Cellular and Molecular Life Sciences

, Volume 74, Issue 22, pp 4209–4229 | Cite as

Structural determinants of a conserved enantiomer-selective carvone binding pocket in the human odorant receptor OR1A1

  • Christiane Geithe
  • Jonas Protze
  • Franziska Kreuchwig
  • Gerd KrauseEmail author
  • Dietmar KrautwurstEmail author
Original Article


Chirality is a common phenomenon within odorants. Most pairs of enantiomers show only moderate differences in odor quality. One example for enantiomers that are easily discriminated by their odor quality is the carvones: humans significantly distinguish between the spearmint-like (R)-(−)-carvone and caraway-like (S)-(+)-carvone enantiomers. Moreover, for the (R)-(−)-carvone, an anosmia is observed in about 8% of the population, suggesting enantioselective odorant receptors (ORs). With only about 15% de-orphaned human ORs, the lack of OR crystal structures, and few comprehensive studies combining in silico and experimental approaches to elucidate structure–function relations of ORs, knowledge on cognate odorant/OR interactions is still sparse. An adjusted homology modeling approach considering OR-specific proline-caused conformations, odorant docking studies, single-nucleotide polymorphism (SNP) analysis, site-directed mutagenesis, and subsequent functional studies with recombinant ORs in a cell-based, real-time luminescence assay revealed 11 amino acid positions to constitute an enantioselective binding pocket necessary for a carvone function in human OR1A1 and murine Olfr43, respectively. Here, we identified enantioselective molecular determinants in both ORs that discriminate between minty and caraway odor. Comparison with orthologs from 36 mammalian species demonstrated a hominid-specific carvone binding pocket with about 100% conservation. Moreover, we identified loss-of-function SNPs associated with the carvone binding pocket of OR1A1. Given carvone enantiomer-specific receptor activation patterns including OR1A1, our data suggest OR1A1 as a candidate receptor for constituting a carvone enantioselective phenotype, which may help to explain mechanisms underlying a (R)-(−)-carvone-specific anosmia in humans.


Structure–function study Molecular modeling Site-directed mutagenesis GPCR Ortholog 



Amino acid


Extracellular loop


G-protein coupled receptor


Key food odorant


Odorant receptor


Single-nucleotide polymorphism


Transmembrane helix



We thank Matthias Kotthoff for the initial experiments, and Julia Fiedler for expert technical assistance.

Supplementary material

18_2017_2576_MOESM1_ESM.pdf (2.6 mb)
Supplementary material 1 (PDF 2677 kb)


