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.

Free backbone carbonyls mediate rhodopsin activation

Abstract

Conserved prolines in the transmembrane helices of G-protein-coupled receptors (GPCRs) are often considered to function as hinges that divide the helix into two segments capable of independent motion. Depending on their potential to hydrogen-bond, the free C=O groups associated with these prolines can facilitate conformational flexibility, conformational switching or stabilization of the receptor structure. To address the role of conserved prolines in family A GPCRs through solid-state NMR spectroscopy, we focus on bovine rhodopsin, a GPCR in the visual receptor subfamily. The free backbone C=O groups on helices H5 and H7 stabilize the inactive rhodopsin structure through hydrogen-bonds to residues on adjacent helices. In response to light-induced isomerization of the retinal chromophore, hydrogen-bonding interactions involving these C=O groups are released, thus facilitating repacking of H5 and H7 onto the transmembrane core of the receptor. These results provide insights into the multiple structural and functional roles of prolines in membrane proteins.

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

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Side (left) and top-down (right) views of the crystal structure of the visual receptor rhodopsin (PDB 1U19 (ref.9)) showing the positions of Pro2155.50, Pro2676.50, Pro2917.38 and Pro3037.50.
Figure 2: REDOR NMR as a probe of hydrogen-bonding changes of carbonyl residues at the i − 4 positions of Pro2155.50, Pro2676.50, Pro2917.38 and Pro3037.50.
Figure 3: 13C-15N REDOR NMR experiments of Meta II in the presence or absence of the C-terminal Gα peptide of transducin.
Figure 4: Receptor activation leads to repacking of helices H5–H7 on the TM core of rhodopsin.

Accession codes

Accessions

Protein Data Bank

References

  1. Sansom, M.S.P. & Weinstein, H. Hinges, swivels and switches: the role of prolines in signalling via transmembrane α-helices. Trends Pharmacol. Sci. 21, 445–451 (2000).

    CAS  Article  Google Scholar 

  2. Cordes, F.S., Bright, J.N. & Sansom, M.S.P. Proline-induced distortions of transmembrane helices. J. Mol. Biol. 323, 951–960 (2002).

    CAS  Article  Google Scholar 

  3. Williams, K.A. & Deber, C.M. Proline residues in transmembrane helices: structural or dynamic role? Biochemistry 30, 8919–8923 (1991).

    CAS  Article  Google Scholar 

  4. von Heijne, G. Proline kinks in transmembrane α-helices. J. Mol. Biol. 218, 499–503 (1991).

    CAS  Article  Google Scholar 

  5. Fu, Q. et al. Structural basis and functional role of intramembrane trimerization of the Fas/CD95 death receptor. Mol. Cell 61, 602–613 (2016).

    CAS  Article  Google Scholar 

  6. Cao, Z. & Bowie, J.U. Shifting hydrogen bonds may produce flexible transmembrane helices. Proc. Natl. Acad. Sci. USA 109, 8121–8126 (2012).

    CAS  Article  Google Scholar 

  7. Smith, S.O. Structure and activation of the visual pigment rhodopsin. Annu. Rev. Biophys. 39, 309–328 (2010).

    CAS  Article  Google Scholar 

  8. 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. Meth. Neurosci. 25, 366–428 (1995).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  10. 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).

    CAS  Article  Google Scholar 

  11. 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).

    CAS  Article  Google Scholar 

  12. Deupi, X. et al. Stabilized G protein binding site in the structure of constitutively active metarhodopsin-II. Proc. Natl. Acad. Sci. USA 109, 119–124 (2012).

    CAS  Article  Google Scholar 

  13. Choe, H.W. et al. Crystal structure of metarhodopsin II. Nature 471, 651–655 (2011).

    CAS  Article  Google Scholar 

  14. 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).

    CAS  Article  Google Scholar 

  15. Schwartz, T.W., Frimurer, T.M., Holst, B., Rosenkilde, M.M. & Elling, C.E. Molecular mechanism of 7TM receptor activation: a global toggle switch model. Annu. Rev. Pharmacol. Toxicol. 46, 481–519 (2006).

    CAS  Article  Google Scholar 

  16. Ganter, U.M., Gärtner, W. & Siebert, F. Rhodopsin-lumirhodopsin phototransition of bovine rhodopsin investigated by Fourier transform infrared difference spectroscopy. Biochemistry 27, 7480–7488 (1988).

    CAS  Article  Google Scholar 

  17. Van Arnam, E.B., Lester, H.A. & Dougherty, D.A. Dissecting the functions of conserved prolines within transmembrane helices of the D2 dopamine receptor. ACS Chem. Biol. 6, 1063–1068 (2011).

    CAS  Article  Google Scholar 

  18. Wess, J., Nanavati, S., Vogel, Z. & Maggio, R. Functional role of proline and tryptophan residues highly conserved among G protein-coupled receptors studied by mutational analysis of the m3 muscarinic receptor. EMBO J. 12, 331–338 (1993).

    CAS  Article  Google Scholar 

  19. Stitham, J., Martin, K.A. & Hwa, J. The critical role of transmembrane prolines in human prostacyclin receptor activation. Mol. Pharmacol. 61, 1202–1210 (2002).

