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.

  • Article
  • Published:

Crystal structure of the ligand-free G-protein-coupled receptor opsin

Abstract

In the G-protein-coupled receptor (GPCR) rhodopsin, the inactivating ligand 11-cis-retinal is bound in the seven-transmembrane helix (TM) bundle and is cis/trans isomerized by light to form active metarhodopsin II. With metarhodopsin II decay, all-trans-retinal is released, and opsin is reloaded with new 11-cis-retinal. Here we present the crystal structure of ligand-free native opsin from bovine retinal rod cells at 2.9 ångström (Å) resolution. Compared to rhodopsin, opsin shows prominent structural changes in the conserved E(D)RY and NPxxY(x)5,6F regions and in TM5–TM7. At the cytoplasmic side, TM6 is tilted outwards by 6–7 Å, whereas the helix structure of TM5 is more elongated and close to TM6. These structural changes, some of which were attributed to an active GPCR state, reorganize the empty retinal-binding pocket to disclose two openings that may serve the entry and exit of retinal. The opsin structure sheds new light on ligand binding to GPCRs and on GPCR activation.

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: Overall fold of opsin.
Figure 2: Structure of opsin and comparison with rhodopsin.
Figure 3: Superposition of conserved E(D)RY regions of rhodopsin and opsin.
Figure 4: Superposition of conserved NPxxY(x) 5,6 F regions of rhodopsin and opsin.
Figure 5: Retinal-binding pocket.
Figure 6: Openings of the retinal-binding pocket in opsin.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The coordinates and structure factors have been deposited in the Protein Data Bank under accession number 3CAP.

References

  1. Lagerstrom, M. C. & Schioth, H. B. Structural diversity of G protein-coupled receptors and significance for drug discovery. Nature Rev. Drug Discov. 7, 339–357 (2008)

    Article  Google Scholar 

  2. Palczewski, K. G protein-coupled receptor rhodopsin. Annu. Rev. Biochem. 75, 743–767 (2006)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Okada, T., Ernst, O. P., Palczewski, K. & Hofmann, K. P. Activation of rhodopsin: new insights from structural and biochemical studies. Trends Biochem. Sci. 26, 318–324 (2001)

    Article  CAS  PubMed  Google Scholar 

  4. Farrens, D. L., Altenbach, C., Yang, K., Hubbell, W. L. & Khorana, H. G. Requirement of rigid-body motion of transmembrane helices for light activation of rhodopsin. Science 274, 768–770 (1996)

    Article  CAS  ADS  PubMed  Google Scholar 

  5. Sheikh, S. P., Zvyaga, T. A., Lichtarge, O., Sakmar, T. P. & Bourne, H. R. Rhodopsin activation blocked by metal-ion-binding sites linking transmembrane helices C and F. Nature 383, 347–350 (1996)

    Article  CAS  ADS  PubMed  Google Scholar 

  6. Fritze, O. et al. Role of the conserved NPxxY(x)5,6F motif in the rhodopsin ground state and during activation. Proc. Natl Acad. Sci. USA 100, 2290–2295 (2003)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  7. Knierim, B., Hofmann, K. P., Ernst, O. P. & Hubbell, W. L. Sequence of late molecular events in the activation of rhodopsin. Proc. Natl Acad. Sci. USA 104, 20290–20295 (2007)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  8. Menon, S. T., Han, M. & Sakmar, T. P. Rhodopsin: structural basis of molecular physiology. Physiol. Rev. 81, 1659–1688 (2001)

    Article  CAS  PubMed  Google Scholar 

  9. Lamb, T. D. & Pugh, E. N. Dark adaptation and the retinoid cycle of vision. Prog. Retin. Eye Res. 23, 307–380 (2004)

    Article  CAS  PubMed  Google Scholar 

  10. McBee, J. K., Palczewski, K., Baehr, W. & Pepperberg, D. R. Confronting complexity: the interlink of phototransduction and retinoid metabolism in the vertebrate retina. Prog. Retin. Eye Res. 20, 469–529 (2001)

    Article  CAS  PubMed  Google Scholar 

  11. Vogel, R. & Siebert, F. Conformations of the active and inactive states of opsin. J. Biol. Chem. 276, 38487–38493 (2001)

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  ADS  PubMed  Google Scholar 

  13. Okada, T. et al. The retinal conformation and its environment in rhodopsin in light of a new 2.2 Å crystal structure. J. Mol. Biol. 342, 571–583 (2004)

