The structural basis of lipopolysaccharide recognition by the TLR4–MD-2 complex


The lipopolysaccharide (LPS) of Gram negative bacteria is a well-known inducer of the innate immune response1. Toll-like receptor (TLR) 4 and myeloid differentiation factor 2 (MD-2) form a heterodimer that recognizes a common ‘pattern’ in structurally diverse LPS molecules. To understand the ligand specificity and receptor activation mechanism of the TLR4–MD-2–LPS complex we determined its crystal structure. LPS binding induced the formation of an m-shaped receptor multimer composed of two copies of the TLR4–MD-2–LPS complex arranged symmetrically. LPS interacts with a large hydrophobic pocket in MD-2 and directly bridges the two components of the multimer. Five of the six lipid chains of LPS are buried deep inside the pocket and the remaining chain is exposed to the surface of MD-2, forming a hydrophobic interaction with the conserved phenylalanines of TLR4. The F126 loop of MD-2 undergoes localized structural change and supports this core hydrophobic interface by making hydrophilic interactions with TLR4. Comparison with the structures of tetra-acylated antagonists bound to MD-2 indicates that two other lipid chains in LPS displace the phosphorylated glucosamine backbone by 5 Å towards the solvent area2,3. This structural shift allows phosphate groups of LPS to contribute to receptor multimerization by forming ionic interactions with a cluster of positively charged residues in TLR4 and MD-2. The TLR4–MD-2–LPS structure illustrates the remarkable versatility of the ligand recognition mechanisms employed by the TLR family4,5, which is essential for defence against diverse microbial infection.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Overall structure of the TLR4–MD-2–LPS complex.
Figure 2: Binding of LPS to TLR4 and MD-2.
Figure 3: The main dimerization interface of the TLR4–MD-2–LPS complex.
Figure 4: Structural comparison of LPS with antagonists.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and the structure factor files have been deposited in the Protein Data Bank ( under accession number 3FXI.


  1. 1

    Beutler, B. & Rietschel, E. T. Innate immune sensing and its roots: the story of endotoxin. Nature Rev. Immunol. 3, 169–176 (2003)

    CAS  Article  Google Scholar 

  2. 2

    Kim, H. M. et al. Crystal structure of the TLR4–MD-2 complex with bound endotoxin antagonist Eritoran. Cell 130, 906–917 (2007)

    CAS  Article  Google Scholar 

  3. 3

    Ohto, U., Fukase, K., Miyake, K. & Satow, Y. Crystal structures of human MD-2 and its complex with antiendotoxic lipid IVa. Science 316, 1632–1634 (2007)

    ADS  CAS  Article  Google Scholar 

  4. 4

    Jin, M. S. et al. Crystal structure of the TLR1–TLR2 heterodimer induced by binding of a tri-acylated lipopeptide. Cell 130, 1071–1082 (2007)

    CAS  Article  Google Scholar 

  5. 5

    Liu, L. et al. Structural basis of toll-like receptor 3 signaling with double-stranded RNA. Science 320, 379–381 (2008)

    ADS  CAS  Article  Google Scholar 

  6. 6

    Raetz, C. R. Biochemistry of endotoxins. Annu. Rev. Biochem. 59, 129–170 (1990)

    CAS  Article  Google Scholar 

  7. 7

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

    CAS  Article  Google Scholar 

  8. 8

    Medzhitov, R., Preston-Hurlburt, P. & Janeway, C. A. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388, 394–397 (1997)

    ADS  CAS  Article  Google Scholar 

  9. 9

    Shimazu, R. et al. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J. Exp. Med. 189, 1777–1782 (1999)

    CAS  Article  Google Scholar 

  10. 10

    Matsushima, N. et al. Comparative sequence analysis of leucine-rich repeats (LRRs) within vertebrate toll-like receptors. BMC Genom. 8, 124 (2007)

    Article  Google Scholar 

  11. 11

    Bell, J. K. et al. The molecular structure of the Toll-like receptor 3 ligand-binding domain. Proc. Natl Acad. Sci. USA 102, 10976–10980 (2005)

    ADS  CAS  Article  Google Scholar 

  12. 12

    Choe, J., Kelker, M. S. & Wilson, I. A. Crystal structure of human toll-like receptor 3 (TLR3) ectodomain. Science 309, 581–585 (2005)

    ADS  CAS  Article  Google Scholar 

  13. 13

    Miyake, K. Roles for accessory molecules in microbial recognition by Toll-like receptors. J. Endotoxin Res. 12, 195–204 (2006)

    CAS  PubMed  Google Scholar 

  14. 14

    Erridge, C., Bennett-Guerrero, E. & Poxton, I. R. Structure and function of lipopolysaccharides. Microbes Infect. 4, 837–851 (2002)

    CAS  Article  Google Scholar 

  15. 15

    Rietschel, E. T. et al. Bacterial endotoxin: molecular relationships of structure to activity and function. FASEB J. 8, 217–225 (1994)

    CAS  Article  Google Scholar 

  16. 16

    Bella, J., Hindle, K. L., McEwan, P. A. & Lovell, S. C. The leucine-rich repeat structure. Cell. Mol. Life Sci. 65, 2307–2333 (2008)

