Skip to main content

Thank you for visiting nature.com. 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.

Na+-mimicking ligands stabilize the inactive state of leukotriene B4 receptor BLT1

Nature Chemical Biology volume 14, pages 262–269 (2018)Cite this article

Subjects

Abstract

Most G-protein-coupled receptors (GPCRs) are stabilized in common in the inactive state by the formation of the sodium ion–centered water cluster with the conserved Asp2.50 inside the seven-transmembrane domain. We determined the crystal structure of the leukotriene B4 (LTB4) receptor BLT1 bound with BIIL260, a chemical bearing a benzamidine moiety. Surprisingly, the amidine group occupies the sodium ion and water locations, interacts with D662.50, and mimics the entire sodium ion–centered water cluster. Thus, BLT1 is fixed in the inactive state, and the transmembrane helices cannot change their conformations to form the active state. Moreover, the benzamidine molecule alone serves as a negative allosteric modulator for BLT1. As the residues involved in the benzamidine binding are widely conserved among GPCRs, the unprecedented inverse-agonist mechanism by the benzamidine moiety could be adapted to other GPCRs. Consequently, the present structure will enable the rational development of inverse agonists specific for each GPCR.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Crystal structure of BLT1 bound with the antagonist BIIL260.
Figure 2: Binding mode of BIIL260 to BLT1.
Figure 3: Structure of the benzamidine moiety binding site of BLT1 and comparison to the high-resolution GPCR structures in the inactive state with the bound sodium ion.
Figure 4: Effects of benzamidine and NaCl on BLT1–LTB4 binding and signal transduction by LTB4.
Figure 5: Effect of the bound benzamidine moiety on the agonist-induced activation of BLT1.
Figure 6: Amino acid conservation of the benzamidine moiety and orthosteric binding sites of GPCRs.

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Canals, M., Sexton, P.M. & Christopoulos, A. Allostery in GPCRs: 'MWC' revisited. Trends Biochem. Sci. 36, 663–672 (2011).

    CAS  PubMed  Google Scholar 

  2. Mahoney, J.P. & Sunahara, R.K. Mechanistic insights into GPCR-G protein interactions. Curr. Opin. Struct. Biol. 41, 247–254 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Katritch, V. et al. Allosteric sodium in class A GPCR signaling. Trends Biochem. Sci. 39, 233–244 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Ballesteros, J.A. & Weinstein, H. Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors. Methods in Neurosciences 25, 366–428 (1995).

    CAS  Google Scholar 

  5. Gao, Z.G. et al. Identification of essential residues involved in the allosteric modulation of the human A3 adenosine receptor. Mol. Pharmacol. 63, 1021–1031 (2003).

    CAS  PubMed  Google Scholar 

  6. Horstman, D.A. et al. An aspartate conserved among G-protein receptors confers allosteric regulation of α2-adrenergic receptors by sodium. J. Biol. Chem. 265, 21590–21595 (1990).

    CAS  PubMed  Google Scholar 

  7. Liu, W. et al. Structural basis for allosteric regulation of GPCRs by sodium ions. Science 337, 232–236 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Miller-Gallacher, J.L. et al. The 2.1 Å resolution structure of cyanopindolol-bound β1-adrenoceptor identifies an intramembrane Na+ ion that stabilises the ligand-free receptor. PLoS One 9, e92727 (2014).

    PubMed  PubMed Central  Google Scholar 

  9. Zhang, C. et al. High-resolution crystal structure of human protease-activated receptor 1. Nature 492, 387–392 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Fenalti, G. et al. Molecular control of δ-opioid receptor signalling. Nature 506, 191–196 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Rasmussen, S.G. et al. Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature 477, 549–555 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Kruse, A.C. et al. Activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature 504, 101–106 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Huang, W. et al. Structural insights into μ-opioid receptor activation. Nature 524, 315–321 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Carpenter, B., Nehmé, R., Warne, T., Leslie, A.G. & Tate, C.G. Structure of the adenosine A2A receptor bound to an engineered G protein. Nature 536, 104–107 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Gutiérrez-de-Terán, H. et al. The role of a sodium ion binding site in the allosteric modulation of the A2A adenosine G protein-coupled receptor. Structure 21, 2175–2185 (2013).