    Article  CAS  PubMed  Google Scholar 

  14. Li, J., Edwards, P. C., Burghammer, M., Villa, C. & Schertler, G. F. Structure of bovine rhodopsin in a trigonal crystal form. J. Mol. Biol. 343, 1409–1438 (2004)

    Article  CAS  PubMed  Google Scholar 

  15. Salom, D. et al. Crystal structure of a photoactivated deprotonated intermediate of rhodopsin. Proc. Natl Acad. Sci. USA 103, 16123–16128 (2006)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  16. Standfuss, J. et al. Crystal structure of a thermally stable rhodopsin mutant. J. Mol. Biol. 372, 1179–1188 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Nakamichi, H. & Okada, T. Local peptide movement in the photoreaction intermediate of rhodopsin. Proc. Natl Acad. Sci. USA 103, 12729–12734 (2006)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  18. Cherezov, V. et al. High-resolution crystal structure of an engineered human β2-adrenergic G protein-coupled receptor. Science 318, 1258–1265 (2007)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  ADS  PubMed  Google Scholar 

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

    Article  CAS  ADS  PubMed  Google Scholar 

  21. Xie, G., Gross, A. K. & Oprian, D. D. An opsin mutant with increased thermal stability. Biochemistry 42, 1995–2001 (2003)

    Article  CAS  PubMed  Google Scholar 

  22. Okada, T., Takeda, K. & Kouyama, T. Highly selective separation of rhodopsin from bovine rod outer segment membranes using combination of divalent cation and alkyl(thio)glucoside. Photochem. Photobiol. 67, 495–499 (1998)

    Article  CAS  PubMed  Google Scholar 

  23. 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 

  24. Ruprecht, J. J., Mielke, T., Vogel, R., Villa, C. & Schertler, G. F. Electron crystallography reveals the structure of metarhodopsin I. EMBO J. 23, 3609–3620 (2004)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Liu, W., Eilers, M., Patel, A. B. & Smith, S. O. Helix packing moments reveal diversity and conservation in membrane protein structure. J. Mol. Biol. 337, 713–729 (2004)

    Article  CAS  PubMed  Google Scholar 

  26. Cohen, G. B., Oprian, D. D. & Robinson, P. R. Mechanism of activation and inactivation of opsin: role of Glu113 and Lys296. Biochemistry 31, 12592–12601 (1992)

    Article  CAS  PubMed  Google Scholar 

  27. 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 Neurosci. 25, 366–428 (1995)

    Article  CAS  Google Scholar 

  28. Ballesteros, J. A. et al. Activation of the β2-adrenergic receptor involves disruption of an ionic lock between the cytoplasmic ends of transmembrane segments 3 and 6. J. Biol. Chem. 276, 29171–29177 (2001)

    Article  CAS  PubMed  Google Scholar 

  29. Filipek, S., Stenkamp, R. E., Teller, D. C. & Palczewski, K. G protein-coupled receptor rhodopsin: a prospectus. Annu. Rev. Physiol. 65, 851–879 (2003)

    Article  CAS  PubMed  Google Scholar 

  30. Crocker, E. et al. Location of Trp265 in metarhodopsin II: implications for the activation mechanism of the visual receptor rhodopsin. J. Mol. Biol. 357, 163–172 (2006)

    Article  CAS  PubMed  Google Scholar 

  31. Shi, L. et al. β2 adrenergic receptor activation. Modulation of the proline kink in transmembrane 6 by a rotamer toggle switch. J. Biol. Chem. 277, 40989–40996 (2002)

    Article  CAS  PubMed  Google Scholar 

  32. Lüdeke, S. et al. The role of Glu181 in the photoactivation of rhodopsin. J. Mol. Biol. 353, 345–356 (2005)

    Article  PubMed  Google Scholar 

  33. Schädel, S. A. et al. Ligand channeling within a G-protein-coupled receptor. The entry and exit of retinals in native opsin. J. Biol. Chem. 278, 24896–24903 (2003)

    Article  PubMed  Google Scholar 

  34. Heck, M. et al. Signaling states of rhodopsin. Formation of the storage form, metarhodopsin III, from active metarhodopsin II. J. Biol. Chem. 278, 3162–3169 (2003)

    Article  CAS  PubMed  Google Scholar 

  35. Bartl, F. J. & Vogel, R. Structural and functional properties of metarhodopsin III: Recent spectroscopic studies on deactivation pathways of rhodopsin. Phys. Chem. Chem. Phys. 9, 1648–1658 (2007)

