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Structural basis for outer membrane lipopolysaccharide insertion

Nature volume 511, pages 52–56 (2014)Cite this article

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Abstract

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.

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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.

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Acknowledgements

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

Authors and Affiliations

  1. Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK,

    Haohao Dong, Yinghong Gu, Zhongshan Wang & Changjiang Dong

  2. Biomedical Sciences Research Complex, School of Chemistry, University of St Andrews, North Haugh, St Andrews KY16 9ST, UK,

    Haohao Dong, Quanju Xiang, Zhongshan Wang & Chuan He

  3. Department of Microbiology, College of Resource and Environment Science, Sichuan Agriculture University, Yaan, 625000, China

    Quanju Xiang

  4. College of Life Sciences, Sichuan University, Chengdu, 610065, China

    Zhongshan Wang & Yizheng Zhang

  5. Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, UK,

    Neil G. Paterson

  6. Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK,

    Phillip J. Stansfeld

  7. School of Electronics and Information, Wuhan Technical College of Communications, No.6 Huangjiahu West Road, Hongshan District, Wuhan, Hubei 430065, China,

    Chuan He

  8. Laboratory of Department of Surgery, the First Affiliated Hospital, Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou, Guangdong 510080, China,

    Wenjian Wang

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  1. Haohao Dong
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Contributions

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.

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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. d–g, 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
Full size table
Extended Data Table 2 The cell growth of LptE and LptD mutants in different conditions
Full size table
Extended Data Table 3 Data collection and structure refinement statisticsa
Full size table

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Dong, H., Xiang, Q., Gu, Y. et al. Structural basis for outer membrane lipopolysaccharide insertion. Nature 511, 52–56 (2014). https://doi.org/10.1038/nature13464

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