    PubMed  Google Scholar 

  16. Yokomizo, T., Izumi, T., Chang, K., Takuwa, Y. & Shimizu, T. A G-protein-coupled receptor for leukotriene B4 that mediates chemotaxis. Nature 387, 620–624 (1997).

    CAS  PubMed  Google Scholar 

  17. Birke, F.W., Meade, C.J., Anderskewitz, R., Speck, G.A. & Jennewein, H.M. In vitro and in vivo pharmacological characterization of BIIL 284, a novel and potent leukotriene B4 receptor antagonist. J. Pharmacol. Exp. Ther. 297, 458–466 (2001).

    CAS  PubMed  Google Scholar 

  18. Rosenbaum, D.M. et al. GPCR engineering yields high-resolution structural insights into β2-adrenergic receptor function. Science 318, 1266–1273 (2007).

    CAS  PubMed  Google Scholar 

  19. Hori, T. et al. Expression, purification and characterization of leukotriene B4 receptor, BLT1 in Pichia pastoris. Protein Expr. Purif. 72, 66–74 (2010).

    CAS  PubMed  Google Scholar 

  20. Hori, T., Nakamura, M., Yokomizo, T., Shimizu, T. & Miyano, M. The leukotriene B4 receptor BLT1 is stabilized by transmembrane helix capping mutations. Biochem. Biophys. Rep. 4, 243–249 (2015).

    PubMed  PubMed Central  Google Scholar 

  21. Hato, M., Yamashita, J. & Shiono, M. Aqueous phase behavior of lipids with isoprenoid type hydrophobic chains. J. Phys. Chem. B 113, 10196–10209 (2009).

    CAS  PubMed  Google Scholar 

  22. Hato, M., Hosaka, T., Tanabe, H., Kitsunai, T. & Yokoyama, S. A new manual dispensing system for in meso membrane protein crystallization with using a stepping motor-based dispenser. J. Struct. Funct. Genomics 15, 165–171 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Lehmann, M., Pasamontes, L., Lassen, S.F. & Wyss, M. The consensus concept for thermostability engineering of proteins. Biochim. Biophys. Acta 1543, 408–415 (2000).

    CAS  PubMed  Google Scholar 

  24. Tate, C.G. A crystal clear solution for determining G-protein-coupled receptor structures. Trends Biochem. Sci. 37, 343–352 (2012).

    CAS  PubMed  Google Scholar 

  25. Wu, B. et al. Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists. Science 330, 1066–1071 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Tan, Q. et al. Structure of the CCR5 chemokine receptor-HIV entry inhibitor maraviroc complex. Science 341, 1387–1390 (2013).

    CAS  PubMed  Google Scholar 

  27. Fredriksson, R., Lagerström, M.C., Lundin, L.G. & Schiöth, H.B. The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharmacol. 63, 1256–1272 (2003).

    CAS  PubMed  Google Scholar 

  28. Zhang, H. et al. Structure of the Angiotensin receptor revealed by serial femtosecond crystallography. Cell 161, 833–844 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Srivastava, A. et al. High-resolution structure of the human GPR40 receptor bound to allosteric agonist TAK-875. Nature 513, 124–127 (2014).

    CAS  PubMed  Google Scholar 

  30. Hanson, M.A. et al. Crystal structure of a lipid G protein-coupled receptor. Science 335, 851–855 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Shao, Z. et al. High-resolution crystal structure of the human CB1 cannabinoid receptor. Nature 540, 602–606 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Palczewski, K. et al. Crystal structure of rhodopsin: A G protein-coupled receptor. Science 289, 739–745 (2000).

