Homology modeling, docking, and molecular dynamics simulation of the receptor GALR2 and its interactions with galanin and a positive allosteric modulator

Original Paper


Galanin receptor type 2 (GALR2) is a class A G-protein-coupled receptor (GPCR), and it has been reported that orthosteric ligands and positive allosteric modulators (PAMs) of GALR2 could potentially be used to treat epilepsy. So far, the X-ray structure of this receptor has not been resolved, and knowledge of the 3D structure of GALR2 may prove informative in attempts to design novel ligands and to explore the mechanism for the allosteric modulation of this receptor. In this study, homology modeling was used to obtain several GALR2 models using known templates. ProSA-web Z-scores and Ramachandran plots as well as pre-screening against a test dataset of known compounds were all utilized to select the best model of GALR2. Molecular dockings of galanin (a peptide) and a nonpeptide ligand were carried out to choose the (GALR2 model)–galanin complex that showed the closest agreement with the corresponding experimental data. Finally, a 50-ns MD simulation was performed to study the interactions between the GALR2 model and the synthetic and endogenous ligands. The results from docking and MD simulation showed that, besides the reported residues, Tyr1604.60, Ile1053.32, Ala2747.35, and Tyr163ECL2 also appear to play important roles in the binding of galanin. The potential allosteric binding pockets in the GALR2 model were then investigated via MD simulation. The results indicated that the mechanism for the allosteric modulation caused by PAMs is the binding of the PAM at pocket III, which is formed by galanin, ECL2, TM2, TM3, and ECL1; this results in the disruption of the Na+-binding site and/or the Na+ ion pathway, leading to GALR2 agonism.


Homology modeling Docking Galanin receptor type 2 Molecular dynamic simulation Positive allosteric modulator 

Supplementary material

894_2016_2944_MOESM1_ESM.doc (12.4 mb)
ESM 1(DOC 12696 kb)


  1. 1.
    George SR, O’Dowd BF, Lee SP (2002) G-protein-coupled receptor oligomerization and its potential for drug discovery. Nat Rev Drug Discov 1(10):808–820CrossRefGoogle Scholar
  2. 2.
    Rask-Andersen M, Almen MS, Schioth HB (2011) Trends in the exploitation of novel drug targets. Nat Rev Drug Discov 10(8):579–590CrossRefGoogle Scholar
  3. 3.
    Overington JP, Al-Lazikani B, Hopkins AL (2006) Opinion: How many drug targets are there? Nat Rev Drug Discov 5(12):993–996Google Scholar
  4. 4.
    Mitsukawa K, Lu X, Bartfai T (2008) Galanin, galanin receptors and drug targets. Cell Mol Life Sci 65(12):1796–1805CrossRefGoogle Scholar
  5. 5.
    Branchek TA, Smith KE, Gerald C, Walker MW (2000) Galanin receptor subtypes. Trends Pharmacol Sci 21(3):109–117CrossRefGoogle Scholar
  6. 6.
    Wang S, Hashemi T, Fried S, Clemmons AL, Hawes BE (1998) Differential intracellular signaling of the GalR1 and GalR2 galanin receptor subtypes. Biochemistry 37(19):6711–6717CrossRefGoogle Scholar
  7. 7.
    Bartfai T, Lu X, Badie-Mahdavi H, Barr AM, Mazarati A, Hua XY, Yaksh T, Haberhauer G, Ceide SC, Trembleau L, Somogyi L, Krock L, Rebek J Jr (2004) Galmic, a nonpeptide galanin receptor agonist, affects behaviors in seizure, pain, and forced-swim tests. Proc Natl Acad Sci USA 101(28):10470–10475Google Scholar
  8. 8.
    McGowan HW, Schuijers JA, Grills BL, McDonald SJ, McDonald AC (2014) Galnon, a galanin receptor agonist, improves intrinsic cortical bone tissue properties but exacerbates bone loss in an ovariectomised rat model. J Musculoskelet Neuronal Interact 14(2):162–172Google Scholar
  9. 9.
