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.

The structural basis of agonist-induced activation in constitutively active rhodopsin

Abstract

G-protein-coupled receptors (GPCRs) comprise the largest family of membrane proteins in the human genome and mediate cellular responses to an extensive array of hormones, neurotransmitters and sensory stimuli. Although some crystal structures have been determined for GPCRs, most are for modified forms, showing little basal activity, and are bound to inverse agonists or antagonists. Consequently, these structures correspond to receptors in their inactive states. The visual pigment rhodopsin is the only GPCR for which structures exist that are thought to be in the active state1,2. However, these structures are for the apoprotein, or opsin, form that does not contain the agonist all-trans retinal. Here we present a crystal structure at a resolution of 3 Å for the constitutively active rhodopsin mutant Glu 113 Gln3,4,5 in complex with a peptide derived from the carboxy terminus of the α-subunit of the G protein transducin. The protein is in an active conformation that retains retinal in the binding pocket after photoactivation. Comparison with the structure of ground-state rhodopsin6 suggests how translocation of the retinal β-ionone ring leads to a rotation of transmembrane helix 6, which is the critical conformational change on activation7. A key feature of this conformational change is a reorganization of water-mediated hydrogen-bond networks between the retinal-binding pocket and three of the most conserved GPCR sequence motifs. We thus show how an agonist ligand can activate its GPCR.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Conformational changes in the retinal binding pocket.
Figure 2: Rearrangement of the heptahelix bundle and rotation of TM6.
Figure 3: Rearrangement of water-mediated hydrogen-bond networks.
Figure 4: Activation of rhodopsin by the agonist all- trans retinal.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors have been deposited at the Protein Data Bank under accession code 2X72.

References

  1. 1

    Park, J. H. et al. Crystal structure of the ligand-free G-protein-coupled receptor opsin. Nature 454, 183–187 (2008)

    ADS  CAS  Article  Google Scholar 

  2. 2

    Scheerer, P. et al. Crystal structure of opsin in its G-protein-interacting conformation. Nature 455, 497–502 (2008)

    ADS  CAS  Article  Google Scholar 

  3. 3

    Zhukovsky, E. A. & Oprian, D. D. Effect of carboxylic acid side chains on the absorption maximum of visual pigments. Science 246, 928–930 (1989)

    ADS  CAS  Article  Google Scholar 

  4. 4

    Sakmar, T. P., Franke, R. R. & Khorana, H. G. Glutamic acid-113 serves as the retinylidene Schiff base counterion in bovine rhodopsin. Proc. Natl Acad. Sci. USA 86, 8309–8313 (1989)

    ADS  CAS  Article  Google Scholar 

  5. 5

    Robinson, P. R., Cohen, G. B., Zhukovsky, E. A. & Oprian, D. D. Constitutively active mutants of rhodopsin. Neuron 9, 719–725 (1992)

    CAS  Article  Google Scholar 

  6. 6

    Li, J. et al. Structure of bovine rhodopsin in a trigonal crystal form. J. Mol. Biol. 343, 1409–1438 (2004)

    CAS  Article  Google Scholar 

  7. 7

    Altenbach, C. et al. High-resolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation. Proc. Natl Acad. Sci. USA 105, 7439–7444 (2008)

    ADS  CAS  Article  Google Scholar 

  8. 8

    Hofmann, K. P. et al. A G protein-coupled receptor at work: the rhodopsin model. Trends Biochem. Sci. 34, 540–552 (2009)

    CAS  Article  Google Scholar 

  9. 9

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

    ADS  CAS  Article  Google Scholar 

  10. 10

    Ruprecht, J. J. et al. Electron crystallography reveals the structure of metarhodopsin I. EMBO J. 23, 3609–3620 (2004)

    CAS  Article  Google Scholar 

  11. 11

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

    ADS  CAS  Article  Google Scholar 

  12. 12

    Hamm, H. E. et al. Site of G protein binding to rhodopsin mapped with synthetic peptides from the alpha subunit. Science 241, 832–835 (1988)

    ADS  CAS  Article  Google Scholar 

  13. 13

    Ballesteros, J. & 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)

    CAS  Article  Google Scholar 

  14. 14

    Standfuss, J., Zaitseva, E., Mahalingam, M. & Vogel, R. Structural impact of the E113Q counterion mutation on the activation and deactivation pathways of the G protein-coupled receptor rhodopsin. J. Mol. Biol. 380, 145–157 (2008)

    CAS  Article  Google Scholar 

  15. 15

    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)

    CAS  Article  Google Scholar 

  16. 16

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

    CAS  Article  Google Scholar 

  17. 17

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

    CAS  Article  Google Scholar 

  18. 18

    Martin, E. L., Rens-Domiano, S., Schatz, P. J. & Hamm, H. E. Potent peptide analogues of a G protein receptor-binding region obtained with a combinatorial library. J. Biol. Chem. 271, 361–366 (1996)

    CAS  Article  Google Scholar 

  19. 19

    Groenendijk, G. W., Jacobs, C. W., Bonting, S. L. & Daemen, F. J. Dark isomerization of retinals in the presence of phosphatidylethanolamine. Eur. J. Biochem. 106, 119–128 (1980)

    CAS  Article  Google Scholar 

  20. 20

    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)

    CAS  Article  Google Scholar 

  21. 21

    Kono, M., Goletz, P. W. & Crouch, R. K. 11-cis- and all-trans-retinols can activate rod opsin: rational design of the visual cycle. Biochemistry 47, 7567–7571 (2008)

    CAS  Article  Google Scholar 

  22. 22

    Ahuja, S. et al. Location of the retinal chromophore in the activated state of rhodopsin. J. Biol. Chem. 284, 10190–10201 (2009)