    CAS  PubMed  Google Scholar 

  33. Thompson, A.A. et al. Structure of the nociceptin/orphanin FQ receptor in complex with a peptide mimetic. Nature 485, 395–399 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Thal, D.M. et al. Crystal structures of the M1 and M4 muscarinic acetylcholine receptors. Nature 531, 335–340 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Kruse, A.C. et al. Structure and dynamics of the M3 muscarinic acetylcholine receptor. Nature 482, 552–556 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Manglik, A. et al. Structural insights into the dynamic process of β2-adrenergic receptor signaling. Cell 161, 1101–1111 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Sabirsh, A., Bywater, R.P., Bristulf, J., Owman, C. & Haeggström, J.Z. Residues from transmembrane helices 3 and 5 participate in leukotriene B4 binding to BLT1. Biochemistry 45, 5733–5744 (2006).

    CAS  PubMed  Google Scholar 

  38. Basu, S. et al. Critical role for polar residues in coupling leukotriene B4 binding to signal transduction in BLT1. J. Biol. Chem. 282, 10005–10017 (2007).

    CAS  PubMed  Google Scholar 

  39. Lam, P.Y. et al. Structure-based design of novel guanidine/benzamidine mimics: potent and orally bioavailable factor Xa inhibitors as novel anticoagulants. J. Med. Chem. 46, 4405–4418 (2003).

    CAS  PubMed  Google Scholar 

  40. Rosenbaum, D.M. et al. Structure and function of an irreversible agonist-β2 adrenoceptor complex. Nature 469, 236–240 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Strange, P.G. Mechanisms of inverse agonism at G-protein-coupled receptors. Trends Pharmacol. Sci. 23, 89–95 (2002).

    CAS  PubMed  Google Scholar 

  42. Khilnani, G. & Khilnani, A.K. Inverse agonism and its therapeutic significance. Indian J. Pharmacol. 43, 492–501 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Okuno, T. et al. Helix 8 of the leukotriene B4 receptor is required for the conformational change to the low affinity state after G-protein activation. J. Biol. Chem. 278, 41500–41509 (2003).

    CAS  PubMed  Google Scholar 

  44. Kuniyeda, K. et al. Identification of the intracellular region of the leukotriene B4 receptor type 1 that is specifically involved in Gi activation. J. Biol. Chem. 282, 3998–4006 (2007).

    CAS  PubMed  Google Scholar 

  45. Inoue, A. et al. TGFα shedding assay: an accurate and versatile method for detecting GPCR activation. Nat. Methods 9, 1021–1029 (2012).

    CAS  PubMed  Google Scholar 

  46. Warne, T. et al. The structural basis for agonist and partial agonist action on a β1-adrenergic receptor. Nature 469, 241–244 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Hollenstein, K. et al. Structure of class B GPCR corticotropin-releasing factor receptor 1. Nature 499, 438–443 (2013).

    CAS  PubMed  Google Scholar 

  48. Wu, H. et al. Structure of a class C GPCR metabotropic glutamate receptor 1 bound to an allosteric modulator. Science 344, 58–64 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Christopher, J.A. et al. Fragment and structure-based drug discovery for a class C GPCR: discovery of the mGlu5 negative allosteric modulator HTL14242 (3-chloro-5-[6-(5-fluoropyridin-2-yl)pyrimidin-4-yl]benzonitrile). J. Med. Chem. 58, 6653–6664 (2015).

    CAS  PubMed  Google Scholar 

  50. Christopoulos, A. & Kenakin, T. G protein-coupled receptor allosterism and complexing. Pharmacol. Rev. 54, 323–374 (2002).

    CAS  PubMed  Google Scholar 

  51. Leach, K., Sexton, P.M. & Christopoulos, A. Allosteric GPCR modulators: taking advantage of permissive receptor pharmacology. Trends Pharmacol. Sci. 28, 382–389 (2007).