  1. 1.
    Dunkel A, Steinhaus M, Kotthoff M, Nowak B, Krautwurst D, Schieberle P, Hofmann T (2014) Nature’s chemical signatures in human olfaction: a foodborne perspective for future biotechnology. Angew Chem Int Ed Engl 53(28):7124–7143CrossRefPubMedGoogle Scholar
  2. 2.
    Krautwurst D, Kotthoff M (2013) A hit map-based statistical method to predict best ligands for orphan olfactory receptors: natural key odorants versus “lock picks”. Methods Mol Biol (Clifton, NJ) 1003:85–97CrossRefGoogle Scholar
  3. 3.
    Olender T, Lancet D, Nebert DW (2008) Update on the olfactory receptor (OR) gene superfamily. Hum Genom 3(1):87–97CrossRefGoogle Scholar
  4. 4.
    Buck L, Axel R (1991) A novel multigene family may encode odorant receptors: a molecular basis for odor recognition. Cell 65(1):175–187CrossRefPubMedGoogle Scholar
  5. 5.
    Kato A, Touhara K (2009) Mammalian olfactory receptors: pharmacology, G protein coupling and desensitization. Cell Mol Life Sci 66(23):3743–3753CrossRefPubMedGoogle Scholar
  6. 6.
    Verbeurgt C, Wilkin F, Tarabichi M, Gregoire F, Dumont JE, Chatelain P (2014) Profiling of olfactory receptor gene expression in whole human olfactory mucosa. PLoS One 9(5):e96333CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Geithe C, Noe F, Kreissl J, Krautwurst D (2017) The broadly tuned odorant receptor OR1A1 is highly selective for 3-methyl-2,4-nonanedione, a key food odorant in aged wines, tea, and other foods. Chem Senses 42(3):181–193CrossRefPubMedGoogle Scholar
  8. 8.
    Malnic B, Hirono J, Sato T, Buck LB (1999) Combinatorial receptor codes for odors. Cell 96(5):713–723CrossRefPubMedGoogle Scholar
  9. 9.
    Nara K, Saraiva LR, Ye X, Buck LB (2011) A large-scale analysis of odor coding in the olfactory epithelium. J Neurosci 31(25):9179–9191CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Charlier L, Topin J, Ronin C, Kim SK, Goddard WA 3rd, Efremov R, Golebiowski J (2012) How broadly tuned olfactory receptors equally recognize their agonists. Human OR1G1 as a test case. Cell Mol Life Sci 69(24):4205–4213CrossRefPubMedGoogle Scholar
  11. 11.
    Laska M (2004) Olfactory discrimination ability of human subjects for enantiomers with an isopropenyl group at the chiral center. Chem Senses 29(2):143–152CrossRefPubMedGoogle Scholar
  12. 12.
    Laska M, Teubner P (1999) Olfactory discrimination ability for homologous series of aliphatic alcohols and aldehydes. Chem Senses 24(3):263–270CrossRefPubMedGoogle Scholar
  13. 13.
    Joshi D, Volkl M, Shepherd GM, Laska M (2006) Olfactory sensitivity for enantiomers and their racemic mixtures—a comparative study in CD-1 mice and spider monkeys. Chem Senses 31(7):655–664CrossRefPubMedGoogle Scholar
  14. 14.
    Laska M, Liesen A, Teubner P (1999) Enantioselectivity of odor perception in squirrel monkeys and humans. Am J Physiol 277(4 Pt 2):R1098–R1103PubMedGoogle Scholar
  15. 15.
    Pelosi P, Viti R (1978) Specific anosmia to l-carvone—minty primary odor. Chem Senses Flavour 3(3):331–337CrossRefGoogle Scholar
  16. 16.
    de March CA, Kim SK, Antonczak S, Goddard WA 3rd, Golebiowski J (2015) G protein-coupled odorant receptors: from sequence to structure. Protein Sci 24(9):1543–1548CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Vaidehi N, Floriano WB, Trabanino R, Hall SE, Freddolino P, Choi EJ, Zamanakos G, Goddard WA 3rd (2002) Prediction of structure and function of G protein-coupled receptors. Proc Natl Acad Sci USA 99(20):12622–12627CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Lai PC, Crasto CJ (2012) Beyond modeling: all-atom olfactory receptor model simulations. Front Genet 3:61CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Charlier L, Topin J, de March CA, Lai PC, Crasto CJ, Golebiowski J (2013) Molecular modelling of odorant/olfactory receptor complexes. Methods Mol Biol (Clifton, NJ) 1003:53–65CrossRefGoogle Scholar
  20. 20.
    Man O, Gilad Y, Lancet D (2004) Prediction of the odorant binding site of olfactory receptor proteins by human-mouse comparisons. Protein Sci 13(1):240–254CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Liu AH, Zhang X, Stolovitzky GA, Califano A, Firestein SJ (2003) Motif-based construction of a functional map for mammalian olfactory receptors. Genomics 81(5):443–456CrossRefPubMedGoogle Scholar
