Stabilized G protein binding site in the structure of constitutively active metarhodopsin-II
10.1073/pnas.1114089108
Crossref journal-article
Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences (341)
Abstract

G protein-coupled receptors (GPCR) are seven transmembrane helix proteins that couple binding of extracellular ligands to conformational changes and activation of intracellular G proteins, GPCR kinases, and arrestins. Constitutively active mutants are ubiquitously found among GPCRs and increase the inherent basal activity of the receptor, which often correlates with a pathological outcome. Here, we have used the M257Y 6.40 constitutively active mutant of the photoreceptor rhodopsin in combination with the specific binding of a C-terminal fragment from the G protein alpha subunit (GαCT) to trap a light activated state for crystallization. The structure of the M257Y/GαCT complex contains the agonist all- trans -retinal covalently bound to the native binding pocket and resembles the G protein binding metarhodopsin-II conformation obtained by the natural activation mechanism; i.e., illumination of the prebound chromophore 11- cis -retinal. The structure further suggests a molecular basis for the constitutive activity of 6.40 substitutions and the strong effect of the introduced tyrosine based on specific interactions with Y223 5.58 in helix 5, Y306 7.53 of the NPxxY motif and R135 3.50 of the E(D)RY motif, highly conserved residues of the G protein binding site.

Bibliography

Deupi, X., Edwards, P., Singhal, A., Nickle, B., Oprian, D., Schertler, G., & Standfuss, J. (2011). Stabilized G protein binding site in the structure of constitutively active metarhodopsin-II. Proceedings of the National Academy of Sciences, 109(1), 119–124.

Authors 7
  1. Xavier Deupi (first)
  2. Patricia Edwards (additional)
  3. Ankita Singhal (additional)
  4. Benjamin Nickle (additional)
  5. Daniel Oprian (additional)
  6. Gebhard Schertler (additional)
  7. Jörg Standfuss (additional)
References 43 Referenced 208
  1. S Ye, et al., Tracking G-protein-coupled receptor activation using genetically encoded infrared probes. Nature 464, 1386–1389 (2010). (10.1038/nature08948) / Nature / Tracking G-protein-coupled receptor activation using genetically encoded infrared probes by Ye S (2010)
  2. K Palczewski, et al., Crystal structure of rhodopsin: A G protein-coupled receptor. Science 289, 739–745 (2000). (10.1126/science.289.5480.739) / Science / Crystal structure of rhodopsin: A G protein-coupled receptor by Palczewski K (2000)
  3. J Li, PC Edwards, M Burghammer, C Villa, GF Schertler, Structure of bovine rhodopsin in a trigonal crystal form. J Mol Biol 343, 1409–1438 (2004). (10.1016/j.jmb.2004.08.090) / J Mol Biol / Structure of bovine rhodopsin in a trigonal crystal form by Li J (2004)
  4. J Standfuss, et al., Crystal structure of a thermally stable rhodopsin mutant. J Mol Biol 372, 1179–1188 (2007). (10.1016/j.jmb.2007.03.007) / J Mol Biol / Crystal structure of a thermally stable rhodopsin mutant by Standfuss J (2007)
  5. H Nakamichi, T Okada, Crystallographic analysis of primary visual photochemistry. Angew Chem Int Ed Engl 45, 4270–4273 (2006). (10.1002/anie.200600595) / Angew Chem Int Ed Engl / Crystallographic analysis of primary visual photochemistry by Nakamichi H (2006)
  6. H Nakamichi, T Okada, Local peptide movement in the photoreaction intermediate of rhodopsin. Proc Natl Acad Sci USA 103, 12729–12734 (2006). (10.1073/pnas.0601765103) / Proc Natl Acad Sci USA / Local peptide movement in the photoreaction intermediate of rhodopsin by Nakamichi H (2006)
  7. JH Park, P Scheerer, KP Hofmann, HW Choe, OP Ernst, Crystal structure of the ligand-free G-protein-coupled receptor opsin. Nature 454, 183–187 (2008). (10.1038/nature07063) / Nature / Crystal structure of the ligand-free G-protein-coupled receptor opsin by Park JH (2008)