    Zhao X, Yun K, Seese RR, Wang Z (2013) Galnon facilitates extinction of morphine-conditioned place preference but also potentiates the consolidation process. PLoS One 8(10), e76395CrossRefGoogle Scholar
  10. 10.
    Wu WP, Hao JX, Lundstrom L, Wiesenfeld-Hallin Z, Langel U, Bartfai T, Xu XJ (2003) Systemic galnon, a low-molecular weight galanin receptor agonist, reduces heat hyperalgesia in rats with nerve injury. Eur J Pharmacol 482(1–3):133–137CrossRefGoogle Scholar
  11. 11.
    Bartfai T, Wang MW (2013) Positive allosteric modulators to peptide GPCRs: a promising class of drugs. Acta Pharmacol Sin 34(7):880–885CrossRefGoogle Scholar
  12. 12.
    Conn PJ, Lindsley CW, Meiler J, Niswender CM (2014) Opportunities and challenges in the discovery of allosteric modulators of GPCRs for treating CNS disorders. Nat Rev Drug Discov 13(9):692–708CrossRefGoogle Scholar
  13. 13.
    Nickols HH, Conn PJ (2014) Development of allosteric modulators of GPCRs for treatment of CNS disorders. Neurobiol Dis 61:55–71CrossRefGoogle Scholar
  14. 14.
    Lu XY, Roberts E, Xia FC, Sanchez-Alavez M, Liu TY, Baldwin R, Wu S, Chang J, Wasterlain CG, Bartfai T (2010) GalR2-positive allosteric modulator exhibits anticonvulsant effects in animal models. Proc Natl Acad Sci USA 107(34):15229–15234Google Scholar
  15. 15.
    Hoyer D (2010) Neuropeptide receptor positive allosteric modulation in epilepsy: galanin modulation revealed. Proc Natl Acad Sci USA 107(34):14943–14944Google Scholar
  16. 16.
    Conn PJ, Christopoulos A, Lindsley CW (2009) Allosteric modulators of GPCRs: a novel approach for the treatment of CNS disorders. Nat Rev Drug Discov 8(1):41–54CrossRefGoogle Scholar
  17. 17.
    Feng ZW, Hu GX, Ma SF, Xie XQ (2015) Computational advances for the development of allosteric modulators and bitopic ligands in G protein-coupled receptors. AAPS J 17(5):1080–1095Google Scholar
  18. 18.
    Feng ZW, Ma SF, Hu GX, Xie XQ (2015) Allosteric binding site and activation mechanism of class C G-protein coupled receptors: metabotropic glutamate receptor family. AAPS J 17(3):737–753Google Scholar
  19. 19.
    Knoflach F, Mutel V, Jolidon S, Kew JN, Malherbe P, Vieira E, Wichmann J, Kemp JA (2001) Positive allosteric modulators of metabotropic glutamate 1 receptor: characterization, mechanism of action, and binding site. Proc Natl Acad Sci USA 98(23):13402–13407Google Scholar
  20. 20.
    Voigtlander U, Johren K, Mohr M, Raasch A, Trankle C, Buller S, Ellis J, Holtje HD, Mohr K (2003) Allosteric site on muscarinic acetylcholine receptors: identification of two amino acids in the muscarinic M2 receptor that account entirely for the M2/M5 subtype selectivities of some structurally diverse allosteric ligands in N-methylscopolamine-occupied receptors. Mol Pharmacol 64(1):21–31CrossRefGoogle Scholar
  21. 21.
    Gerlach LO, Skerlj RT, Bridger GJ, Schwartz TW (2001) Molecular interactions of cyclam and bicyclam non-peptide antagonists with the CXCR4 chemokine receptor. J Biol Chem 276(17):14153–14160Google Scholar
  22. 22.
    Dror RO, Green HF, Valant C, Borhani DW, Valcourt JR, Pan AC, Arlow DH, Canals M, Lane JR, Rahmani R, Baell JB, Sexton PM, Christopoulos A, Shaw DE (2013) Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs. Nature 503(7475):295–299Google Scholar
  23. 23.