  22. 22.
    Schmiedeberg K, Shirokova E, Weber H-P, Schilling B, Meyerhof W, Krautwurst D (2007) Structural determinants of odorant recognition by the human olfactory receptors OR1A1 and OR1A2. J Struct Biol 159(3):400–412CrossRefPubMedGoogle Scholar
  23. 23.
    Abaffy T, Malhotra A, Luetje CW (2007) The molecular basis for ligand specificity in a mouse olfactory receptor: a network of functionally important residues. J Biol Chem 282(2):1216–1224CrossRefPubMedGoogle Scholar
  24. 24.
    Baud O, Etter S, Spreafico M, Bordoli L, Schwede T, Vogel H, Pick H (2011) The mouse eugenol odorant receptor: structural and functional plasticity of a broadly tuned odorant binding pocket. Biochemistry 50(5):843–853CrossRefPubMedGoogle Scholar
  25. 25.
    Katada S, Hirokawa T, Oka Y, Suwa M, Touhara K (2005) Structural basis for a broad but selective ligand spectrum of a mouse olfactory receptor: mapping the odorant-binding site. J Neurosci 25(7):1806–1815CrossRefPubMedGoogle Scholar
  26. 26.
    Gelis L, Wolf S, Hatt H, Neuhaus EM, Gerwert K (2012) Prediction of a ligand-binding niche within a human olfactory receptor by combining site-directed mutagenesis with dynamic homology modeling. Angew Chem Int Ed Engl 51(5):1274–1278CrossRefPubMedGoogle Scholar
  27. 27.
    Sekharan S, Ertem Mehmed Z, Zhuang H, Block E, Matsunami H, Zhang R, Wei Jennifer N, Pan Y, Batista Victor S (2014) QM/MM model of the mouse olfactory receptor MOR244-3 validated by site-directed mutagenesis experiments. Biophys J 107(5):L5–L8CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Li S, Ahmed L, Zhang R, et al (2016) Smelling sulfur: copper and silver regulate the response of human odorant receptor OR2T11 to low-molecular-weight thiols. J Am Chem Soc 138(40):13281–13288. doi: 10.1021/jacs.6b06983 CrossRefGoogle Scholar
  29. 29.
    Bakalyar HA, Reed RR (1990) Identification of a specialized adenylyl cyclase that may mediate odorant detection. Science 250(4986):1403–1406CrossRefPubMedGoogle Scholar
  30. 30.
    Levy NS, Bakalyar HA, Reed RR (1991) Signal transduction in olfactory neurons. J Steroid Biochem Mol Biol 39(4B):633–637CrossRefPubMedGoogle Scholar
  31. 31.
    Wong ST, Trinh K, Hacker B, Chan GC, Lowe G, Gaggar A, Xia Z, Gold GH, Storm DR (2000) Disruption of the type III adenylyl cyclase gene leads to peripheral and behavioral anosmia in transgenic mice. Neuron 27(3):487–497CrossRefPubMedGoogle Scholar
  32. 32.
    Zou DJ, Chesler AT, Le Pichon CE, Kuznetsov A, Pei X, Hwang EL, Firestein S (2007) Absence of adenylyl cyclase 3 perturbs peripheral olfactory projections in mice. J Neurosci 27(25):6675–6683CrossRefPubMedGoogle Scholar
  33. 33.
    Pace U, Hanski E, Salomon Y, Lancet D (1985) Odorant-sensitive adenylate cyclase may mediate olfactory reception. Nature 316(6025):255–258CrossRefPubMedGoogle Scholar
  34. 34.
    Krautwurst D (2008) Human olfactory receptor families and their odorants. Chem Biodivers 5(6):842–852CrossRefPubMedGoogle Scholar
  35. 35.
    Krautwurst D, Yau KW, Reed RR (1998) Identification of ligands for olfactory receptors by functional expression of a receptor library. Cell 95(7):917–926CrossRefPubMedGoogle Scholar
  36. 36.
    Touhara K (2007) Deorphanizing vertebrate olfactory receptors: recent advances in odorant-response assays. Neurochem Int 51(2–4):132–139CrossRefPubMedGoogle Scholar
  37. 37.
    Zhuang H, Matsunami H (2007) Synergism of accessory factors in functional expression of mammalian odorant receptors. J Biol Chem 282(20):15284–15293CrossRefPubMedGoogle Scholar
  38. 38.
    Zhuang H, Matsunami H (2008) Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells. Nat Protoc 3(9):1402–1413CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Geithe C, Krautwurst D (2015) Chirality matters and SNPs make the difference—genetic variations on enantiomer-specific odorant receptors for carvone. In: Taylor AJ, Mottram DS (eds) Flavour science: proceedings of the XIV Weurman flavour research symposium, vol 14. 14. Context Products Ltd., Leicestershire, pp 297–302Google Scholar
  40. 40.