  8. P Scheerer, et al., Crystal structure of opsin in its G-protein-interacting conformation. Nature 455, 497–502 (2008). (10.1038/nature07330) / Nature / Crystal structure of opsin in its G-protein-interacting conformation by Scheerer P (2008)
  9. J Standfuss, et al., The structural basis of agonist-induced activation in constitutively active rhodopsin. Nature 471, 656–660 (2011). (10.1038/nature09795) / Nature / The structural basis of agonist-induced activation in constitutively active rhodopsin by Standfuss J (2011)
  10. EA Zhukovsky, DD Oprian, Effect of carboxylic acid side chains on the absorption maximum of visual pigments. Science 246, 928–930 (1989). (10.1126/science.2573154) / Science / Effect of carboxylic acid side chains on the absorption maximum of visual pigments by Zhukovsky EA (1989)
  11. HW Choe, et al., Crystal structure of metarhodopsin II. Nature 471, 651–655 (2011). (10.1038/nature09789) / Nature / Crystal structure of metarhodopsin II by Choe HW (2011)
  12. MJ Smit, et al., Pharmacogenomic and structural analysis of constitutive G protein-coupled receptor activity. Annu Rev Pharmacol Toxicol 47, 53–87 (2007). (10.1146/annurev.pharmtox.47.120505.105126) / Annu Rev Pharmacol Toxicol / Pharmacogenomic and structural analysis of constitutive G protein-coupled receptor activity by Smit MJ (2007)
  13. VR Rao, GB Cohen, DD Oprian, Rhodopsin mutation G90D and a molecular mechanism for congenital night blindness. Nature 367, 639–642 (1994). (10.1038/367639a0) / Nature / Rhodopsin mutation G90D and a molecular mechanism for congenital night blindness by Rao VR (1994)
  14. PR Robinson, GB Cohen, EA Zhukovsky, DD Oprian, Constitutively active mutants of rhodopsin. Neuron 9, 719–725 (1992). (10.1016/0896-6273(92)90034-B) / Neuron / Constitutively active mutants of rhodopsin by Robinson PR (1992)
  15. J Standfuss, E Zaitseva, M Mahalingam, R Vogel, 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). (10.1016/j.jmb.2008.04.055) / J Mol Biol / Structural impact of the E113Q counterion mutation on the activation and deactivation pathways of the G protein-coupled receptor rhodopsin by Standfuss J (2008)
  16. G Xie, AK Gross, DD Oprian, An opsin mutant with increased thermal stability. Biochemistry 42, 1995–2001 (2003). (10.1021/bi020611z) / Biochemistry / An opsin mutant with increased thermal stability by Xie G (2003)
  17. M Han, TP Sakmar, Assays for activation of recombinant expressed opsins by all-trans-retinals. Methods Enzymol 315, 251–267 (2000). (10.1016/S0076-6879(00)15848-3) / Methods Enzymol / Assays for activation of recombinant expressed opsins by all-trans-retinals by Han M (2000)
  18. HE Hamm, et al., Site of G protein binding to rhodopsin mapped with synthetic peptides from the alpha subunit. Science 241, 832–835 (1988). (10.1126/science.3136547) / Science / Site of G protein binding to rhodopsin mapped with synthetic peptides from the alpha subunit by Hamm HE (1988)
  19. EL Martin, S Rens-Domiano, PJ Schatz, HE Hamm, Potent peptide analogues of a G protein receptor-binding region obtained with a combinatorial library. J Biol Chem 271, 361–366 (1996). (10.1074/jbc.271.1.361) / J Biol Chem / Potent peptide analogues of a G protein receptor-binding region obtained with a combinatorial library by Martin EL (1996)
  20. PJ Reeves, N Callewaert, R Contreras, HG Khorana, 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). (10.1073/pnas.212519299) / Proc Natl Acad Sci USA / 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 by Reeves PJ (2002)
  21. PW Hildebrand, et al., A ligand channel through the G protein coupled receptor opsin. PLoS One 4, e4382 (2009). (10.1371/journal.pone.0004382) / PLoS One / A ligand channel through the G protein coupled receptor opsin by Hildebrand PW (2009)
  22. O Fritze, 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). (10.1073/pnas.0435715100) / Proc Natl Acad Sci USA / Role of the conserved NPxxY(x)5,6F motif in the rhodopsin ground state and during activation by Fritze O (2003)
