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.

Structure of the chemokine receptor CXCR1 in phospholipid bilayers

Abstract

CXCR1 is one of two high-affinity receptors for the CXC chemokine interleukin-8 (IL-8), a major mediator of immune and inflammatory responses implicated in many disorders, including tumour growth1,2,3. IL-8, released in response to inflammatory stimuli, binds to the extracellular side of CXCR1. The ligand-activated intracellular signalling pathways result in neutrophil migration to the site of inflammation2. CXCR1 is a class A, rhodopsin-like G-protein-coupled receptor (GPCR), the largest class of integral membrane proteins responsible for cellular signal transduction and targeted as drug receptors4,5,6,7. Despite its importance, the molecular mechanism of CXCR1 signal transduction is poorly understood owing to the limited structural information available. Recent structural determination of GPCRs has advanced by modifying the receptors with stabilizing mutations, insertion of the protein T4 lysozyme and truncations of their amino acid sequences8, as well as addition of stabilizing antibodies and small molecules9 that facilitate crystallization in cubic phase monoolein mixtures10. The intracellular loops of GPCRs are crucial for G-protein interactions11, and activation of CXCR1 involves both amino-terminal residues and extracellular loops2,12,13. Our previous nuclear magnetic resonance studies indicate that IL-8 binding to the N-terminal residues is mediated by the membrane, underscoring the importance of the phospholipid bilayer for physiological activity14. Here we report the three-dimensional structure of human CXCR1 determined by NMR spectroscopy. The receptor is in liquid crystalline phospholipid bilayers, without modification of its amino acid sequence and under physiological conditions. Features important for intracellular G-protein activation and signal transduction are revealed. The structure of human CXCR1 in a lipid bilayer should help to facilitate the discovery of new compounds that interact with GPCRs and combat diseases such as breast cancer.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Structure determination of CXCR1.
Figure 2: Three-dimensional structure of CXCR1.
Figure 3: Structural comparison of CXCR1 and CXCR4.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates for residues 29–324 of CXCR1 and NMR restraints have been deposited in the Protein Data Bank (PDB) under accession 2LNL. Assigned NMR frequencies have been deposited in the Biological Magnetic Resonance Bank (BMRB) under accession 18170.

References

  1. Holmes, W. E., Lee, J., Kuang, W. J., Rice, G. C. & Wood, W. I. Structure and functional expression of a human interleukin-8 receptor. Science 253, 1278–1280 (1991)

    Article  ADS  CAS  PubMed  Google Scholar 

  2. Sallusto, F. & Baggiolini, M. Chemokines and leukocyte traffic. Nature Immunol. 9, 949–952 (2008)

    Article  CAS  Google Scholar 

  3. Waugh, D. J. & Wilson, C. The interleukin-8 pathway in cancer. Clin. Cancer Res. 14, 6735–6741 (2008)

    Article  CAS  PubMed  Google Scholar 

  4. Rajagopal, S., Rajagopal, K. & Lefkowitz, R. J. Teaching old receptors new tricks: biasing seven-transmembrane receptors. Nature Rev. Drug Discov. 9, 373–386 (2010)

    Article  CAS  Google Scholar 

  5. Goncalves, J. A., Ahuja, S., Erfani, S., Eilers, M. & Smith, S. O. Structure and function of G protein-coupled receptors using NMR spectroscopy. Prog. Nucl. Magn. Reson. Spectrosc. 57, 159–180 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Rosenbaum, D. M., Rasmussen, S. G. & Kobilka, B. K. The structure and function of G-protein-coupled receptors. Nature 459, 356–363 (2009)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  7. Katritch, V., Cherezov, V. & Stevens, R. C. Diversity and modularity of G protein-coupled receptor structures. Trends Pharmacol. Sci. 33, 17–27 (2012)

    Article  CAS  PubMed  Google Scholar 

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

    Article  ADS  CAS  PubMed  Google Scholar 

  9. Rasmussen, S. G. et al. Crystal structure of the human β2 adrenergic G-protein-coupled receptor. Nature 450, 383–387 (2007)

    Article  ADS  CAS  PubMed  Google Scholar 

  10. Landau, E. M. & Rosenbusch, J. P. Lipidic cubic phases: a novel concept for the crystallization of membrane proteins. Proc. Natl Acad. Sci. USA 93, 14532–14535 (1996)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  11. Oldham, W. M. & Hamm, H. E. Heterotrimeric G protein activation by G-protein-coupled receptors. Nature. Rev. Mol. Cell Biol. 9, 60–71 (2008)