    Shang Y, LeRouzic V, Schneider S, Bisignano P, Pasternak GW, Filizola M (2014) Mechanistic insights into the allosteric modulation of opioid receptors by sodium ions. Biochemistry 53(31):5140–5149CrossRefGoogle Scholar
  24. 24.
    Livingston KE, Traynor JR (2014) Disruption of the Na+ ion binding site as a mechanism for positive allosteric modulation of the mu-opioid receptor. Proc Natl Acad Sci USA 111(51):18369–18374Google Scholar
  25. 25.
    Rodriguez D, Ranganathan A, Carlsson J (2015) Discovery of GPCR ligands by molecular docking screening: novel opportunities provided by crystal structures. Curr Top Med Chem 15(24):2484–2503CrossRefGoogle Scholar
  26. 26.
    Costanzi S, Wang K (2014) The GPCR crystallography boom: providing an invaluable source of structural information and expanding the scope of homology modeling. Adv Exp Med Biol 796:3–13CrossRefGoogle Scholar
  27. 27.
    Lundstrom L, Sollenberg UE, Bartfai T, Langel U (2007) Molecular characterization of the ligand binding site of the human galanin receptor type 2, identifying subtype selective interactions. J Neurochem 103(5):1774–1784CrossRefGoogle Scholar
  28. 28.
    Wennerberg AB, Cooke RM, Carlquist M, Rigler R, Campbell ID (1990) A 1H NMR study of the solution conformation of the neuropeptide galanin. Biochem Biophys Res Commun 166(3):1102–1109Google Scholar
  29. 29.
    Morris MB, Ralston GB, Biden TJ, Browne CL, King GF, Iismaa TP (1995) Structural and biochemical studies of human galanin: NMR evidence for nascent helical structures in aqueous solution. Biochemistry 34(14):4538–4545CrossRefGoogle Scholar
  30. 30.
    Parthiban M, Shanmughavel P (2007) Three dimensional modeling of N-terminal region of galanin and its interaction with the galanin receptor. Bioinformation 2(3):119–125CrossRefGoogle Scholar
  31. 31.
    Jurkowski W, Yazdi S, Elofsson A (2013) Ligand binding properties of human galanin receptors. Mol Membr Biol 30(2):206–216CrossRefGoogle Scholar
  32. 32.
    Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, Le Trong I, Teller DC, Okada T, Stenkamp RE, Yamamoto M, Miyano M (2000) Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289(5480):739–745CrossRefGoogle Scholar
  33. 33.
    Manglik A, Kruse AC, Kobilka TS, Thian FS, Mathiesen JM, Sunahara RK, Pardo L, Weis WI, Kobilka BK, Granier S (2012) Crystal structure of the micro-opioid receptor bound to a morphinan antagonist. Nature 485(7398):321–326CrossRefGoogle Scholar
  34. 34.
    Cherezov V, Rosenbaum DM, Hanson MA, Rasmussen SG, Thian FS, Kobilka TS, Choi HJ, Kuhn P, Weis WI, Kobilka BK, Stevens RC (2007) High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor. Science 318(5854):1258–1265CrossRefGoogle Scholar
  35. 35.
    Fenalti G, Giguere PM, Katritch V, Huang XP, Thompson AA, Cherezov V, Roth BL, Stevens RC (2014) Molecular control of delta-opioid receptor signalling. Nature 506(7487):191–196CrossRefGoogle Scholar
  36. 36.
    Warne T, Moukhametzianov R, Baker JG, Nehme R, Edwards PC, Leslie AG, Schertler GF, Tate CG (2011) The structural basis for agonist and partial agonist action on a beta(1)-adrenergic receptor. Nature 469(7329):241–244CrossRefGoogle Scholar
  37. 37.
    Wu H, Wang C, Gregory KJ, Han GW, Cho HP, Xia Y, Niswender CM, Katritch V, Meiler J, Cherezov V, Conn PJ, Stevens RC (2014) Structure of a class C GPCR metabotropic glutamate receptor 1 bound to an allosteric modulator. Science 344(6179):58–64CrossRefGoogle Scholar
  38. 38.