  23. R Vogel, 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). (10.1016/j.jmb.2008.05.022) / J Mol Biol / Functional role of the “ionic lock”—An interhelical hydrogen-bond network in family A heptahelical receptors by Vogel R (2008)
  24. JA Goncalves, et al., Highly conserved tyrosine stabilizes the active state of rhodopsin. Proc Natl Acad Sci USA 107, 19861–19866 (2010). (10.1073/pnas.1009405107) / Proc Natl Acad Sci USA / Highly conserved tyrosine stabilizes the active state of rhodopsin by Goncalves JA (2010)
  25. M Mahalingam, R Vogel, The all-trans-15-syn-retinal chromophore of metarhodopsin III is a partial agonist and not an inverse agonist. Biochemistry 45, 15624–15632 (2006). (10.1021/bi061970n) / Biochemistry / The all-trans-15-syn-retinal chromophore of metarhodopsin III is a partial agonist and not an inverse agonist by Mahalingam M (2006)
  26. S Ahuja, et al., Location of the retinal chromophore in the activated state of rhodopsin*. J Biol Chem 284, 10190–10201 (2009). (10.1074/jbc.M805725200) / J Biol Chem / Location of the retinal chromophore in the activated state of rhodopsin* by Ahuja S (2009)
  27. PJ Spooner, et al., Conformational similarities in the beta-ionone ring region of the rhodopsin chromophore in its ground state and after photoactivation to the metarhodopsin-I intermediate. Biochemistry 42, 13371–13378 (2003). (10.1021/bi0354029) / Biochemistry / Conformational similarities in the beta-ionone ring region of the rhodopsin chromophore in its ground state and after photoactivation to the metarhodopsin-I intermediate by Spooner PJ (2003)
  28. S Ahuja, et al., Helix movement is coupled to displacement of the second extracellular loop in rhodopsin activation. Nat Struct Mol Biol 16, 168–175 (2009). (10.1038/nsmb.1549) / Nat Struct Mol Biol / Helix movement is coupled to displacement of the second extracellular loop in rhodopsin activation by Ahuja S (2009)
  29. SG Rasmussen, et al., Structure of a nanobody-stabilized active state of the beta(2) adrenoceptor. Nature 469, 175–180 (2011). (10.1038/nature09648) / Nature / Structure of a nanobody-stabilized active state of the beta(2) adrenoceptor by Rasmussen SG (2011)
  30. SG Rasmussen, et al., Crystal structure of the beta(2) adrenergic receptor-Gs protein complex. Nature 477, 549–555 (2011). (10.1038/nature10361) / Nature / Crystal structure of the beta(2) adrenergic receptor-Gs protein complex by Rasmussen SG (2011)
  31. GB Cohen, DD Oprian, PR Robinson, Mechanism of activation and inactivation of opsin: Role of Glu113 and Lys296. Biochemistry 31, 12592–12601 (1992). (10.1021/bi00165a008) / Biochemistry / Mechanism of activation and inactivation of opsin: Role of Glu113 and Lys296 by Cohen GB (1992)
  32. XJ Yao, et al., The effect of ligand efficacy on the formation and stability of a GPCR-G protein complex. Proc Natl Acad Sci USA 106, 9501–9506 (2009). (10.1073/pnas.0811437106) / Proc Natl Acad Sci USA / The effect of ligand efficacy on the formation and stability of a GPCR-G protein complex by Yao XJ (2009)
  33. C Altenbach, AK Kusnetzow, OP Ernst, KP Hofmann, WL Hubbell, High-resolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation. Proc Natl Acad Sci USA 105, 7439–7444 (2008). (10.1073/pnas.0802515105) / Proc Natl Acad Sci USA / High-resolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation by Altenbach C (2008)
  34. DL Farrens, C Altenbach, K Yang, WL Hubbell, HG Khorana, Requirement of rigid-body motion of transmembrane helices for light activation of rhodopsin. Science 274, 768–770 (1996). (10.1126/science.274.5288.768) / Science / Requirement of rigid-body motion of transmembrane helices for light activation of rhodopsin by Farrens DL (1996)
