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Helix movement is coupled to displacement of the second extracellular loop in rhodopsin activation

Abstract

The second extracellular loop (EL2) of rhodopsin forms a cap over the binding site of its photoreactive 11-cis retinylidene chromophore. A crucial question has been whether EL2 forms a reversible gate that opens upon activation or acts as a rigid barrier. Distance measurements using solid-state 13C NMR spectroscopy between the retinal chromophore and the β4 strand of EL2 show that the loop is displaced from the retinal binding site upon activation, and there is a rearrangement in the hydrogen-bonding networks connecting EL2 with the extracellular ends of transmembrane helices H4, H5 and H6. NMR measurements further reveal that structural changes in EL2 are coupled to the motion of helix H5 and breaking of the ionic lock that regulates activation. These results provide a comprehensive view of how retinal isomerization triggers helix motion and activation in this prototypical G protein–coupled receptor.

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Figure 1: Structural changes involving the conserved Cys110-Cys187 disulfide link on activation of rhodopsin.
Figure 2: Two-dimensional 13C DARR NMR spectra of retinal-EL2 interactions.
Figure 3: A view of the extracellular side of rhodopsin from the crystal structure6.
Figure 4: One dimensional 13C cross-polarization magic angle spinning (CP-MAS) spectra of rhodopsin and meta II labeled with 13Cζ-tyrosine.
Figure 5: Two-dimensional DARR NMR of Tyr(Cζ)-Met(Cε) contacts in rhodopsin and the M288L rhodopsin mutant.
Figure 6: Crystal structure of rhodopsin20 highlighting EL2 and H5.

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References

  1. Samson, M. et al. The second extracellular loop of CCR5 is the major determinant of ligand specificity. J. Biol. Chem. 272, 24934–24941 (1997).

    Article  CAS  Google Scholar 

  2. Shi, L. & Javitch, J.A. The second extracellular loop of the dopamine D-2 receptor lines the binding-site crevice. Proc. Natl. Acad. Sci. USA 101, 440–445 (2004).

    Article  CAS  Google Scholar 

  3. Klco, J.M., Wiegand, C.B., Narzinski, K. & Baranski, T.J. Essential role for the second extracellular loop in C5a receptor activation. Nat. Struct. Mol. Biol. 12, 320–326 (2005).

    Article  CAS  Google Scholar 

  4. Scarselli, M., Li, B., Kim, S.K. & Wess, J. Multiple residues in the second extracellular loop are critical for M-3 muscarinic acetylcholine receptor activation. J. Biol. Chem. 282, 7385–7396 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. 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  Google Scholar 

  7. Karnik, S.S. & Khorana, H.G. Assembly of functional rhodopsin requires a disulfide bond between cysteine residues 110 and 187. J. Biol. Chem. 265, 17520–17524 (1990).

    CAS  PubMed  Google Scholar 

  8. Hwa, J., Klein-Seetharaman, J. & Khorana, H.G. Structure and function in rhodopsin: mass spectrometric identification of the abnormal intradiscal disulfide bond in misfolded retinitis pigmentosa mutants. Proc. Natl. Acad. Sci. USA 98, 4872–4876 (2001).

    Article  CAS  Google Scholar 

  9. Steinberg, G., Ottolenghi, M. & Sheves, M. pKa of the protonated Schiff base of bovine rhodopsin: a study with artificial pigments. Biophys. J. 64, 1499–1502 (1993).

    Article  CAS  Google Scholar 

  10. Sakmar, T.P., Franke, R.R. & Khorana, H.G. The role of the retinylidene Schiff base counterion in rhodopsin in determining wavelength absorbance and Schiff base pKa. Proc. Natl. Acad. Sci. USA 88, 3079–3083 (1991).

    Article  CAS  Google Scholar 

  11. 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  Google Scholar 

  12. Rader, A.J. et al. Identification of core amino acids stabilizing rhodopsin. Proc. Natl. Acad. Sci. USA 101, 7246–7251 (2004).

    Article  CAS  Google Scholar 

  13. Holst, B. & Schwartz, T.W. Molecular mechanism of agonism and inverse agonism in the melanocortin receptors—Zn2+ as a structural and functional probe. Ann. NY Acad. Sci. 994, 1–11 (2003).

    Article  CAS  Google Scholar 

  14. 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  Google Scholar 

  15. Matsumoto, H. & Yoshizawa, T. Recognition of opsin to longitudinal length of retinal isomers in formation of rhodopsin. Vision Res. 18, 607–609 (1978).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. Herzfeld, J. et al. Solid-state 13C NMR study of tyrosine protonation in dark-adapted bacteriorhodopsin. Biochemistry 29, 5567–5574 (1990).

    Article  CAS  Google Scholar 

  18. DeLange, F. et al. Tyrosine structural changes detected during the photoactivation of rhodopsin. J. Biol. Chem. 273, 23735–23739 (1998).