    Geithe C, Krautwurst D (2015) Chirality matters—enantioselective orthologous odorant receptors for related terpenoid structures. In: Engel K-H, Takeoka G (eds) Importance of chirality to flavor compounds, vol 1212. ACS Symposium Series, vol 1212. American Chemical Society, Washington, DC, pp 161–181CrossRefGoogle Scholar
  41. 41.
    Binkowski B, Fan F, Wood K (2009) Engineered luciferases for molecular sensing in living cells. Curr Opin Biotechnol 20(1):14–18CrossRefPubMedGoogle Scholar
  42. 42.
    Saito H, Chi Q, Zhuang H, Matsunami H, Mainland JD (2009) Odor coding by a Mammalian receptor repertoire. Sci Signal 2(60):ra9CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Adipietro KA, Mainland JD, Matsunami H (2012) Functional evolution of mammalian odorant receptors. PLoS Genet 8(7):e1002821CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Mainland JD, Keller A, Li YR, Zhou T, Trimmer C, Snyder LL, Moberly AH, Adipietro KA, Liu WL, Zhuang H, Zhan S, Lee SS, Lin A, Matsunami H (2014) The missense of smell: functional variability in the human odorant receptor repertoire. Nat Neurosci 17(1):114–120CrossRefPubMedGoogle Scholar
  45. 45.
    Saito H, Kubota M, Roberts RW, Chi Q, Matsunami H (2004) RTP family members induce functional expression of mammalian odorant receptors. Cell 119(5):679–691CrossRefPubMedGoogle Scholar
  46. 46.
    Shirokova E, Schmiedeberg K, Bedner P, Niessen H, Willecke K, Raguse JD, Meyerhof W, Krautwurst D (2005) Identification of specific ligands for orphan olfactory receptors. G protein-dependent agonism and antagonism of odorants. J Biol Chem 280(12):11807–11815CrossRefPubMedGoogle Scholar
  47. 47.
    Jones DT, Reed RR (1989) Golf: an olfactory neuron specific-G protein involved in odorant signal transduction. Science 244(4906):790–795CrossRefPubMedGoogle Scholar
  48. 48.
    Li F, Ponissery-Saidu S, Yee KK, Wang H, Chen ML, Iguchi N, Zhang G, Jiang P, Reisert J, Huang L (2013) Heterotrimeric G protein subunit Ggamma13 is critical to olfaction. J Neurosci 33(18):7975–7984CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Crooks GE, Hon G, Chandonia JM, Brenner SE (2004) WebLogo: a sequence logo generator. Genome Res 14(6):1188–1190CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Schneider TD, Stephens RM (1990) Sequence logos: a new way to display consensus sequences. Nucleic Acids Res 18(20):6097–6100CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Safran M, Chalifa-Caspi V, Shmueli O, Olender T, Lapidot M, Rosen N, Shmoish M, Peter Y, Glusman G, Feldmesser E, Adato A, Peter I, Khen M, Atarot T, Groner Y, Lancet D (2003) Human gene-centric databases at the Weizmann Institute of Science: GeneCards, UDB, CroW 21 and HORDE. Nucleic Acids Res 31(1):142–146CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Worth CL, Kleinau G, Krause G (2009) Comparative sequence and structural analyses of G-protein-coupled receptor crystal structures and implications for molecular models. PLoS One 4(9):e7011CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Worth CL, Kreuchwig A, Kleinau G, Krause G (2011) GPCR-SSFE: a comprehensive database of G-protein-coupled receptor template predictions and homology models. BMC Bioinform 12:185CrossRefGoogle Scholar
  54. 54.
    Worth CL, Kreuchwig F, Tiemann JKS, et al (2017) GPCR-SSFE 2.0-a fragment-based molecular modeling web tool for class A G-protein coupled receptors. Nucleic Acids Res. doi: 10.1093/nar/gkx399 PubMedPubMedCentralGoogle Scholar
  55. 55.
    Ballesteros JA, Weinstein H (1995) Integrated methods for the construction of three-dimensional models and computational probing of structure–function relations in G protein-coupled receptors. In: Stuart CS (ed) Methods in neurosciences, vol 25. Academic Press, New York, pp 366–428Google Scholar
  56. 56.
    Pilpel Y, Lancet D (1999) The variable and conserved interfaces of modeled olfactory receptor proteins. Protein Sci 8(5):969–977CrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    Genomes Project Consortium, Abecasis GR, Auton A, Brooks LD, DePristo MA, Durbin RM, Handsaker RE, Kang HM, Marth GT, McVean GA (2012) An integrated map of genetic variation from 1,092 human genomes. Nature 491(7422):56–65CrossRefGoogle Scholar
  58. 58.
    Venkatakrishnan AJ, Deupi X, Lebon G, Tate CG, Schertler GF, Babu MM (2013) Molecular signatures of G-protein-coupled receptors. Nature 494(7436):185–194CrossRefPubMedGoogle Scholar
  59. 59.
    Takai Y, Touhara K (2015) Enantioselective recognition of menthol by mouse odorant receptors. Biosci Biotechnol Biochem 79(12):1980–1986CrossRefPubMedGoogle Scholar
  60. 60.
    Lai PC, Guida B, Shi J, Crasto CJ (2014) Preferential binding of an odor within olfactory receptors: a precursor to receptor activation. Chem Senses 39(2):107–123CrossRefPubMedPubMedCentralGoogle Scholar
  61. 61.
    Lai PC, Singer MS, Crasto CJ (2005) Structural activation pathways from dynamic olfactory receptor-odorant interactions. Chem Senses 30(9):781–792CrossRefPubMedGoogle Scholar
  62. 62.
    de March CA, Yu Y, Ni MJ, Adipietro KA, Matsunami H, Ma M, Golebiowski J (2015) Conserved residues control activation of mammalian G protein-coupled odorant receptors. J Am Chem Soc 137(26):8611–8616CrossRefPubMedPubMedCentralGoogle Scholar
  63. 63.
    Cvicek V, Goddard WA 3rd, Abrol R (2016) Structure-based sequence alignment of the transmembrane domains of all human GPCRs: phylogenetic, structural and functional implications. PLoS Comput Biol 12(3):e1004805CrossRefPubMedPubMedCentralGoogle Scholar
  64. 64.
    Launay G, Teletchea S, Wade F, Pajot-Augy E, Gibrat JF, Sanz G (2012) Automatic modeling of mammalian olfactory receptors and docking of odorants. Protein Eng Des Select 25(8):377–386CrossRefGoogle Scholar
  65. 65.
    Park JH, Morizumi T, Li Y, Hong JE, Pai EF, Hofmann KP, Choe HW, Ernst OP (2013) Opsin, a structural model for olfactory receptors? Angew Chem Int Ed Engl 52(42):11021–11024CrossRefPubMedGoogle Scholar
  66. 66.
    Kobilka BK (2011) Structural insights into adrenergic receptor function and pharmacology. Trends Pharmacol Sci 32(4):213–218CrossRefPubMedPubMedCentralGoogle Scholar
  67. 67.
    McIntyre JC, Hege MM, Berbari NF (2016) Trafficking of ciliary G protein-coupled receptors. Methods Cell Biol 132:35–54CrossRefPubMedGoogle Scholar
  68. 68.
    Nemet I, Ropelewski P, Imanishi Y (2015) Rhodopsin Trafficking and mistrafficking: signals, molecular components, and mechanisms. Prog Mol Biol Transl Sci 132:39–71CrossRefPubMedGoogle Scholar
  69. 69.
    Young B, Wertman J, Dupre DJ (2015) Regulation of GPCR anterograde trafficking by molecular chaperones and motifs. Prog Mol Biol Transl Sci 132:289–305CrossRefPubMedGoogle Scholar
  70. 70.
    Jiang Y, Matsunami H (2015) Mammalian odorant receptors: functional evolution and variation. Curr Opin Neurobiol 34C:54–60CrossRefGoogle Scholar
  71. 71.
    Trimmer C, Mainland JD (2017) Simplifying the odor landscape. Chem Senses 42(3):177–179CrossRefPubMedGoogle Scholar
  72. 72.
    HORDE. The Human Olfactory Receptor Data Exploratorium (HORDE) (2011) The Weizmann Institute. Accessed 16 March 2015
  73. 73.
    Kurland MD, Newcomer MB, Peterlin Z, Ryan K, Firestein S, Batista VS (2010) Discrimination of saturated aldehydes by the rat I7 olfactory receptor. Biochemistry 49(30):6302–6304CrossRefPubMedPubMedCentralGoogle Scholar
  74. 74.
    Anselmi C, Buonocore A, Centini M, Facino RM, Hatt H (2011) The human olfactory receptor 17–40: requisites for fitting into the binding pocket. Comput Biol Chem 35(3):159–168CrossRefPubMedGoogle Scholar
  75. 75.
    Bavan S, Sherman B, Luetje CW, Abaffy T (2014) Discovery of novel ligands for mouse olfactory receptor MOR42-3 using an in silico screening approach and in vitro validation. PLoS One 9(3):e92064CrossRefPubMedPubMedCentralGoogle Scholar
  76. 76.
    Baud O, Yuan S, Veya L, Filipek S, Vogel H, Pick H (2015) Exchanging ligand-binding specificity between a pair of mouse olfactory receptor paralogs reveals odorant recognition principles. Sci Rep 5:14948CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer International Publishing AG 2017

Authors and Affiliations

  1. 1.Deutsche Forschungsanstalt für Lebensmittelchemie Leibniz Institut (DFA)FreisingGermany
  2. 2.Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)BerlinGermany
  3. 3.Max-Delbrück-Centrum für Molekulare Medizin (MDC)BerlinGermany

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