    Article  CAS  PubMed  Google Scholar 

  36. Farrens, D. L. & Khorana, H. G. Structure and function in rhodopsin. Measurement of the rate of metarhodopsin II decay by fluorescence spectroscopy. J. Biol. Chem. 270, 5073–5076 (1995)

    Article  CAS  PubMed  Google Scholar 

  37. Hofmann, K. P., Pulvermüller, A., Buczylko, J., Van Hooser, P. & Palczewski, K. The role of arrestin and retinoids in the regeneration pathway of rhodopsin. J. Biol. Chem. 267, 15701–15706 (1992)

    CAS  PubMed  Google Scholar 

  38. Jäger, S., Palczewski, K. & Hofmann, K. P. Opsin/all-trans-retinal complex activates transducin by different mechanisms than photolyzed rhodopsin. Biochemistry 35, 2901–2908 (1996)

    Article  PubMed  Google Scholar 

  39. Sachs, K., Maretzki, D., Meyer, C. K. & Hofmann, K. P. Diffusible ligand all-trans-retinal activates opsin via a palmitoylation-dependent mechanism. J. Biol. Chem. 275, 6189–6194 (2000)

    Article  CAS  PubMed  Google Scholar 

  40. De Lean, A., Stadel, J. M. & Lefkowitz, R. J. A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled beta-adrenergic receptor. J. Biol. Chem. 255, 7108–7117 (1980)

    CAS  PubMed  Google Scholar 

  41. Scheer, A., Fanelli, F., Costa, T., De Benedetti, P. G. & Cotecchia, S. The activation process of the α1B-adrenergic receptor: potential role of protonation and hydrophobicity of a highly conserved aspartate. Proc. Natl Acad. Sci. USA 94, 808–813 (1997)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  42. Kefalov, V. J., Crouch, R. K. & Cornwall, M. C. Role of noncovalent binding of 11-cis-retinal to opsin in dark adaptation of rod and cone photoreceptors. Neuron 29, 749–755 (2001)

    Article  CAS  PubMed  Google Scholar 

  43. Sachs, K., Maretzki, D. & Hofmann, K. P. Assays for activation of opsin by all-trans-retinal. Methods Enzymol. 315, 238–251 (2000)

    Article  CAS  PubMed  Google Scholar 

  44. Jancarik, J. & Kim, S.-H. Sparse matrix sampling: a screening method for crystallization of proteins. J. Appl. Crystallogr. 24, 409–411 (1991)

    Article  CAS  Google Scholar 

  45. Surya, A., Foster, K. W. & Knox, B. E. Transducin activation by the bovine opsin apoprotein. J. Biol. Chem. 270, 5024–5031 (1995)

    Article  CAS  PubMed  Google Scholar 

  46. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    Article  CAS  PubMed  Google Scholar 

  47. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  Google Scholar 

  50. Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993)

    Article  CAS  Google Scholar 

  51. Hooft, R. W., Vriend, G., Sander, C. & Abola, E. E. Errors in protein structures. Nature 381, 272 (1996)

    Article  CAS  ADS  PubMed  Google Scholar 

  52. DeLano, W. L. The PyMOL Molecular Graphics System. <http://www.pymol.org> (2002)

Download references

Acknowledgements

We thank J. Engelmann and C. Koch for technical assistance; Y. Li for help in the early stage of the project; Y. J. Kim for help with data collection; M. Sommer, C. Enenkel and M. Heck for critically reading the manuscript; and N. Krauss for support and sharing his expertise in crystallography. We thank U. Müller and the scientific staff of the Protein Structure Factory and the Freie Universität Berlin at beamlines BL 14.1 and BL 14.2 at BESSY for continuous support of the project. This work was supported by the Deutsche Forschungsgemeinschaft Sfb449 (to O.P.E.), Sfb740 (to O.P.E. and K.P.H.), DFG-KOSEF international cooperation ER 294/1-1 (to O.P.E.) and F01-2004-000-10054-0 (to H.-W.C.). H-.W.C. was supported by CBNU funds for overseas research 2006–2007.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Klaus Peter Hofmann, Hui-Woog Choe or Oliver Peter Ernst.

Supplementary information

Supplementary Information

This file contains Supplementary Table 1, Supplementary Discussion and Supplementary Figure 1. Supplementary Table 1 includes X-ray data collection and refinement statistics. A model of rhodopsin regeneration shown in Supplementary Figure 1 is discussed. (PDF 468 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Park, J., Scheerer, P., Hofmann, K. et al. Crystal structure of the ligand-free G-protein-coupled receptor opsin. Nature 454, 183–187 (2008). https://doi.org/10.1038/nature07063

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature07063

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