    CAS  Article  Google Scholar 

  17. 17

    Galanos, C. et al. Synthetic and natural Escherichia coli free lipid A express identical endotoxic activities. Eur. J. Biochem. 148, 1–5 (1985)

    CAS  Article  Google Scholar 

  18. 18

    Kobayashi, M. et al. Regulatory roles for MD-2 and TLR4 in ligand-induced receptor clustering. J. Immunol. 176, 6211–6218 (2006)

    CAS  Article  Google Scholar 

  19. 19

    Kawasaki, K., Nogawa, H. & Nishijima, M. Identification of mouse MD-2 residues important for forming the cell surface TLR4–MD-2 complex recognized by anti-TLR4–MD-2 antibodies, and for conferring LPS and taxol responsiveness on mouse TLR4 by alanine-scanning mutagenesis. J. Immunol. 170, 413–420 (2003)

    CAS  Article  Google Scholar 

  20. 20

    Re, F. & Strominger, J. L. Separate functional domains of human MD-2 mediate Toll-like receptor 4-binding and lipopolysaccharide responsiveness. J. Immunol. 171, 5272–5276 (2003)

    CAS  Article  Google Scholar 

  21. 21

    Visintin, A., Latz, E., Monks, B. G., Espevik, T. & Golenbock, D. T. Lysines 128 and 132 enable lipopolysaccharide binding to MD-2, leading to Toll-like receptor-4 aggregation and signal transduction. J. Biol. Chem. 278, 48313–48320 (2003)

    CAS  Article  Google Scholar 

  22. 22

    Teghanemt, A. et al. Novel roles in human MD-2 of phenylalanines 121 and 126 and tyrosine 131 in activation of Toll-like receptor 4 by endotoxin. J. Biol. Chem. 283, 1257–1266 (2008)

    CAS  Article  Google Scholar 

  23. 23

    Jin, M. S. & Lee, J. O. Structures of the Toll-like receptor family and its ligand complexes. Immunity 29, 182–191 (2008)

    CAS  Article  Google Scholar 

  24. 24

    Rietschel, E. T. et al. The chemical structure of bacterial endotoxin in relation to bioactivity. Immunobiology 187, 169–190 (1993)

    CAS  Article  Google Scholar 

  25. 25

    Teghanemt, A., Zhang, D., Levis, E. N., Weiss, J. P. & Gioannini, T. L. Molecular basis of reduced potency of underacylated endotoxins. J. Immunol. 175, 4669–4676 (2005)

    CAS  Article  Google Scholar 

  26. 26

    Rossignol, D. P. & Lynn, M. TLR4 antagonists for endotoxemia and beyond. Curr. Opin. Investig. Drugs 6, 496–502 (2005)

    CAS  PubMed  Google Scholar 

  27. 27

    Mata-Haro, V. et al. The vaccine adjuvant monophosphoryl lipid A as a TRIF-biased agonist of TLR4. Science 316, 1628–1632 (2007)

    ADS  CAS  Article  Google Scholar 

  28. 28

    Ulmer, A. J. et al. Biological activity of synthetic phosphonooxyethyl analogs of lipid A and lipid A partial structures. Infect. Immun. 60, 3309–3314 (1992)

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Walsh, C. et al. Elucidation of the MD-2/TLR4 interface required for signaling by lipid IVa. J. Immunol. 181, 1245–1254 (2008)

    CAS  Article  Google Scholar 

  30. 30

    McCoy, A. J., Grosse-Kunstleve, R. W., Storoni, L. C. & Read, R. J. Likelihood-enhanced fast translation functions. Acta Crystallogr. D 61, 458–464 (2005)

    Article  Google Scholar 

  31. 31

    Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Cryst. 26, 795–800 (1993)

    CAS  Article  Google Scholar 

  32. 32

    Brünger, A. T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  Google Scholar 

  33. 33

    Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

    Article  Google Scholar 

  34. 34

    Lovell, S. C. et al. Structure validation by Cα geometry: φ, ψ and Cβ deviation. Proteins 50, 437–450 (2003)

    CAS  Article  Google Scholar 

Download references


We thank the staff of beamline 4A at the Pohang Accelerator Laboratory and beamline ID23-2 at ESRF for help with data collection. We thank J. Gross for critical reading of the manuscript. J.-O.L and co-workers are funded by the Creative Research Initiative (Center for Membrane Receptor Research) from the Ministry of Education, Science and Technology of Korea.

Author Contributions B.S.P., D.H.S. and H.M.K. performed the experiments. B.S.P. and J.-O.L. designed the experiments and analysed the data. B.S.P., H.L. and J.-O.L. wrote the paper. J.-O.L. managed the project and had overall responsibility for data interpretation and writing the manuscript. All authors discussed and commented on the manuscript.

Author information



Corresponding author

Correspondence to Jie-Oh Lee.

Supplementary information

Supplementary Information

This file contains a Supplementary Discussion, Supplementary Figures 1-11 with Legends, Supplementary Tables 1-2 and Supplementary References (PDF 3855 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Park, B., Song, D., Kim, H. et al. The structural basis of lipopolysaccharide recognition by the TLR4–MD-2 complex. Nature 458, 1191–1195 (2009).

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