Crystal structure of opsin in its G-protein-interacting conformation

Article metrics

Abstract

Opsin, the ligand-free form of the G-protein-coupled receptor rhodopsin, at low pH adopts a conformationally distinct, active G-protein-binding state known as Ops*. A synthetic peptide derived from the main binding site of the heterotrimeric G protein—the carboxy terminus of the α-subunit (GαCT)—stabilizes Ops*. Here we present the 3.2 Å crystal structure of the bovine Ops*–GαCT peptide complex. GαCT binds to a site in opsin that is opened by an outward tilt of transmembrane helix (TM) 6, a pairing of TM5 and TM6, and a restructured TM7–helix 8 kink. Contacts along the inner surface of TM5 and TM6 induce an α-helical conformation in GαCT with a C-terminal reverse turn. Main-chain carbonyl groups in the reverse turn constitute the centre of a hydrogen-bonded network, which links the two receptor regions containing the conserved E(D)RY and NPxxY(x)5,6F motifs. On the basis of the Ops*–GαCT structure and known conformational changes in Gα, we discuss signal transfer from the receptor to the G protein nucleotide-binding site.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Overall structure of Ops*–GαCT complex.
Figure 2: Comparison of Ops*–GαCT and dark-state rhodopsin structures.
Figure 3: Interactions of Arg 135 of the conserved E(D)RY motif.
Figure 4: Stabilizing effects of GαCT on Ops*.
Figure 5: Conceptual model for signal transmission from the active receptor to the G protein by the Gα C terminus.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

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

References

  1. 1

    Pierce, K. L., Premont, R. T. & Lefkowitz, R. J. Seven-transmembrane receptors. Nature Rev. Mol. Cell Biol. 3, 639–650 (2002)

  2. 2

    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)

  3. 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)

  4. 4

    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)

  5. 5

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

  6. 6

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

  7. 7

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

  8. 8

    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)

  9. 9

    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)

  10. 10

    Murakami, M. & Kouyama, T. Crystal structure of squid rhodopsin. Nature 453, 363–367 (2008)

  11. 11

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

  12. 12

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

  13. 13

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

  14. 14

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

  15. 15

    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)

  16. 16

    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)

  17. 17

    Altenbach, C., Kusnetzow, A. K., Ernst, O. P., Hofmann, K. P. & Hubbell, W. L. High-resolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation. Proc. Natl Acad. Sci. USA 105, 7439–7444 (2008)

  18. 18

    Park, J. H., Scheerer, P., Hofmann, K. P., Choe, H.-W. & Ernst, O. P. Crystal structure of the ligand-free G-protein-coupled receptor opsin. Nature 454, 183–187 (2008)

  19. 19

    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)

  20. 20

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

  21. 21

    Kisselev, O. G., Ermolaeva, M. V. & Gautam, N. A farnesylated domain in the G protein γ subunit is a specific determinant of receptor coupling. J. Biol. Chem. 269, 21399–21402 (1994)

  22. 22

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

  23. 23

    Herrmann, R. et al. Sequence of interactions in receptor-G protein coupling. J. Biol. Chem. 279, 24283–24290 (2004)

  24. 24

    Kisselev, O. G. et al. Light-activated rhodopsin induces structural binding motif in G protein α subunit. Proc. Natl Acad. Sci. USA 95, 4270–4275 (1998)

  25. 25

    Koenig, B. W. et al. Structure and orientation of a G protein fragment in the receptor bound state from residual dipolar couplings. J. Mol. Biol. 322, 441–461 (2002)

  26. 26

    Arnis, S. & Hofmann, K. P. Two different forms of metarhodopsin II: Schiff base deprotonation precedes proton uptake and signaling state. Proc. Natl Acad. Sci. USA 90, 7849–7853 (1993)

  27. 27

    Arnis, S., Fahmy, K., Hofmann, K. P. & Sakmar, T. P. A conserved carboxylic acid group mediates light-dependent proton uptake and signaling by rhodopsin. J. Biol. Chem. 269, 23879–23881 (1994)

  28. 28

    Acharya, S., Saad, Y. & Karnik, S. S. Transducin-α C-terminal peptide binding site consists of C-D and E-F loops of rhodopsin. J. Biol. Chem. 272, 6519–6524 (1997)

  29. 29

    Janz, J. M. & Farrens, D. L. Rhodopsin activation exposes a key hydrophobic binding site for the transducin α-subunit C terminus. J. Biol. Chem. 279, 29767–29773 (2004)

  30. 30

    Cai, K., Itoh, Y. & Khorana, H. G. Mapping of contact sites in complex formation between transducin and light-activated rhodopsin by covalent crosslinking: use of a photoactivatable reagent. Proc. Natl Acad. Sci. USA 98, 4877–4882 (2001)

  31. 31

    Lambright, D. G. et al. The 2.0 Å crystal structure of a heterotrimeric G protein. Nature 379, 311–319 (1996)

  32. 32

    Ridge, K. D. et al. Conformational changes associated with receptor stimulated guanine nucleotide exchange in a heterotrimeric G-protein α-subunit: NMR analysis of GTPγ S-bound states. J. Biol. Chem. 281, 7635–7648 (2006)