    Wu H, Wacker D, Mileni M, Katritch V, Han GW, Vardy E, Liu W, Thompson AA, Huang XP, Carroll FI, Mascarella SW, Westkaemper RB, Mosier PD, Roth BL, Cherezov V, Stevens RC (2012) Structure of the human kappa-opioid receptor in complex with JDTic. Nature 485(7398):327–332CrossRefGoogle Scholar
  39. 39.
    Wang C, Wu H, Katritch V, Han GW, Huang XP, Liu W, Siu FY, Roth BL, Cherezov V, Stevens RC (2013) Structure of the human smoothened receptor bound to an antitumour agent. Nature 497(7449):338–343CrossRefGoogle Scholar
  40. 40.
    Thompson AA, Liu W, Chun E, Katritch V, Wu H, Vardy E, Huang XP, Trapella C, Guerrini R, Calo G, Roth BL, Cherezov V, Stevens RC (2012) Structure of the nociceptin/orphanin FQ receptor in complex with a peptide mimetic. Nature 485(7398):395–399CrossRefGoogle Scholar
  41. 41.
    Lebon G, Warne T, Edwards PC, Bennett K, Langmead CJ, Leslie AG, Tate CG (2011) Agonist-bound adenosine A2A receptor structures reveal common features of GPCR activation. Nature 474(7352):521–525CrossRefGoogle Scholar
  42. 42.
    Zhang J, Zhang K, Gao ZG, Paoletta S, Zhang D, Han GW, Li T, Ma L, Zhang W, Muller CE, Yang H, Jiang H, Cherezov V, Katritch V, Jacobson KA, Stevens RC, Wu B, Zhao Q (2014) Agonist-bound structure of the human P2Y12 receptor. Nature 509(7498):119–122CrossRefGoogle Scholar
  43. 43.
    Isberg V, de Graaf C, Bortolato A, Cherezov V, Katritch V, Marshal FH, Mordalski S, Pin JP, Stevens RC, Vriend G, Gloriam DE (2015) Generic GPCR residue numbers—aligning topology maps while minding the gaps. Trends Pharmacol Sci 36(1):22–31Google Scholar
  44. 44.
    Marti-Renom MA, Stuart AC, Fiser A, Sanchez R, Melo F, Sali A (2000) Comparative protein structure modeling of genes and genomes. Annu Rev Biophys Biomol Struct 29:291–325CrossRefGoogle Scholar
  45. 45.
    Barany-Wallje E, Andersson A, Graslund A, Maler L (2004) NMR solution structure and position of transportan in neutral phospholipid bicelles. FEBS Lett 567(2–3):265–269CrossRefGoogle Scholar
  46. 46.
    Wiederstein M, Sippl MJ (2007) ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Res 35(Web Server issue):W407–W410CrossRefGoogle Scholar
  47. 47.
    Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26(2):283–291CrossRefGoogle Scholar
  48. 48.
    Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25(13):1605–1612Google Scholar
  49. 49.
    Jain AN (1996) Scoring noncovalent protein-ligand interactions: a continuous differentiable function tuned to compute binding affinities. J Comput Aided Mol Des 10(5):427–440CrossRefGoogle Scholar
  50. 50.
    Pierce BG, Wiehe K, Hwang H, Kim BH, Vreven T, Weng ZP (2014) ZDOCK server: interactive docking prediction of protein–protein complexes and symmetric multimers. Bioinformatics 30(12):1771–1773Google Scholar
  51. 51.
    Pedretti A, Villa L, Vistoli G (2002) VEGA: a versatile program to convert, handle and visualize molecular structure on Windows-based PCs. J Mol Graph Model 21(1):47–49CrossRefGoogle Scholar
  52. 52.