    Article  CAS  Google Scholar 

  12. Crump, M. P. et al. Solution structure and basis for functional activity of stromal cell-derived factor-1; dissociation of CXCR4 activation from binding and inhibition of HIV-1. EMBO J. 16, 6996–7007 (1997)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Rajagopalan, L. & Rajarathnam, K. Ligand selectivity and affinity of chemokine receptor CXCR1. J. Biol. Chem. 279, 30000–30008 (2004)

    Article  CAS  PubMed  Google Scholar 

  14. Park, S. H., Casagrande, F., Cho, L., Albrecht, L. & Opella, S. J. Interactions of interleukin-8 with the human chemokine receptor CXCR1 in phospholipid bilayers by NMR spectroscopy. J. Mol. Biol. 414, 194–203 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Das, B. B. et al. Structure determination of a membrane protein in proteoliposomes. J. Am. Chem. Soc. 134, 2047–2056 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. McDermott, A. Structure and dynamics of membrane proteins by magic angle spinning solid-state NMR. Annu. Rev. Biophys. 38, 385–403 (2009)

    Article  MathSciNet  CAS  PubMed  Google Scholar 

  17. Opella, S. J. & Marassi, F. M. Structure determination of membrane proteins by NMR spectroscopy. Chem. Rev. 104, 3587–3606 (2004)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Edidin, M. Rotational and translational diffusion in membranes. Annu. Rev. Biophys. Bioeng. 3, 179–201 (1974)

    Article  CAS  PubMed  Google Scholar 

  19. Das, R. & Baker, D. Macromolecular modeling with rosetta. Annu. Rev. Biochem. 77, 363–382 (2008)

    Article  CAS  PubMed  Google Scholar 

  20. Shen, Y. et al. Consistent blind protein structure generation from NMR chemical shift data. Proc. Natl Acad. Sci. USA 105, 4685–4690 (2008)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  21. Yarov-Yarovoy, V., Schonbrun, J. & Baker, D. Multipass membrane protein structure prediction using Rosetta. Proteins 62, 1010–1025 (2006)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Park, S. H. et al. High-resolution NMR spectroscopy of a GPCR in aligned bicelles. J. Am. Chem. Soc. 128, 7402–7403 (2006)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Park, S. H. et al. Optimization of purification and refolding of the human chemokine receptor CXCR1 improves the stability of proteoliposomes for structure determination. Biochim. Biophys. Acta 1818, 584–591 (2012)

    Article  CAS  PubMed  Google Scholar 

  24. Park, S. H. et al. Local and global dynamics of the G protein-coupled receptor CXCR1. Biochemistry 50, 2371–2380 (2011)

    Article  CAS  PubMed  Google Scholar 

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

    Article  ADS  CAS  PubMed  Google Scholar 

  26. Schwieters, C. D., Kuszewski, J. J., Tjandra, N. & Clore, G. M. The Xplor-NIH NMR molecular structure determination package. J. Magn. Reson. 160, 65–73 (2003)

    Article  ADS  CAS  PubMed  Google Scholar 

  27. Krebs, A., Edwards, P. C., Villa, C., Li, J. & Schertler, G. F. The three-dimensional structure of bovine rhodopsin determined by electron cryomicroscopy. J. Biol. Chem. 278, 50217–50225 (2003)

    Article  CAS  PubMed  Google Scholar 

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

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  29. Mesleh, M. F. et al. Dipolar waves map the structure and topology of helices in membrane proteins. J. Am. Chem. Soc. 125, 8928–8935 (2003)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Ginestier, C. et al. CXCR1 blockade selectively targets human breast cancer stem cells in vitro and in xenografts. J. Clin. Invest. 120, 485–497 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Casagrande, F., Maier, K., Kiefer, H., Opella, S. J. & Park, S. H. in Production of Membrane Proteins (ed. Robinson, A. S. ) 297–316 (Wiley-VCH Verlag GmbH & Co. KGaA, 2011)

    Book  Google Scholar 

  32. Murphy, P. M. & Tiffany, H. L. Cloning of complementary DNA encoding a functional human interleukin-8 receptor. Science 253, 1280–1283 (1991)

    Article  ADS  CAS  PubMed  Google Scholar 

  33. Wishart, D. S. et al. 1H, 13C and 15N chemical shift referencing in biomolecular NMR. J. Biomol. NMR 6, 135–140 (1995)