    Article  CAS  Google Scholar 

  19. Patel, A.B. et al. Coupling of retinal isomerization to the activation of rhodopsin. Proc. Natl. Acad. Sci. USA 101, 10048–10053 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  22. 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).

    Article  CAS  Google Scholar 

  23. Patel, A.B. et al. Changes in interhelical hydrogen bonding upon rhodopsin activation. J. Mol. Biol. 347, 803–812 (2005).

    Article  CAS  Google Scholar 

  24. Imai, H. et al. Single amino acid residue as a functional determinant of rod and cone visual pigments. Proc. Natl. Acad. Sci. USA 94, 2322–2326 (1997).

    Article  CAS  Google Scholar 

  25. Jäger, F. et al. Interactions of the β-ionone ring with the protein in the visual pigment rhodopsin control the activation mechanism. An FTIR and fluorescence study on artificial vertebrate rhodopsins. Biochemistry 33, 7389–7397 (1994).

    Article  Google Scholar 

  26. Ganter, U.M., Schmid, E.D., Perez-Sala, D., Rando, R.R. & Siebert, F. Removal of the 9-methyl group of retinal inhibits signal transduction in the visual process. A Fourier transform infrared and biochemical investigation. Biochemistry 28, 5954–5962 (1989).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  28. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  30. Yan, E.C.Y. et al. Function of extracellular loop 2 in rhodopsin: glutamic acid 181 modulates stability and absorption wavelength of metarhodopsin II. Biochemistry 41, 3620–3627 (2002).

    Article  CAS  Google Scholar 

  31. Janz, J.M. & Farrens, D.L. Role of the retinal hydrogen bond network in rhodopsin Schiff base stability and hydrolysis. J. Biol. Chem. 279, 55886–55894 (2004).

    Article  CAS  Google Scholar 

  32. Furutani, Y., Shichida, Y. & Kandori, H. Structural changes of water molecules during the photoactivation processes in bovine rhodopsin. Biochemistry 42, 9619–9625 (2003).

    Article  CAS  Google Scholar 

  33. Davidson, F.F., Loewen, P.C. & Khorana, H.G. Structure and function in rhodopsin: replacement by alanine of cysteine residues 110 and 187, components of a conserved disulfide bond in rhodopsin, affects the light-activated metarhodopsin II state. Proc. Natl. Acad. Sci. USA 91, 4029–4033 (1994).

    Article  CAS  Google Scholar 

  34. Janz, J.M., Fay, J.F. & Farrens, D.L. Stability of dark state rhodopsin is mediated by a conserved ion pair in intradiscal loop E-2. J. Biol. Chem. 278, 16982–16991 (2003).

    Article  CAS  Google Scholar 

  35. Goodwin, J.A., Hulme, E.C., Langmead, C.J. & Tehan, B.G. Roof and floor of the muscarinic binding pocket: variations in the binding modes of orthosteric ligands. Mol. Pharmacol. 72, 1484–1496 (2007).

    Article  CAS  Google Scholar 

  36. Javitch, J.A., Fu, D. & Chen, J. Residues in the fifth membrane-spanning segment of the dopamine D2 receptor exposed in the binding-site crevice. Biochemistry 34, 16433–16439 (1995).

    Article  CAS  Google Scholar 

  37. Struthers, M., Yu, H.B. & Oprian, D.D. G protein-coupled receptor activation: analysis of a highly constrained, “straitjacketed” rhodopsin. Biochemistry 39, 7938–7942 (2000).

    Article  CAS  Google Scholar 

  38. Han, S.J. et al. Identification of an agonist-induced conformational change occurring adjacent to the ligand-binding pocket of the M-3 muscarinic acetylcholine receptor. J. Biol. Chem. 280, 34849–34858 (2005).

    Article  CAS  Google Scholar 

  39. Elling, C.E. et al. Metal ion site engineering indicates a global toggle switch model for seven-transmembrane receptor activation. J. Biol. Chem. 281, 17337–17346 (2006).

    Article  CAS  Google Scholar 

  40. Doi, T., Molday, R.S. & Khorana, H.G. Role of the intradiscal domain in rhodopsin assembly and function. Proc. Natl. Acad. Sci. USA 87, 4991–4995 (1990).

    Article  CAS  Google Scholar 

  41. Yan, E.C.Y. et al. Photointermediates of the rhodopsin S186A mutant as a probe of the hydrogen-bond network in the chromophore pocket and the mechanism of counterion switch. J. Phys. Chem. C 111, 8843–8848 (2007).

    Article  CAS  Google Scholar 

  42. 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  Google Scholar 

  43. 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  Google Scholar 

  44. Sheikh, S.P. et al. Similar structures and shared switch mechanisms of the β2-adrenoceptor and the parathyroid hormone receptor—Zn(II) bridges between helices III and VI block activation. J. Biol. Chem. 274, 17033–17041 (1999).

