in Escherichia coli. Mol. Microbiol. 45, 1289–1302 (2002)

    CAS  PubMed  Google Scholar 

  17. 17

    Ruiz, N., Falcone, B., Kahne, D. & Silhavy, T. J. Chemical conditionality: a genetic strategy to probe organelle assembly. Cell 121, 307–317 (2005)

    CAS  PubMed  Google Scholar 

  18. 18

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

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Whitney, J. C. et al. Structural basis for alginate secretion across the bacterial outer membrane. Proc. Natl Acad. Sci. USA 108, 13083–13088 (2011)

    ADS  CAS  PubMed  Google Scholar 

  20. 20

    Okuda, S., Freinkman, E. & Kahne, D. Cytoplasmic ATP hydrolysis powers transport of lipopolysaccharide across the periplasm in E. coli. Science 338, 1214–1217 (2012)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Bos, M. P. & Tommassen, J. The LptD chaperone LptE is not directly involved in lipopolysaccharide transport in Neisseria meningitidis. J. Biol. Chem. 286, 28688–28696 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    van den Berg, B., Black, P. N., Clemons, W. M., Jr & Rapoport, T. A. Crystal structure of the long-chain fatty acid transporter FadL. Science 304, 1506–1509 (2004)

    ADS  CAS  PubMed  Google Scholar 

  23. 23

    Hong, H., Patel, D. R., Tamm, L. K. & van den Berg, B. The outer membrane protein OmpW forms an eight-stranded beta-barrel with a hydrophobic channel. J. Biol. Chem. 281, 7568–7577 (2006)

    CAS  Google Scholar 

  24. 24

    Khan, M. A. & Bishop R. E Molecular mechanism for the lateral lipid diffusion between the outer membrane external leaflet and a beta-barrel hydrocarbon ruler. Biochemistry 48, 9745–9756 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Noinaj, N. et al. Structural insight into the biogenesis of β-barrel membrane proteins. Nature 501, 385–390 (2013)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    Chng, S. S. et al. Disulfide rearrangement triggered by translocon assembly controls lipopolysaccharide export. Science 337, 1665–1668 (2012)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Ruiz, N., Chng, S. S., Hiniker, A., Kahne, D. & Silhavy, T. J. Nonconsecutive disulfide bond formation in an essential integral outer membrane protein. Proc. Natl Acad. Sci. USA 107, 12245–12250 (2010)

    ADS  CAS  PubMed  Google Scholar 

  28. 28

    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)

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Suits, M. D., Sperandeo, P., Deho, G., Polissi, A. & Jia, Z. Novel structure of the conserved gram-negative lipopolysaccharide transport protein A and mutagenesis analysis. J. Mol. Biol. 380, 476–488 (2008)

    CAS  PubMed  Google Scholar 

  30. 30

    Sperandeo, P. et al. New insights into the Lpt machinery for lipopolysaccharide transport to the cell surface: LptA-LptC interaction and LptA stability as sensors of a properly assembled transenvelope complex. J. Bacteriol. 193, 1042–1053 (2011)

    CAS  PubMed  Google Scholar 

  31. 31

    Kabsch, W. Xds. Acta Crystallogr. D 66, 125–132 (2010)

    CAS  Google Scholar 

  32. 32

    Vonrhein, C., Blanc, E., Roversi, P. & Bricogne, G. Automated structure solution with autoSHARP. Methods Mol. Biol. 364, 215–230 (2007)

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33

    Cowtan, K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. D 62, 1002–1011 (2006)

    PubMed  Google Scholar 

  34. 34

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

    Article  Google Scholar 

  35. 35

    Winn, M. D., Murshudov, G. N. & Papiz, M. Z. Macromolecular TLS refinement in REFMAC at moderate resolutions. Methods Enzymol. 374, 300–321 (2003)

    CAS  PubMed  Google Scholar 

  36. 36

    Liu, H. & Naismith, J. H. An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnol. 8, 91 (2008)

    PubMed  PubMed Central  Google Scholar 

  37. 37

    Pronk, S. et al. GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics 29, 845–854 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Monticelli, L., Sorin, E. J., Tieleman, D. P., Pande, V. S. & Colombo, G. Molecular simulation of multistate peptide dynamics: a comparison between microsecond timescale sampling and multiple shorter trajectories. J. Comput. Chem. 29, 1740–1752 (2008)