    CAS  PubMed  Google Scholar 

  52. Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Evans, P.R. An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr. D Biol. Crystallogr. 67, 282–292 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Foadi, J. et al. Clustering procedures for the optimal selection of data sets from multiple crystals in macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 69, 1617–1632 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Karplus, P.A. & Diederichs, K. Linking crystallographic model and data quality. Science 336, 1030–1033 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  59. Morris, G.M. et al. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J. Comput. Chem. 30, 2785–2791 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are grateful to Y. Nakamura, H. Tanabe, H. Ago, K. Ida and our colleagues at the RIKEN Center for Life Science Technologies for technical advice and useful discussions on sample preparation, crystallization, data processing, and structural refinement. The synchrotron radiation experiments were performed at BL32XU of SPring-8, with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (Proposal No. 2016B2726). Some of the vectors for the TGFα-shedding assay were kindly donated by J. Aoki and A. Inoue, at Tohoku University. This work was supported by the Platform Project for Supporting Drug Discovery and Life Science Research funded by Japan Agency for Medical Research and Development (AMED) (S.Y.), by Takeda Science Foundation (T.S.), by MEXT/JSPS KAKENHI Grant Numbers 23770133 (T.H.), 15KK0320, 16K08596 (T.O.), 26460061 (M.N.), 15H05897, 15H05904, and 15H04708 (T.Y.), and by Grants-in-Aid from the Foundation of Strategic Research Projects in Private Universities from MEXT (S1311011, S1411007) (T.Y.) and (2013-2017) (M.M.). M.M. received support for another GPCR project from Takeda Pharmaceutical Co. BIIL260 and BIIL284 were kindly provided by Boehringer Ingelheim Pharma GmbH & Co. KG.

Author information

Authors and Affiliations

  1. RIKEN Structural Biology Laboratory, Tsurumi-ku, Yokohama, Kanagawa, Japan

    Tetsuya Hori & Shigeyuki Yokoyama

  2. RIKEN SPring-8 Center, Sayo, Hyogo, Japan

    Tetsuya Hori, Kunio Hirata, Keitaro Yamashita, Yoshiaki Kawano, Masaki Yamamoto & Masashi Miyano

  3. Department of Biochemistry, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan

    Toshiaki Okuno & Takehiko Yokomizo

  4. Japan Science and Technology Agency, Precursory Research for Embryonic Science and Technology (PRESTO), Kawaguchi, Saitama, Japan

    Kunio Hirata

  5. Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, Tsurumi-ku, Yokohama, Japan

    Masakatsu Hato

  6. Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

    Motonao Nakamura & Takao Shimizu

  7. Department of Life Science, Faculty of Science, Okayama University of Science, Kita-ku, Okayama, Japan

    Motonao Nakamura

  8. Department of Lipid Signaling, National Center for Global Health and Medicine, Shinjuku, Tokyo, Japan

    Takao Shimizu

  9. Department of Chemistry and Biological Science, College of Science and Engineering, Aoyama Gakuin University, Chuo-ku, Sagamihara, Kanagawa, Japan

    Masashi Miyano

Authors
  1. Tetsuya Hori
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Toshiaki Okuno
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kunio Hirata
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Keitaro Yamashita
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yoshiaki Kawano
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Masaki Yamamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Masakatsu Hato
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Motonao Nakamura
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Takao Shimizu
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Takehiko Yokomizo
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Masashi Miyano
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Shigeyuki Yokoyama
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

T.H., T.S., T.Y., M.M., and S.Y. designed the research. T.H. performed all experiments from sample preparation to structure determination and functional analyses, except that T.O. performed the expression of BLT1 mutants using HEK293 cells and the signal assays. K.H., K.Y., Y.K., and M.Y. assisted with the data collection at BL32XU of SPring-8. M.H. constructed the in-house crystallization device and assisted with the crystallization experiment. T.O., M.N., T.S., T.Y., M.M., and S.Y. assisted with the LTB4 binding assay. M.M. supervised the expression, purification, and crystallization in the early stage of the project. S.Y. supervised the purification, crystallization, and structure determination and analysis. T.H. and S.Y. wrote the manuscript, and all of the other authors commented on it.

Corresponding author

Correspondence to Shigeyuki Yokoyama.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–5, Supplementary Figures 1–14 (PDF 34380 kb)

Life Sciences Reporting Summary (PDF 129 kb)

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hori, T., Okuno, T., Hirata, K. et al. Na+-mimicking ligands stabilize the inactive state of leukotriene B4 receptor BLT1. Nat Chem Biol 14, 262–269 (2018). https://doi.org/10.1038/nchembio.2547

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.2547

This article is cited by

Search

Advanced search

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