  33. 33

    Ernst, O. P. et al. Mutation of the fourth cytoplasmic loop of rhodopsin affects binding of transducin and peptides derived from the carboxyl-terminal sequences of transducin α and γ subunits. J. Biol. Chem. 275, 1937–1943 (2000)

  34. 34

    Edwards, M. D. et al. Pivotal role of the glycine-rich TM3 helix in gating the MscS mechanosensitive channel. Nature Struct. Mol. Biol. 12, 113–119 (2005)

  35. 35

    Hildebrand, P. W. et al. Hydrogen-bonding and packing features of membrane proteins: functional implications. Biophys. J. 94, 1945–1953 (2008)

  36. 36

    Cohen, G. B., Oprian, D. D. & Robinson, P. R. Mechanism of activation and inactivation of opsin: role of Glu 113 and Lys 296. Biochemistry 31, 12592–12601 (1992)

  37. 37

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

  38. 38

    Fahmy, K. & Sakmar, T. P. Regulation of the rhodopsin-transducin interaction by a highly conserved carboxylic acid group. Biochemistry 32, 7229–7236 (1993)

  39. 39

    Franke, R. R., König, B., Sakmar, T. P., Khorana, H. G. & Hofmann, K. P. Rhodopsin mutants that bind but fail to activate transducin. Science 250, 123–125 (1990)

  40. 40

    Hofmann, K. P., Spahn, C. M., Heinrich, R. & Heinemann, U. Building functional modules from molecular interactions. Trends Biochem. Sci. 31, 497–508 (2006)

  41. 41

    Meyer, C. K. et al. Signaling states of rhodopsin. Retinal provides a scaffold for activating proton transfer switches. J. Biol. Chem. 275, 19713–19718 (2000)

  42. 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)

  43. 43

    Oldham, W. M., Van Eps, N., Preininger, A. M., Hubbell, W. L. & Hamm, H. E. Mechanism of the receptor-catalyzed activation of heterotrimeric G proteins. Nature Struct. Mol. Biol. 13, 772–777 (2006)

  44. 44

    Natochin, M., Moussaif, M. & Artemyev, N. O. Probing the mechanism of rhodopsin-catalyzed transducin activation. J. Neurochem. 77, 202–210 (2001)

  45. 45

    Marin, E. P., Krishna, A. G. & Sakmar, T. P. Disruption of the α5 helix of transducin impairs rhodopsin-catalyzed nucleotide exchange. Biochemistry 41, 6988–6994 (2002)

  46. 46

    Herrmann, R., Heck, M., Henklein, P., Hofmann, K. P. & Ernst, O. P. Signal transfer from GPCRs to G proteins: Role of the Gα N-terminal region in rhodopsin-transducin coupling. J. Biol. Chem. 281, 30234–30241 (2006)

  47. 47

    Nanoff, C. et al. The carboxyl terminus of the Gα-subunit is the latch for triggered activation of heterotrimeric G proteins. Mol. Pharmacol. 69, 397–405 (2006)

  48. 48

    Johnston, C. A. & Siderovski, D. P. Structural basis for nucleotide exchange on Gαi subunits and receptor coupling specificity. Proc. Natl Acad. Sci. USA 104, 2001–2006 (2007)

  49. 49

    Heck, M. & Hofmann, K. P. Maximal rate and nucleotide dependence of rhodopsin-catalyzed transducin activation: initial rate analysis based on a double displacement mechanism. J. Biol. Chem. 276, 10000–10009 (2001)

  50. 50

    Herrmann, R. et al. Rhodopsin-transducin coupling: role of the Gα C-terminus in nucleotide exchange catalysis. Vision Res. 46, 4582–4593 (2006)

  51. 51

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

  52. 52

    Murakami, M., Kitahara, R., Gotoh, T. & Kouyama, T. Crystallization and crystal properties of squid rhodopsin. Acta Crystallogr. F 63, 475–479 (2007)

  53. 53

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

  54. 54

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

  55. 55

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

  56. 56

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

  57. 57

    Emsley, P. & Cowtan, K. Coot: Model-Building Tools for Molecular Graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

  58. 58

    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)

  59. 59

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

  60. 60

    McDonald, I. K. & Thornton, J. M. Satisfying hydrogen bonding potential in proteins. J. Mol. Biol. 238, 777–793 (1994)

  61. 61

    Wallace, A. C., Laskowski, R. A. & Thornton, J. M. LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. Protein Eng. 8, 127–134 (1995)

  62. 62

    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; P. Henklein for peptide synthesis; C. Enenkel and M. Sommer for critically reading the manuscript; 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.), and CBNU funds for overseas research 2006–2007 (to H.-W.C.) and a fellowship of the Leibniz Graduate School of Molecular Biophysics, Berlin (to Y.J.K.).

Author information

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

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-9 with Legends, Supplementary Tables 1-2 and Supplementary References (PDF 3737 kb)

PowerPoint slides

PowerPoint slide for Fig. 1

PowerPoint slide for Fig. 2

PowerPoint slide for Fig. 3

PowerPoint slide for Fig. 4

PowerPoint slide for Fig. 5

Rights and permissions

Reprints and Permissions

About this article

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.