    Article  CAS  PubMed  Google Scholar 

  34. Morcombe, C. R. & Zilm, K. W. Chemical shift referencing in MAS solid state NMR. J. Magn. Reson. 162, 479–486 (2003)

    Article  ADS  CAS  PubMed  Google Scholar 

  35. Park, S. H., Das, B. B., De Angelis, A. A., Scrima, M. & Opella, S. J. Mechanically, magnetically, and “rotationally aligned” membrane proteins in phospholipid bilayers give equivalent angular constraints for NMR structure determination. J. Phys. Chem. B 114, 13995–14003 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Sharma, D. & Rajarathnam, K. 13C NMR chemical shifts can predict disulfide bond formation. J. Biomol. NMR 18, 165–171 (2000)

    Article  CAS  PubMed  Google Scholar 

  37. Cornilescu, G., Delaglio, F. & Bax, A. Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J. Biomol. NMR 13, 289–302 (1999)

    Article  CAS  PubMed  Google Scholar 

  38. Shen, Y., Delaglio, F., Cornilescu, G. & Bax, A. TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. J. Biomol. NMR 44, 213–223 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Denny, J. K., Wang, J., Cross, T. A. & Quine, J. R. PISEMA powder patterns and PISA wheels. J. Magn. Reson. 152, 217–226 (2001)

    Article  ADS  CAS  PubMed  Google Scholar 

  40. Schwieters, C. D. & Clore, G. M. Internal coordinates for molecular dynamics and minimization in structure determination and refinement. J. Magn. Reson. 152, 288–302 (2001)

    Article  ADS  CAS  PubMed  Google Scholar 

  41. Kuszewski, J., Gronenborn, A. M. & Clore, G. M. Improvements and extensions in the conformational database potential for the refinement of NMR and X-ray structures of proteins and nucleic acids. J. Magn. Reson. 125, 171–177 (1997)

    Article  ADS  CAS  PubMed  Google Scholar 

  42. Kuszewski, J., Gronenborn, A. M. & Clore, G. M. Improving the packing and accuracy of NMR structures with a pseudopotential for the radius of gyration. J. Am. Chem. Soc. 121, 2337–2338 (1999)

    Article  CAS  Google Scholar 

  43. Nilges, M., Clore, G. M. & Gronenborn, A. M. Determination of three-dimensional structures of proteins from interproton distance data by dynamical simulated annealing from a random array of atoms. FEBS Lett. 239, 129–136 (1988)

    Article  CAS  PubMed  Google Scholar 

  44. Laskowski, R. A., Rullmannn, J. A., MacArthur, M. W., Kaptein, R. & Thornton, J. M. AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR 8, 477–486 (1996)

    Article  CAS  PubMed  Google Scholar 

  45. DeLano, W. L. PyMOl. www.pymol.org (2005)

  46. Clore, G. M. & Garrett, D. S. R-factor, free R, and complete cross-validation for dipolar coupling refinement of NMR structures. J. Am. Chem. Soc. 121, 9008–9012 (1999)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This research was supported by grants R01EB005161, R01GM075877, R21GM94727, R21GM075917, P01AI074805 and P41EB002031 from the National Institutes of Health (NIH). Further support came from Cambridge Isotope Laboratories. F.C. was supported by fellowships from the Swiss National Science Foundation (PBBSP3-123151) and the Novartis Foundation.

Author information

Authors and Affiliations

Authors

Contributions

S.J.O. designed the study. S.P. optimized CXCR1 purification, refolding and NMR sample preparation. B.B.D. performed the NMR experiments. H.J.N. assisted in NMR data analysis. H.K. developed initial protocols for CXCR1 purification, refolding and functional assays. K.M. assisted in the revision of these methods for NMR experiments. A.A.D. tested samples for their suitability for NMR experiments. F.C., M.C. and K.M. expressed and purified CXCR1. F.M.M. and Y.T. performed the structure calculations. S.J.O., S.P., B.B.D., F.M.M. and Y.T. prepared the figures and wrote the paper.

Corresponding author

Correspondence to Stanley J. Opella.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains detailed Supplementary Methods, Supplementary References, Supplementary Table 1, which shows NMR structural statistics, Supplementary Table 2, which lists NMR experimental parameters and Supplementary Figures 1-11, which display additional experimental data. (PDF 3577 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Park, S., Das, B., Casagrande, F. et al. Structure of the chemokine receptor CXCR1 in phospholipid bilayers. Nature 491, 779–783 (2012). https://doi.org/10.1038/nature11580

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature11580

This article is cited by

Comments

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.

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