    Article  CAS  Google Scholar 

  45. Olah, M.E., Jacobson, K.A. & Stiles, G.L. Role of the 2nd extracellular loop of adenosine receptors in agonist and antagonist binding—analysis of Chimeric A1/A3-adenosine receptors. J. Biol. Chem. 269, 24692–24698 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Wurch, T., Colpaert, F.C. & Pauwels, P.J. Chimeric receptor analysis of the ketanserin binding site in the human 5-hydroxytryptamine1D receptor: importance of the second extracellular loop and fifth transmembrane domain in antagonist binding. Mol. Pharmacol. 54, 1088–1096 (1998).

    Article  CAS  Google Scholar 

  47. Conner, M. et al. Systematic analysis of the entire second extracellular loop of the V-1a vasopressin receptor—key residues, conserved throughout a G-protein-coupled receptor family, identified. J. Biol. Chem. 282, 17405–17412 (2007).

    Article  CAS  Google Scholar 

  48. Pfleger, K.D.G., Pawson, A.J. & Millar, R.P. Changes to gonadotropin-releasing hormone (GnRH) receptor extracellular loops differentially affect GnRH analog binding and activation: evidence for distinct ligand-stabilized receptor conformations. Endocrinology 149, 3118–3129 (2008).

    Article  CAS  Google Scholar 

  49. 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).

    Article  CAS  Google Scholar 

  50. 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  Google Scholar 

  51. Madabushi, S. et al. Evolutionary trace of G protein-coupled receptors reveals clusters of residues that determine global and class-specific functions. J. Biol. Chem. 279, 8126–8132 (2004).

    Article  CAS  Google Scholar 

  52. Holst, B., Elling, C.E. & Schwartz, T.W. Partial agonism through a zinc-ion switch constructed between transmembrane domains III and VII in the tachykinin NK1 receptor. Mol. Pharmacol. 58, 263–270 (2000).

    Article  CAS  Google Scholar 

  53. 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, 13413–13418 (2002).

    Article  CAS  Google Scholar 

  54. Reeves, P.J., Thurmond, R.L. & Khorana, H.G. Structure and function in rhodopsin: high level expression of a synthetic bovine opsin gene and its mutants in stable mammalian cell lines. Proc. Natl. Acad. Sci. USA 93, 11487–11492 (1996).

    Article  CAS  Google Scholar 

  55. Dulbecco, R. & Freeman, G. Plaque production by the polyoma virus. Virology 8, 396–397 (1959).

    Article  CAS  Google Scholar 

  56. Eilers, M., Reeves, P.J., Ying, W.W., Khorana, H.G. & Smith, S.O. Magic angle spinning NMR of the protonated retinylidene schiff base nitrogen in rhodopsin: expression of 15N-lysine and 13C-glycine labeled opsin in a stable cell line. Proc. Natl. Acad. Sci. USA 96, 487–492 (1999).

    Article  CAS  Google Scholar 

  57. Lugtenburg, J. The synthesis of 13C-labeled retinals. Pure Appl. Chem. 57, 753–762 (1985).

    Article  CAS  Google Scholar 

  58. Crocker, E. et al. Dipolar assisted rotational resonance NMR of tryptophan and tyrosine in rhodopsin. J. Biomol. NMR 29, 11–20 (2004).

    Article  CAS  Google Scholar 

  59. Han, M., Groesbeek, M., Smith, S.O. & Sakmar, T.P. Role of the C9 methyl group in rhodopsin activation: characterization of mutant opsins with the artificial chromophore 11-cis-9-demethylretinal. Biochemistry 37, 538–545 (1998).

    Article  CAS  Google Scholar 

  60. Fahmy, K. et al. Protonation states of membrane-embedded carboxylic acid groups in rhodopsin and metarhodopsin II: a Fourier-transform infrared spectroscopy study of site-directed mutants. Proc. Natl. Acad. Sci. USA 90, 10206–10210 (1993).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by the US National Insitutes of Health (NIH)–National Science Foundation instrumentation grants (S10 RR13889 and DBI-9977553), a grant from the NIH to S.O.S. (GM-41412), and a grant from the US-Israel Binational Science Foundation to M.S. We thank C.A. Opefi for help with the M288A and M288L mutants and gratefully acknowledge the W.M. Keck Foundation for support of the NMR facilities in the Center of Structural Biology at Stony Brook. M.S. acknowledges support from the Kimmelman Center for Biomolecular Structure and Assembly.

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Ahuja, S., Hornak, V., Yan, E. et al. Helix movement is coupled to displacement of the second extracellular loop in rhodopsin activation. Nat Struct Mol Biol 16, 168–175 (2009). https://doi.org/10.1038/nsmb.1549

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