    CAS  Article  Google Scholar 

  20. 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).

    CAS  Article  Google Scholar 

  21. Deupi, X. & Standfuss, J. Structural insights into agonist-induced activation of G-protein-coupled receptors. Curr. Opin. Struct. Biol. 21, 541–551 (2011).

    CAS  Article  Google Scholar 

  22. Deupi, X. Relevance of rhodopsin studies for GPCR activation. Biochim. Biophys. Acta 1837, 674–682 (2014).

    CAS  Article  Google Scholar 

  23. Gullion, T. & Schaefer, J. Rotational-echo double-resonance NMR. J. Magn. Reson. 81, 196–200 (1989).

    CAS  Google Scholar 

  24. Saito, H. Conformation-dependent C-13 chemical-shifts: a new means of conformational characterization as obtained by high-resolution solid-state C-13 NMR. Magn. Reson. Chem. 24, 835–852 (1986).

    CAS  Article  Google Scholar 

  25. Gu, Z.T., Zambrano, R. & McDermott, A. Hydrogen-bonding of carboxyl groups in solid state amino acids and peptides: comparison of carbon chemical shielding, infrared frequencies, and structures. J. Am. Chem. Soc. 116, 6368–6372 (1994).

    CAS  Article  Google Scholar 

  26. Szilagyi, L. Chemical-shifts in proteins come of age. Prog. Nucl. Magn. Reson. Spectrosc. 27, 325–443 (1995).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  28. Beck, M., Sakmar, T.P. & Siebert, F. Spectroscopic evidence for interaction between transmembrane helices 3 and 5 in rhodopsin. Biochemistry 37, 7630–7639 (1998).

    CAS  Article  Google Scholar 

  29. 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).

    CAS  Article  Google Scholar 

  30. White, S.H. & Wimley, W.C. Membrane protein folding and stability: physical principles. Annu. Rev. Biophys. Biomol. Struct. 28, 319–365 (1999).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  32. Sung, C.H., Davenport, C.M. & Nathans, J. Rhodopsin mutations responsible for autosomal dominant retinitis pigmentosa: clustering of functional classes along the polypeptide chain. J. Biol. Chem. 268, 26645–26649 (1993).

    CAS  PubMed  Google Scholar 

  33. Wigley, W.C. et al. A protein sequence that can encode native structure by disfavoring alternate conformations. Nat. Struct. Biol. 9, 381–388 (2002).

    CAS  PubMed  Google Scholar 

  34. Goncalves, J.A. et al. Highly conserved tyrosine stabilizes the active state of rhodopsin. Proc. Natl. Acad. Sci. USA 107, 19861–19866 (2010).

    CAS  Article  Google Scholar 

  35. 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).

    CAS  Article  Google Scholar 

  36. Isogai, S. et al. Backbone NMR reveals allosteric signal transduction networks in the β1-adrenergic receptor. Nature 530, 237–241 (2016).

    CAS  Article  Google Scholar 

  37. Goncalves, J. et al. Magic angle spinning nuclear magnetic resonance spectroscopy of G protein-coupled receptors. Methods Enzymol. 522, 365–389 (2013).

    CAS  Article  Google Scholar 

  38. Gullion, T. & Schaefer, J. in Advances in Magnetic Resonance Vol. 13 (ed. Warren, W.S.) 57–84 (Academic Press, 1989).

  39. Eilers, M., Ying, W., Reeves, P.J., Khorana, H.G. & Smith, S.O. Magic angle spinning nuclear magnetic resonance of isotopically labeled rhodopsin. Methods Enzymol. 343, 212–222 (2002).

    Article  Google Scholar 

  40. Jastrzebska, B., Goc, A., Golczak, M. & Palczewski, K. Phospholipids are needed for the proper formation, stability, and function of the photoactivated rhodopsin-transducin complex. Biochemistry 48, 5159–5170 (2009).

    CAS  Article  Google Scholar 

  41. 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).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Institutes of Health (NIH) grant GM41412 (to S.O.S.) We thank H. Sasaki and X. Zhou (Institute of Protein Research, Osaka University) for expression and purification of several of the 15N-13C-labeled rhodopsin samples, and J. Goncalves for preliminary experiments with the Gα peptide. We thank J. Nathans (Johns Hopkins University) for providing the HEK293S cell line.

Author information

Authors and Affiliations

Authors

Contributions

M.E., P.J.R. and S.O.S. conceived the study; N.K., A.P. and M.E. prepared samples; M.E. and M.Z. collected and analyzed NMR data; A.P. and O.B.S.-R. analyzed the protein database for proline interactions; C.A.O. constructed rhodopsin mutants; and N.K., A.P., P.J.R. and S.O.S. wrote the manuscript.

Corresponding author

Correspondence to Steven O Smith.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Notes 1 and 2 (PDF 924 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kimata, N., Pope, A., Sanchez-Reyes, O. et al. Free backbone carbonyls mediate rhodopsin activation. Nat Struct Mol Biol 23, 738–743 (2016). https://doi.org/10.1038/nsmb.3257

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.3257

Further reading

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