    Dolinsky TJ, Nielsen JE, McCammon JA, Baker NA (2004) PDB2PQR: an automated pipeline for the setup of Poisson–Boltzmann electrostatics calculations. Nucleic Acids Res 32(Web Server issue):W665–W667Google Scholar
  53. 53.
    Hsin J, Arkhipov A, Yin Y, Stone JE, Schulten K (2008) Using VMD: an introductory tutorial. Curr Protoc Bioinformatics 5:5.7Google Scholar
  54. 54.
    Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML (1983) Comparison of simple potential functions for simulating liquid water. J Chem Phys 79(2):926–935CrossRefGoogle Scholar
  55. 55.
    Kalé L, Skeel R, Bhandarkar M, Brunner R, Gursoy A, Krawetz N, Phillips J, Shinozaki A, Varadarajan K, Schulten K (1999) NAMD2: greater scalability for parallel molecular dynamics. J Comput Phys 151(1):283–312CrossRefGoogle Scholar
  56. 56.
    Brooks BR, Bruccoleri RE, Olafson BD, States DJ, Swaminathan S, Karplus M (1983) CHARMM: a program for macromolecular energy, minimization, and dynamics calculations. J Comput Chem 4(2):187–217CrossRefGoogle Scholar
  57. 57.
    MacKerell AD, Bashford D, Bellott M, Dunbrack RL, Evanseck JD, Field MJ, Fischer S, Gao J, Guo H, Ha S, Joseph-McCarthy D, Kuchnir L, Kuczera K, Lau FTK, Mattos C, Michnick S, Ngo T, Nguyen DT, Prodhom B, Reiher WE, Roux B, Schlenkrich M, Smith JC, Stote R, Straub J, Watanabe M, Wiórkiewicz-Kuczera J, Yin D, Karplus M (1998) All-atom empirical potential for molecular modeling and dynamics studies of proteins. J Phys Chem B 102(18):3586–3616CrossRefGoogle Scholar
  58. 58.
    Essmann U, Perera L, Berkowitz ML, Darden T, Lee H, Pedersen LG (1995) A smooth particle mesh Ewald method. J Chem Phys 103(19):8577–8593CrossRefGoogle Scholar
  59. 59.
    Kabsch W, Sander C (1983) Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22(12):2577–2637CrossRefGoogle Scholar
  60. 60.
    Khoddami M, Nadri H, Moradi A, Sakhteman A (2015) Homology modeling, molecular dynamic simulation, and docking based binding site analysis of human dopamine (D4) receptor. J Mol Model 21(2):36Google Scholar
  61. 61.
    Cai Z, Ouyang Q, Zeng D, Nguyen KN, Modi J, Wang L, White AG, Rogers BE, Xie XQ, Anderson CJ (2014) (64)Cu-labeled somatostatin analogues conjugated with cross-bridged phosphonate-based chelators via strain-promoted click chemistry for pet imaging: in silico through in vivo studies. J Med Chem 57(14):6019–6029CrossRefGoogle Scholar
  62. 62.
    Floren A, Land T, Langel U (2000) Galanin receptor subtypes and ligand binding. Neuropeptides 34(6):331–337CrossRefGoogle Scholar
  63. 63.
    Bisignano P, Burford NT, Shang Y, Marlow B, Livingston KE, Fenton AM, Rockwell K, Budenholzer L, Traynor JR, Gerritz SW, Alt A, Filizola M (2015) Ligand-based discovery of a new scaffold for allosteric modulation of the mu-opioid receptor. J Chem Inf Model 55(9):1836–1843CrossRefGoogle Scholar
  64. 64.
    Burford NT, Livingston KE, Canals M, Ryan MR, Budenholzer LM, Han Y, Shang Y, Herbst JJ, O’Connell J, Banks M, Zhang L, Filizola M, Bassoni DL, Wehrman TS, Christopoulos A, Traynor JR, Gerritz SW, Alt A (2015) Discovery, synthesis, and molecular pharmacology of selective positive allosteric modulators of the delta-opioid receptor. J Med Chem 58(10):4220–4229CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  1. 1.College of PharmacyThird Military Medical UniversityChongqingChina

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