    CAS  PubMed  Google Scholar 

  39. 39

    Stansfeld, P. J., Jefferys, E. E. & Sansom, M. S. Multiscale simulations reveal conserved patterns of lipid interactions with aquaporins. Structure 21, 810–819 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Stansfeld, P. J. & Sansom, M. S. P. From coarse grained to atomistic: a serial multiscale approach to membrane protein simulations. J. Chem. Theory Comput. 7, 1157–1166 (2011)

    CAS  PubMed  Google Scholar 

  41. 41

    Oostenbrink, C., Villa, A., Mark, A. E. & Van Gunsteren, W. F. A biomolecular force field based on the free enthalpy of hydration and solvation: The GROMOS force-field parameter sets 53A5 and 53A6. J. Comput. Chem. 25, 1656–1676 (2004)

    CAS  PubMed  Google Scholar 

  42. 42

    Bussi, G., Donadio, D. & Parrinello, M. Canonical sampling through velocity rescaling. J. Chem. Phys. 7, 014101 (2007)

    ADS  Google Scholar 

  43. 43

    Parrinello, M. & Giaquinta, P. V. Analytical solution of a new integral-equation for triplet correlations in hard-sphere fluids. J. Chem. Phys. 74, 1990–1997 (1981)

    ADS  MathSciNet  CAS  Google Scholar 

  44. 44

    Colombo, G., Marrink, S. J. & Mark, A. E. Simulation of MscL gating in a bilayer under stress. Biophys. J. 84, 2331–2337 (2003)

    CAS  PubMed  PubMed Central  Google Scholar 

Download references


We thank T. J. Silhavy for providing AM689 and AM661 cells, J. Naismith and C. Whitfield for support, and R. Field, A. Johnston and X. Tang for critical reading of the manuscript. We thank the staff at I24, I02, I03 and I04 of Diamond Light Source UK for beam time (proposal mx7641) and their assistance with data collection. C.D. is a Wellcome Trust career development fellow (083501/Z/07/Z). Q.X. and Z.W. are the receipts of the Chinese overseas study scholarships of The China Scholarship Council.

Author information




C.D. and W.W. conceived and designed the experiments. H.D., Q.X., C.H. expressed, purified and crystallized the LptD–LptE complex, and Y.G. and Z.W. did the mutagenesis and the functional assays. C.D., N.G.P., H.D. and W.W. undertook data collection and structure determination. Q.X., Z.W. and Y.Z. generated the constructs for the protein expression and P.J.S. performed the molecular dynamics simulation. C.D., H.D., W.W. and N.G.P. wrote the manuscript.

Corresponding authors

Correspondence to Wenjian Wang or Changjiang Dong.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

The atomic coordinate and the structure factor of the LptD–LptE is deposited at the Protein Data Bank under access code 4N4R.

Extended data figures and tables

Extended Data Figure 1 Amino acid sequence alignment of LptD.

The C-terminal domain of LptD forms a 26-stranded β-barrel and is highly conserved. Two cysteine residues, C173 and C727, are conserved and involved in disulphide bond formation. Salmxx0, S. typhimurium, accession GI25008880; Ecolxx1, E. coli, accession GI 2507089; Vibcxx3, Vibrio cholera, accession GI67477419; Pseaxx4, Pseudomonas aeruginosa, accession GI25008883; Neimxx2: Neisseria meningitidis, accession GI304336980.

Extended Data Figure 2 Amino acid sequence alignment of LptE.

The C-terminal residues of LptE from different bacteria are highly variable. Although the sequence identity of LptE is low, secondary and tertiary structure is conserved. Salmxx0, S. typhimurium, accession GI81523600; Ecolix1, E. coli, accession GI259491800; Vibrcx3, V. cholera, accession GI469684423; Neismx2, Neisseria bacilliformis, accession GI389606221; Pseudae, P. aeruginosa, GI15599183. Hydrophobic residues Y98, M100, V104, F126, V144, V89, M147, L77 and V108 of LptE may be involved in lipid A binding.

Extended Data Figure 3 Side view of the LptD barrel and LptD–LptE complex.

a, b, The aromatic residues that line the barrel outer wall are shown as sticks. c, d, The LptD–LptE complex in the outer membrane. E, OM and P represent extracellular side, outer membrane and periplasmic side, respectively. Panels a and c rotate 180° along the y axis to b and d, respectively.