  35. G Lebon, K Bennett, A Jazayeri, CG Tate, Thermostabilisation of an agonist-bound conformation of the human adenosine A(2A) receptor. J Mol Biol 409, 298–310 (2011). (10.1016/j.jmb.2011.03.075) / J Mol Biol / Thermostabilisation of an agonist-bound conformation of the human adenosine A(2A) receptor by Lebon G (2011)
  36. DA Shapiro, K Kristiansen, DM Weiner, WK Kroeze, BL Roth, Evidence for a model of agonist-induced activation of 5-hydroxytryptamine 2A serotonin receptors that involves the disruption of a strong ionic interaction between helices 3 and 6. J Biol Chem 277, 11441–11449 (2002). (10.1074/jbc.M111675200) / J Biol Chem / Evidence for a model of agonist-induced activation of 5-hydroxytryptamine 2A serotonin receptors that involves the disruption of a strong ionic interaction between helices 3 and 6 by Shapiro DA (2002)
  37. RA Bakker, et al., Constitutively active mutants of the histamine H1 receptor suggest a conserved hydrophobic asparagine-cage that constrains the activation of class A G protein-coupled receptors. Mol Pharmacol 73, 94–103 (2008). (10.1124/mol.107.038547) / Mol Pharmacol / Constitutively active mutants of the histamine H1 receptor suggest a conserved hydrophobic asparagine-cage that constrains the activation of class A G protein-coupled receptors by Bakker RA (2008)
  38. M Mahalingam, K Martinez-Mayorga, MF Brown, R Vogel, Two protonation switches control rhodopsin activation in membranes. Proc Natl Acad Sci USA 105, 17795–17800 (2008). (10.1073/pnas.0804541105) / Proc Natl Acad Sci USA / Two protonation switches control rhodopsin activation in membranes by Mahalingam M (2008)
  39. X Deupi, J Standfuss, Structural insights into agonist-induced activation of G-protein-coupled receptors. Curr Opin Struct Biol 21, 541–551 (2011). (10.1016/j.sbi.2011.06.002) / Curr Opin Struct Biol / Structural insights into agonist-induced activation of G-protein-coupled receptors by Deupi X (2011)
  40. JM Kim, et al., Structural origins of constitutive activation in rhodopsin: Role of the K296/E113 salt bridge. Proc Natl Acad Sci USA 101, 12508–12513 (2004). (10.1073/pnas.0404519101) / Proc Natl Acad Sci USA / Structural origins of constitutive activation in rhodopsin: Role of the K296/E113 salt bridge by Kim JM (2004)
  41. X Deupi, BK Kobilka, Energy landscapes as a tool to integrate GPCR structure, dynamics, and function. Physiology (Bethesda) 25, 293–303 (2010). / Physiology (Bethesda) / Energy landscapes as a tool to integrate GPCR structure, dynamics, and function by Deupi X (2010)
  42. EA Zhukovsky, PR Robinson, DD Oprian, Transducin activation by rhodopsin without a covalent bond to the 11-cis-retinal chromophore. Science 251, 558–560 (1991). (10.1126/science.1990431) / Science / Transducin activation by rhodopsin without a covalent bond to the 11-cis-retinal chromophore by Zhukovsky EA (1991)
  43. T Matsuyama, T Yamashita, H Imai, Y Shichida, Covalent bond between ligand and receptor required for efficient activation in rhodopsin. J Biol Chem 285, 8114–8121 (10.1074/jbc.M109.063875) / J Biol Chem / Covalent bond between ligand and receptor required for efficient activation in rhodopsin by Matsuyama T
Dates
Type When
Created 13 years, 8 months ago (Dec. 24, 2011, midnight)
Deposited 3 years, 2 months ago (June 7, 2022, 4:21 a.m.)
Indexed 1 month, 4 weeks ago (July 2, 2025, 2:34 p.m.)
Issued 13 years, 8 months ago (Dec. 23, 2011)
Published 13 years, 8 months ago (Dec. 23, 2011)
Published Online 13 years, 8 months ago (Dec. 23, 2011)
Published Print 13 years, 7 months ago (Jan. 3, 2012)
Funders 0

None

@article{Deupi_2011, title={Stabilized G protein binding site in the structure of constitutively active metarhodopsin-II}, volume={109}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.1114089108}, DOI={10.1073/pnas.1114089108}, number={1}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Deupi, Xavier and Edwards, Patricia and Singhal, Ankita and Nickle, Benjamin and Oprian, Daniel and Schertler, Gebhard and Standfuss, Jörg}, year={2011}, month=dec, pages={119–124} }