    CAS  Article  Google Scholar 

  23. 23

    Warne, T. et al. Structure of a β1-adrenergic G-protein-coupled receptor. Nature 454, 486–491 (2008)

    ADS  CAS  Article  Google Scholar 

  24. 24

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

    ADS  CAS  Article  Google Scholar 

  25. 25

    Jaakola, V. P. et al. The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist. Science 322, 1211–1217 (2008)

    ADS  CAS  Article  Google Scholar 

  26. 26

    Shi, L. et al. Beta2 adrenergic receptor activation. Modulation of the proline kink in transmembrane 6 by a rotamer toggle switch. J. Biol. Chem. 277, 40989–40996 (2002)

    CAS  Article  Google Scholar 

  27. 27

    Schwartz, T. W. et al. Molecular mechanism of 7TM receptor activation–a global toggle switch model. Annu. Rev. Pharmacol. Toxicol. 46, 481–519 (2006)

    CAS  Article  Google Scholar 

  28. 28

    Pardo, L. et al. The role of internal water molecules in the structure and function of the rhodopsin family of G protein-coupled receptors. ChemBioChem 8, 19–24 (2007)

    CAS  Article  Google Scholar 

  29. 29

    Vogel, R. et al. Functional role of the “ionic lock”–an interhelical hydrogen-bond network in family A heptahelical receptors. J. Mol. Biol. 380, 648–655 (2008)

    CAS  Article  Google Scholar 

  30. 30

    Reeves, P. J., Callewaert, N., Contreras, R. & Khorana, H. G. Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proc. Natl Acad. Sci. USA 99, 13419–13424 (2002)

    ADS  CAS  Article  Google Scholar 

  31. 31

    Reeves, P. J., Kim, J. M. & Khorana, H. G. Structure and function in rhodopsin: a tetracycline-inducible system in stable mammalian cell lines for high-level expression of opsin mutants. Proc. Natl Acad. Sci. USA 99, 13413 (2002)

    ADS  CAS  Article  Google Scholar 

  32. 32

    Reeves, P. J., Callewaert, N., Contreras, R. & Khorana, H. G. Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proc. Natl Acad. Sci. USA 99, 13419 (2002)

    ADS  CAS  Article  Google Scholar 

  33. 33

    Sakmar, T. P., Franke, R. R. & Khorana, H. G. Glutamic acid-113 serves as the retinylidene Schiff base counterion in bovine rhodopsin. Proc. Natl Acad. Sci. USA 86, 8309 (1989)

    ADS  CAS  Article  Google Scholar 

  34. 34

    Zhukovsky, E. A. & Oprian, D. D. Effect of carboxylic acid side chains on the absorption maximum of visual pigments. Science 246, 928 (1989)

    ADS  CAS  Article  Google Scholar 

  35. 35

    Standfuss, J., Zaitseva, E., Mahalingam, M. & Vogel, R. Structural impact of the E113Q counterion mutation on the activation and deactivation pathways of the G protein-coupled receptor rhodopsin. J. Mol. Biol. 380, 145 (2008)

    CAS  Article  Google Scholar 

  36. 36

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

    CAS  Article  Google Scholar 

  37. 37

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

    Article  Google Scholar 

  38. 38

    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 

  39. 39

    Park, J. H. et al. Crystal structure of the ligand-free G-protein-coupled receptor opsin. Nature 454, 183 (2008)

    ADS  CAS  Article  Google Scholar 

  40. 40

    Scheerer, P. et al. Crystal structure of opsin in its G-protein-interacting conformation. Nature 455, 497 (2008)

    ADS  CAS  Article  Google Scholar 

  41. 41

    Li, J. et al. Structure of bovine rhodopsin in a trigonal crystal form. J. Mol. Biol. 343, 1409 (2004)

    CAS  Article  Google Scholar 

  42. 42

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

    Article  Google Scholar 

  43. 43

    Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002)

    Article  Google Scholar 

Download references

Acknowledgements

We thank X. Deupi and R. Vogel for discussions and reading of the manuscript. We thank R. Crouch for the kind gift of 11-cis retinal. P. J. Reeves we thank for providing the pACMVtetO vector and the HEK293S-GnTI cells, and for his advice on creating stable cell lines and tetracycline-inducible expression. We also thank the staff at the macromolecular crystallography beamlines at the European Synchrotron Radiation Facility, the Diamond Light Source and the Swiss Light Source. The work was financially supported by NIH grant EY007965 (to D.D.O.), the Human Frontier Science Project programme grant RG/0052 (to D.D.O. and G.F.X.S.), the European Commission FP6 specific targeted research project LSH-2003-1.1.0-1 (to G.F.X.S.), the Marie Curie Intra European Fellowship MEIF-CT-2006-039171 (to J.S.) and the EMBO long-term fellowship ALTF 198-2006 (to J.S.).

Author information

Affiliations

Authors

Contributions

The project was initiated by D.D.O. and G.F.X.S. J.S. performed cloning, initial expression and purification using essential experimental protocols and materials contributed by A.D. and D.D.O. Receptor activation and retinal binding studies were contributed by A.D. and G.X. Initial crystallization was by J.S., who also collected data and refined the structures. P.C.E. optimized expression and crystallization, performed crystal cryo-cooling and coordinated data collection. M.F. investigated the stability of mutant proteins. Manuscript preparation was performed by J.S. and D.D.O. The overall project management was by G.F.X.S.

Corresponding author

Correspondence to Gebhard F. X. Schertler.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains Supplementary Figures 1-5 with legends, Supplementary Tables 1-2 and additional references. (PDF 503 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Standfuss, J., Edwards, P., D’Antona, A. et al. The structural basis of agonist-induced activation in constitutively active rhodopsin. Nature 471, 656–660 (2011). https://doi.org/10.1038/nature09795

Download citation

Further reading

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