Extended Data Figure 4 LptE superimposition with other homologues and the potential hydrophobic residues of LptE for LPS binding.

The S. typhimurium LptE structure from the LptD–LptE complex is very similar to other structures of LptE. S. typhimurium LptE is in purple, and LptEs of Caulobacter crescentus, N. meningitidis and Nitrosomonas europaea are in blue, orange and yellow respectively. a, LptE superimposition with LptE of C. crescentus CB15 (4KWY) with r.m.s.d. of 3.046 over 115 Cα. b, LptE superimposition with LptE of N. europaea (2JXP) with r.m.s.d. of 2.3177 over 128 Cα. c, LptE superimposition with LptE of N. meningitidis (3BF2) with r.m.s.d. of 1.6533 over 115 Cα. d, The potential hydrophobic residues of LptE for LPS binding are between the long α2 helix and the four β-strands.

Extended Data Figure 5 The LptD and LptE interactions.

a, Schematic representation of LptD and LptE interactions. b, LptE interaction with Lp8 of LptD. c, The interior Lp8 of LptD interacts with the lumen residues from strands β13 to β17 of LptD. d, The interior Lp4 of LptD interacts with lumen residues from strands β4 to β6 of LptD. e, C-terminal residues E773 to M786 of LptD interact with the lumen residues from strands β20 to β25 of LptD.

Extended Data Figure 6 Three proline residues at LptD and the proposed disulphide bond formed by the double mutation N232C and N757C, and LptD–LptE simulation stability and the separation of β1 and β26.

a, P231, P246 and P261 are located at strands β1, β2 and β3 of LptD, respectively. b, The double cysteine mutant N232C/N757C may form a disulphide bond at the lumen of the barrel, crosslinking strands β1 and β26, and preventing the lateral opening that is key to the proposed mechanism of LPS insertion. This double mutation is fatal to E. coli. c, The structural fluctuations of LptD–LptE complex over 100 ns at 323 K. Regions of high stability are coloured blue, while the more mobile domains are coloured red. Overall the barrel architecture is stable in the bilayer. d, By applying a negative constant pressure to the membrane plane it is possible to simulate the opening of the lateral gate between the strands β1 and β26. e, The LptD–LptE complex is shown from the periplasmic side at the start and 50 ns points of a simulation with a −70 bar pressure coupling. In addition to opening of the lateral gate, the extracellular region of the barrel also separates to potentially accommodate the large O-antigen and core oligosaccharide of LPS.

Extended Data Figure 7 LptD–LptE complex is resistant to proteolysis and forms crystals, with diffraction images from the LptD–LptE complex crystals, and the 2Fo − Fc electron density map of selected regions of the LptD–LptE complex contoured at 1σ.

a, Limited protease digestion of the LptD–LptE complex was carried out for 3 h at room temperature using α-chymotrypsin. This protease removes the N-terminal domain, residues 25–211 of LptD and C-terminal residues 170–194 of LptE. Band 1, the oxidized LptD. Band 2, the reduced LptD and non-cleaved. Band 3, the cleaved LptD. Band 4, the non-cleaved LptE. Band 5, the cleaved LptE. This is similar to that of the LptD–LptE complex of E. coli by trypsin digestion14. The bands have been confirmed by mass spectroscopy. b, c, The diffraction is anisotropic, with Mn(I/s.d.)>2 resolution limits of 2.95, 4.03 and 2.80 Å along 3 reciprocal axes. The dark circle is a resolution ring at 4 Å. b, Diffraction image collected at 0 degree. c, Image collected at 90 degree. Detector edge is at 2.8 Å. dg, The electron density map of the LptD–LptE complex is clear at 2.86 Å. d, e, Show the LptD electron density map of some aromatic residues at the barrel wall. f, A typical region of the electron density map from LptD. g, A typical region of the electron density map from LptE.

Extended Data Table 1 Primers for LptE and LptD site-directed mutagenesis
Extended Data Table 2 The cell growth of LptE and LptD mutants in different conditions
Extended Data Table 3 Data collection and structure refinement statisticsa

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Dong, H., Xiang, Q., Gu, Y. et al. Structural basis for outer membrane lipopolysaccharide insertion. Nature 511, 52–56 (2014).

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing