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Homology Model-Assisted Elucidation of Binding Sites in GPCRs

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Membrane Protein Structure and Dynamics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 914))

Abstract

G protein-coupled receptors (GPCRs) are important mediators of cell signaling and a major family of drug targets. Despite recent breakthroughs, experimental elucidation of GPCR structures remains a formidable challenge. Homology modeling of 3D structures of GPCRs provides a practical tool for elucidating the structural determinants governing the interactions of these important receptors with their ligands. The working model of the binding site can then be used for virtual screening of additional ligands that may fit this site, for determining and comparing specificity profiles of related receptors, and for structure-based design of agonists and antagonists. The current review presents the protocol and enumerates the steps for modeling and validating the residues involved in ligand binding. The main stages include (a) modeling the receptor structure using an automated fragment-based approach, (b) predicting potential binding pockets, (c) docking known binders, (d) analyzing predicted interactions and comparing with positions that have been shown to bind ligands in other receptors, (e) validating the structural model by mutagenesis.

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References

  1. Heilker R et al (2009) G-protein-coupled receptor-focused drug discovery using a target class platform approach. Drug Discov Today 14:231–240

    Article  PubMed  CAS  Google Scholar 

  2. Rosenbaum DM, Rasmussen SGF, Kobilka BK (2009) The structure and function of G-protein-coupled receptors. Nature 459:356–363

    Article  PubMed  CAS  Google Scholar 

  3. Topiol S, Sabio M (2009) X-ray structure breakthroughs in the GPCR transmembrane region. Biochem Pharmacol 78:11–20

    Article  PubMed  CAS  Google Scholar 

  4. Sprang SR (2011) Cell signalling: binding the receptor at both ends. Nature 469:172–173

    Article  PubMed  CAS  Google Scholar 

  5. Xu F et al (2011) Structure of an agonist-bound human A2A adenosine receptor. Science 332(6027):322–327

    Article  PubMed  CAS  Google Scholar 

  6. Mobarec JC, Sanchez R, Filizola M (2009) Modern homology modeling of G-protein coupled receptors: which structural template to use? J Med Chem 52:5207–5216

    Article  PubMed  CAS  Google Scholar 

  7. Yarnitzky T, Levit A, Niv MY (2010) Homology modeling of G-protein-coupled receptors with X-ray structures on the rise. Curr Opin Drug Discov Devel 13:317–325

    PubMed  CAS  Google Scholar 

  8. Senderowitz H, Marantz Y (2009) G Protein-coupled receptors: target-based in silico screening. Curr Pharm Des 15:4049–4068

    Article  PubMed  CAS  Google Scholar 

  9. de Graaf C, Rognan D (2009) Customizing G Protein-coupled receptor models for structure-based virtual screening. Curr Pharm Des 15:4026–4048

    Article  PubMed  Google Scholar 

  10. Reisert J, Restrepo D (2009) Molecular tuning of odorant receptors and its implication for odor signal processing. Chem Senses 34:535–545

    Article  PubMed  CAS  Google Scholar 

  11. Cui M et al (2006) The heterodimeric sweet taste receptor has multiple potential ligand binding sites. Curr Pharm Des 12:4591–4600

    Article  PubMed  CAS  Google Scholar 

  12. Brockhoff A et al (2010) Structural requirements of bitter taste receptor activation. Proc Natl Acad Sci U S A 107:11110–11115

    Article  PubMed  CAS  Google Scholar 

  13. Lagerstrom MC, Schioth HB (2008) Structural diversity of G protein-coupled receptors and significance for drug discovery. Nat Rev Drug Discov 7:339–357

    Article  PubMed  Google Scholar 

  14. Harmar AJ et al (2009) IUPHAR-DB: the IUPHAR database of G protein-coupled receptors and ion channels. Nucleic Acids Res 37:D680–D685

    Article  PubMed  CAS  Google Scholar 

  15. Petrel C et al (2004) Positive and negative allosteric modulators of the Ca2+-sensing receptor interact within overlapping but not identical binding sites in the transmembrane domain. J Biol Chem 279:18990–18997

    Article  PubMed  CAS  Google Scholar 

  16. Bhattacharya S et al (2010) Allosteric antagonist binding sites in class B GPCRs: corticotropin receptor 1. J Comput Aided Mol Des 24:659–674

    Article  PubMed  CAS  Google Scholar 

  17. Niv MY et al (2006) Modeling activated states of GPCRs: the rhodopsin template. J Comput Aided Mol Des 20:437–448

    Article  PubMed  CAS  Google Scholar 

  18. Niv MY, Filizola M (2008) Influence of oligomerization on the dynamics of G-protein-coupled receptors as assessed by normal mode analysis. Proteins 71:575–586

    Article  PubMed  CAS  Google Scholar 

  19. Ivanov AA, Barak D, Jacobson KA (2009) Evaluation of homology modeling of G-protein-coupled receptors in light of the A(2A) adenosine receptor crystallographic structure. J Med Chem 52:3284–3292

    Article  PubMed  CAS  Google Scholar 

  20. Slack JP et al (2010) Modulation of bitter taste perception by a small molecule hTAS2R antagonist. Curr Biol 20:1104–1109

    Article  PubMed  CAS  Google Scholar 

  21. Biarnes X et al (2010) Insights into the binding of phenyltiocarbamide (PTC) agonist to its target human TAS2R38 bitter receptor. PLoS One 5:e12394

    Article  PubMed  Google Scholar 

  22. Vaidehi N, Pease JE, Horuk R (2009) Modeling small molecule-compound binding to G-protein-coupled receptors. Methods Enzymol 460:263–288

    Article  PubMed  CAS  Google Scholar 

  23. Simms J et al (2009) Homology modeling of GPCRs. Methods Mol Biol 552:97–113

    Article  PubMed  CAS  Google Scholar 

  24. Zhang Y (2009) I-TASSER: fully automated protein structure prediction in CASP8. Proteins 77(Suppl 9):100–113

    Article  PubMed  CAS  Google Scholar 

  25. 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. Methods Neurosci 25:366–428

    Article  CAS  Google Scholar 

  26. Okuno Y et al (2008) GLIDA: GPCR ligand database for chemical genomics drug discovery database and tools update. Nucleic Acids Res 36:D907–D912

    Article  PubMed  CAS  Google Scholar 

  27. Ivetac A, McCammon JA (2010) Mapping the druggable allosteric space of G-protein-coupled receptors: a fragment-based molecular dynamics approach. Chem Biol Drug Des 76:201–217

    PubMed  CAS  Google Scholar 

  28. Beukers MW et al (1999) TinyGRAP database: a bioinformatics tool to mine G-protein-coupled receptor mutant data. Trends Pharmacol Sci 20:475–477

    Article  PubMed  CAS  Google Scholar 

  29. Horn F, Lau AL, Cohen FE (2004) Automated extraction of mutation data from the literature: application of MuteXt to G protein-coupled receptors and nuclear hormone receptors. Bioinformatics 20:557–568

    Article  PubMed  CAS  Google Scholar 

  30. Costanzi S (2008) On the applicability of GPCR homology models to computer-aided drug discovery: a comparison between in silico and crystal structures of the beta2-adrenergic receptor. J Med Chem 51:2907–2914

    Article  PubMed  CAS  Google Scholar 

  31. Reynolds KA, Katritch V, Abagyan R (2009) Identifying conformational changes of the beta(2) adrenoceptor that enable accurate prediction of ligand/receptor interactions and screening for GPCR modulators. J Comput Aided Mol Des 23:273–288

    Article  PubMed  CAS  Google Scholar 

  32. Laurie AT, Jackson RM (2005) Q-SiteFinder: an energy-based method for the prediction of protein-ligand binding sites. Bioinformatics 21:1908–1916

    Article  PubMed  CAS  Google Scholar 

  33. Cherezov V et al (2007) High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor. Science 318:1258–1265

    Article  PubMed  CAS  Google Scholar 

  34. Hanson MA et al (2008) A specific cholesterol binding site is established by the 2.8 A structure of the human beta2-adrenergic receptor. Structure 16:897–905

    Article  PubMed  CAS  Google Scholar 

  35. Floriano WB et al (2006) Modeling the human PTC bitter-taste receptor interactions with bitter tastants. J Mol Model 12:931–941

    Article  PubMed  CAS  Google Scholar 

  36. Miguet L, Zhang Z, Grigorov MG (2006) Computational studies of ligand-receptor interactions in bitter taste receptors. J Recept Signal Transduct Res 26:611–630

    Article  PubMed  CAS  Google Scholar 

  37. Rasmussen SG et al (2011) Structure of a nanobody-stabilized active state of the beta(2) adrenoceptor. Nature 469:175–180

    Article  PubMed  CAS  Google Scholar 

  38. Horn F et al (2003) GPCRDB information system for G protein-coupled receptors. Nucleic Acids Res 31:294–297

    Article  PubMed  CAS  Google Scholar 

  39. Jones G et al (1997) Development and validation of a genetic algorithm for flexible docking. J Mol Biol 267:727–748

    Article  PubMed  CAS  Google Scholar 

  40. Friesner RA et al (2004) Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J Med Chem 47:1739–1749

    Article  PubMed  CAS  Google Scholar 

  41. Halgren TA et al (2004) Glide: a new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screening. J Med Chem 47:1750–1759

    Article  PubMed  CAS  Google Scholar 

  42. Venkatachalam CM et al (2003) LigandFit: a novel method for the shape-directed rapid docking of ligands to protein active sites. J Mol Graph Model 21:289–307

    Article  PubMed  CAS  Google Scholar 

  43. Wu G et al (2003) Detailed analysis of grid-based molecular docking: a case study of CDOCKER-A CHARMm-based MD docking algorithm. J Comput Chem 24:1549–1562

    Article  PubMed  CAS  Google Scholar 

  44. Wallace AC, Laskowski RA, Thornton JM (1995) LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. Protein Eng 8:127–134

    Article  PubMed  CAS  Google Scholar 

  45. Fang G et al (1999) PCR-mediated recombination: a general method applied to construct chimeric infectious molecular clones of plasma-derived HIV-1 RNA. Nat Med 5:239–242

    Article  PubMed  CAS  Google Scholar 

  46. Reichling C, Meyerhof W, Behrens M (2008) Functions of human bitter taste receptors depend on N-glycosylation. J Neurochem 106:1138–1148

    Article  PubMed  CAS  Google Scholar 

  47. Behrens M et al (2004) Molecular cloning and characterisation of DESC4, a new transmembrane serine protease. Cell Mol Life Sci 61:2866–2877

    Article  PubMed  CAS  Google Scholar 

  48. Cunningham BC, Wells JA (1989) High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis. Science 244:1081–1085

    Article  PubMed  CAS  Google Scholar 

  49. Ueda T et al (2003) Functional interaction between T2R taste receptors and G-protein alpha subunits expressed in taste receptor cells. J Neurosci 23:7376–7380

    PubMed  CAS  Google Scholar 

  50. Di Virgilio F, Steinberg TH, Silverstein SC (1990) Inhibition of Fura-2 sequestration and secretion with organic anion transport blockers. Cell Calcium 11:57–62

    Article  PubMed  Google Scholar 

  51. Bufe B et al (2002) The human TAS2R16 receptor mediates bitter taste in response to beta-glucopyranosides. Nat Genet 32:397–401

    Article  PubMed  CAS  Google Scholar 

  52. Behrens M et al (2004) The human taste receptor hTAS2R14 responds to a variety of different bitter compounds. Biochem Biophys Res Commun 319:479–485

    Article  PubMed  CAS  Google Scholar 

  53. Kuhn C et al (2004) Bitter taste receptors for saccharin and acesulfame K. J Neurosci 24:10260–10265

    Article  PubMed  CAS  Google Scholar 

  54. Sela I et al (2010) G protein coupled receptors-in silico drug discovery and design. Curr Top Med Chem 10:638–656

    Article  PubMed  CAS  Google Scholar 

  55. Brockhoff A et al (2007) Broad tuning of the human bitter taste receptor hTAS2R46 to various sesquiterpene lactones, clerodane and labdane diterpenoids, strychnine, and denatonium. J Agric Food Chem 55:6236–6243

    Article  PubMed  CAS  Google Scholar 

  56. de Graaf C et al (2008) Molecular modeling of the second extracellular loop of G-protein coupled receptors and its implication on structure-based virtual screening. Proteins 71:599–620

    Article  PubMed  Google Scholar 

  57. Shi L, Javitch JA (2004) The second extracellular loop of the dopamine D2 receptor lines the binding-site crevice. Proc Natl Acad Sci U S A 101:440–445

    Article  PubMed  CAS  Google Scholar 

  58. Mustafi D, Palczewski K (2009) Topology of class A G protein-coupled receptors: insights gained from crystal structures of rhodopsins, adrenergic and adenosine receptors. Mol Pharmacol 75:1–12

    Article  PubMed  CAS  Google Scholar 

  59. Bokoch MP et al (2010) Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor. Nature 463:108–112

    Article  PubMed  CAS  Google Scholar 

  60. Ahuja S et al (2009) Helix movement is coupled to displacement of the second extracellular loop in rhodopsin activation. Nat Struct Mol Biol 16:168–175

    Article  PubMed  CAS  Google Scholar 

  61. Shi L, Javitch JA (2002) The binding site of aminergic G protein-coupled receptors: the transmembrane segments and second extracellular loop. Annu Rev Pharmacol Toxicol 42:437–467

    Article  PubMed  CAS  Google Scholar 

  62. Coetsee M et al (2008) Identification of Tyr(290(6.58)) of the human gonadotropin-releasing hormone (GnRH) receptor as a contact residue for both GnRH I and GnRH II: importance for high-affinity binding and receptor activation. Biochemistry 47:10305–10313

    Article  PubMed  CAS  Google Scholar 

  63. Pronin AN et al (2004) Identification of ligands for two human bitter T2R receptors. Chem Senses 29:583–593

    Article  PubMed  CAS  Google Scholar 

  64. Sakurai T et al (2010) Characterization of the beta-D-glucopyranoside binding site of the human bitter taste receptor hTAS2R16. J Biol Chem 285(36):28373–28378

    Article  PubMed  CAS  Google Scholar 

  65. Hall SE et al (2009) Elucidation of binding sites of dual antagonists in the human chemokine receptors CCR2 and CCR5. Mol Pharmacol 75:1325–1336

    Article  PubMed  CAS  Google Scholar 

  66. Wacker D et al (2010) Conserved binding mode of human beta2 adrenergic receptor inverse agonists and antagonist revealed by X-ray crystallography. J Am Chem Soc 132:11443–11445

    Article  PubMed  CAS  Google Scholar 

  67. Jaakola V-P et al (2008) The 2.6 Angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist. Science 322:1211–1217

    Article  PubMed  CAS  Google Scholar 

  68. Goodwin JA et al (2007) Roof and floor of the muscarinic binding pocket: variations in the binding modes of orthosteric ligands. Mol Pharmacol 72:1484–1496

    Article  PubMed  CAS  Google Scholar 

  69. Rodriguez GJ et al (2010) Evolution-guided discovery and recoding of allosteric pathway specificity determinants in psychoactive bioamine receptors. Proc Natl Acad Sci U S A 107:7787–7792

    Article  PubMed  CAS  Google Scholar 

  70. Gloriam DE et al (2009) Definition of the G protein-coupled receptor transmembrane bundle binding pocket and calculation of receptor similarities for drug design. J Med Chem 52:4429–4442

    Article  PubMed  CAS  Google Scholar 

  71. Wu B et al (2010) Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists. Science 330:1066–1071

    Article  PubMed  CAS  Google Scholar 

  72. Warne T et al (2011) The structural basis for agonist and partial agonist action on a beta(1)-adrenergic receptor. Nature 469:241–244

    Article  PubMed  CAS  Google Scholar 

  73. Chien EY et al (2010) Structure of the human dopamine D3 receptor in complex with a D2/D3 selective antagonist. Science 330:1091–1095

    Article  PubMed  CAS  Google Scholar 

  74. Jaakola VP et al (2010) Ligand binding and subtype selectivity of the human A(2A) adenosine receptor: identification and characterization of essential amino acid residues. J Biol Chem 285:13032–13044

    Article  PubMed  CAS  Google Scholar 

  75. Hofmann KP et al (2009) A G protein-coupled receptor at work: the rhodopsin model. Trends Biochem Sci 34:540–552

    Article  PubMed  CAS  Google Scholar 

  76. de Graaf C, Rognan D (2008) Selective structure-based virtual screening for full and partial agonists of the beta2 adrenergic receptor. J Med Chem 51:4978–4985

    Article  PubMed  Google Scholar 

  77. Vaidehi N (2010) Dynamics and flexibility of G-protein-coupled receptor conformations and their relevance to drug design. Drug Discov Today 15(21–22):951–957

    Article  PubMed  CAS  Google Scholar 

  78. Bhattacharya S, Vaidehi N (2010) Computa­tional mapping of the conformational transitions in agonist selective pathways of a G-protein coupled receptor. J Am Chem Soc 132:5205–5214

    Article  PubMed  CAS  Google Scholar 

  79. Provasi D, Filizola M (2010) Putative active states of a prototypic g-protein-coupled receptor from biased molecular dynamics. Biophys J 98:2347–2355

    Article  PubMed  CAS  Google Scholar 

  80. Ishikawa M et al (2010) Investigation of the histamine H3 receptor binding site. Design and synthesis of hybrid agonists with a lipophilic side chain. J Med Chem 53:6445–6456

    Article  PubMed  CAS  Google Scholar 

  81. Shapiro DA et al (2002) 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

    Article  PubMed  CAS  Google Scholar 

  82. Jiang Q et al (1997) Mutagenesis reveals structure-activity parallels between human A2A adenosine receptors and biogenic amine G protein-coupled receptors. J Med Chem 40:2588–2595

    Article  PubMed  CAS  Google Scholar 

  83. Kim J et al (1995) Site-directed mutagenesis identifies residues involved in ligand recognition in the human A2a adenosine receptor. J Biol Chem 270:13987–13997

    Article  PubMed  CAS  Google Scholar 

  84. Balaraman GS, Bhattacharya S, Vaidehi N (2010) Structural insights into conformational stability of wild-type and mutant beta1-adrenergic receptor. Biophys J 99:568–577

    Article  PubMed  CAS  Google Scholar 

  85. Hilser VJ (2010) An ensemble view of allostery. Science 327:653–654

    Article  PubMed  CAS  Google Scholar 

  86. Michino M et al (2009) Community-wide assessment of GPCR structure modelling and ligand docking: GPCR Dock 2008. Nat Rev Drug Discov 8:455–463

    Article  PubMed  CAS  Google Scholar 

  87. Kufareva I et al (2011) Status of GPCR modeling and docking as reflected by community-wide GPCR dock 2010 Assessment, Structure 19:1108–1126

    Article  PubMed  CAS  Google Scholar 

  88. Jacobson KA and Costanzi S (2012) New insights for drug design from the X-ray crystallographic structures of GPCRs. Mol Pharmacol doi:10.1124/mol.112.079335

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We thank the Niedersachsen-Israeli research foundation (M.Y.N.)and the German Research Foundation, Deutsche Forschungs­gemeinschaft (ME1024/2-3) (W.M.) and (ME 2014/8-1) (W.M., M.B. and M.Y.N), for funding and Dr. Talia Yarnitzky and Dr. Merav Fichman for helpful comments.

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Authors and Affiliations

  1. Institute of Biochemistry, Food Science, and Nutrition, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University, Rehovot, Israel

    Anat Levit & Masha Y. Niv

  2. Israel Institute for Biological Research, Ness Ziona, Israel

    Dov Barak

  3. Department of Molecular Genetics, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany

    Maik Behrens & Wolfgang Meyerhof

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  1. Anat Levit
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  2. Dov Barak
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  3. Maik Behrens
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  4. Wolfgang Meyerhof
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  5. Masha Y. Niv
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Corresponding author

Correspondence to Masha Y. Niv .

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Editors and Affiliations

  1. , Division of Immunology, Beckman Research Institute of the City o, E. Duarte Road 1500, Duarte, 91010, USA

    Nagarajan Vaidehi

  2. , Department of Structural Biology, University of Pittsburgh School of Medic, Fifth Avenue 3501, Pittsburgh, 15261, USA

    Judith Klein-Seetharaman

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Levit, A., Barak, D., Behrens, M., Meyerhof, W., Niv, M.Y. (2012). Homology Model-Assisted Elucidation of Binding Sites in GPCRs. In: Vaidehi, N., Klein-Seetharaman, J. (eds) Membrane Protein Structure and Dynamics. Methods in Molecular Biology, vol 914. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-023-6_11

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  • DOI: https://doi.org/10.1007/978-1-62703-023-6_11

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