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Binding site characterization of G protein-coupled receptor by alanine-scanning mutagenesis using molecular dynamics and binding free energy approach: application to C-C chemokine receptor-2 (CCR2)

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Abstract

The C-C chemokine receptor 2 (CCR2) was proved as a multidrug target in many diseases like diabetes, inflammation and AIDS, but rational drug design on this target is still lagging behind as the information on the exact binding site and the crystal structure is not yet available. Therefore, for a successful structure-based drug design, an accurate receptor model in ligand-bound state is necessary. In this study, binding-site residues of CCR2 was determined using in silico alanine scanning mutagenesis and the interactions between TAK-779 and the developed homology model of CCR2. Molecular dynamic simulation and Molecular Mechanics-Generalized Born Solvent Area method was applied to calculate binding free energy difference between the template and mutated protein. Upon mutating 29 amino acids of template protein and comparison of binding free energy with wild type, six residues were identified as putative hot spots of CCR2.

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References

  1. Gu L, Tseng SC, Rollins BJ (1999) Monocyte chemoattractant protein-1. Chemokines 72: 7–29. doi:10.1159/000058723

    Article  CAS  Google Scholar 

  2. Newton RC, Vaddi K (1997) Biological responses to C-C chemokines. Methods Enzymol 287: 174–186. doi:10.1016/S0076-6879

    Article  PubMed  CAS  Google Scholar 

  3. Baggiolini M, Dewald B, Moser B (1994) Interleukin-8 and related chemotactic cytokines-CXC and CC chemokines. Adv Immunol 55: 97–179. doi:10.1016/S00652776

    Article  PubMed  CAS  Google Scholar 

  4. Murphy PM, Baggiolini M, Charo IF, Hébert CA, Horuk R, Matsushima K, Miller LH, Oppenheim JJ, Power CA (2000) International union of pharmacology. XXII. Nomenclature for chemokine receptors. Pharmacol Rev 52: 145–176. doi:10.1124/pr.54.2.227

    PubMed  CAS  Google Scholar 

  5. Mellado M, Rodriguez-Frade JM, Aragay A, Del Real G, Martin AM, Vila-Coro AJ, Serrano A, Mayor F Jr, Martinez-A C (1998) The chemokine monocyte chemotactic protein 1 triggers janus kinase 2 activation and tyrosine phosphorylation of the CCR2B receptor. J Immunol 161: 805–813

    PubMed  CAS  Google Scholar 

  6. Gong X, Gong W, Kuhns DB, Ben-Baruch A, Howard OM, Wang JM (1997) Monocyte chemotactic protein-2 (MCP-2) uses CCR1 and CCR2B as its functional receptors. J Biol Chem 272: 11682–11685. doi:10.1074/jbc.272.18.11682

    Article  PubMed  CAS  Google Scholar 

  7. Combadiere C, Ahuja SK, Van Damme J, Tiffany HL, Gao JL, Murphy PM (1995) Monocyte chemoattractant protein-3 is a functional ligand for CC chemokine receptors 1 and 2B. J Biol Chem 270: 29671–29675. doi:10.1074/jbc.270.50.29671

    Article  PubMed  CAS  Google Scholar 

  8. Charo IF, Ransohoff RM (2006) The many roles of chemokines and chemokine receptors in inflammation. New Engl J Med 354: 610–621. doi:10.1056/NEJMra052723

    Article  PubMed  CAS  Google Scholar 

  9. Lumeng CN, DeYoung SM, Bodzin JL, Saltiel AR (2007) Increased inflammatory properties of adipose tissue macrophages recruited during diet-induced obesity. Diabetes 56: 16–23. doi:10.2337/db06-1076

    Article  PubMed  CAS  Google Scholar 

  10. Sartipy P, Loskutoff DJ (2003) Monocyte chemoattractant protein 1 in obesity and insulin resistance. Proc Natl Acad Sci USA 100: 7265–7270. doi:10.1073/pnas.1133870100

    Article  PubMed  CAS  Google Scholar 

  11. Lumeng CN, Bodzin JL, Saltiel AR (2007) Obesity induces a phenotypic switch in adipose tissue macrophage polarization. J Clin Invest 117: 175–184. doi:10.1172/JCI29881

    Article  PubMed  CAS  Google Scholar 

  12. Abbadie C, Lindia JA, Cumiskey AM, Peterson LB, Mudgett JS, Bayne EK, DeMartino JA, MacIntyre DE, Forrest MJ (2003) Impaired neuropathic pain responses in mice lacking the chemokine receptor CCR2. Sci Signal 100: 7947–7952. doi:10.1073/pnas.1331358100

    CAS  Google Scholar 

  13. Ogata H, Takeya M, Yoshimura T, Takagi K, Takahashi K (1999) The role of monocyte chemoattractant protein-1 (MCP-1) in the pathogenesis of collagen-induced arthritis in rats. J Pathol 182: 106–114. doi:10.1002/(SICI)1096-9896

    Article  Google Scholar 

  14. Butora G, Jiao R, Parsons WH, Vicario PP, Jin H, Ayala JM, Cascieri MA, Yang L (2007) 3-Amino-1-alkyl-cyclopentane carboxamides as small molecule antagonists of the human and murine CC chemokine receptor 2. Bioorg Med Chem Lett 17: 3636–3641. doi:10.1016/j.bmcl.2007.04.053

    Article  PubMed  CAS  Google Scholar 

  15. Yang L, Butora G, Jiao RX, Pasternak A, Zhou C, Parsons WH, Mills SG, Vicario PP, Ayala JM, Cascieri MA, MacCoss M (2007) Discovery of 3-piperidinyl-1-cyclopentanecarboxamide as a novel scaffold for highly potent CC chemokine receptor 2 antagonists. J Med Chem 50: 2609–2611. doi:10.1021/jm070166b

    Article  PubMed  CAS  Google Scholar 

  16. Pinkerton AB, Huang D, Cube RV, Hutchinson JH, Struthers M, Ayala JM, Vicario PP, Patel SR, Wisniewski T, DeMartino JA, Vernier J (2007) Diaryl substituted pyrazoles as potent CCR2 receptor antagonists. Bioorg Med Chem Lett 17: 807–813. doi:10.1016/j.bmcl.2006.10.060

    Article  PubMed  CAS  Google Scholar 

  17. Pasternak A, Goble SD, Doss GA, Tsou NN, Butora G, Vicario PP, Ayala JM, Struthers M, DeMartino JA, Mills SG, Yang L (2008) Conformational studies of 3-amino-1-alkyl-cyclopentane carboxamide CCR2 antagonists leading to new spirocyclic antagonists. Bioorg Med Chem Lett 18: 1374–1377. doi:10.1016/j.bmcl.2008.01.016

    Article  PubMed  CAS  Google Scholar 

  18. Yang L, Jiao RX, Moyes C, Morriello G, Butora G, Shankaran K, Pasternak A, Goble SD, Zhou C, MacCoss M, Cumiskey AM, Peterson L, Forrest M, Ayala JM, Jin H, DeMartino J, Mills SG (2007) The discovery of MK-0812, a potent and selective CCR2 antagonist. American Chemical Society Meeting, Chicago

    Google Scholar 

  19. Xia M, Sui Z (2009) Recent developments in CCR2 antagonists. Expert Opin Ther Patents 19: 295–303. doi:10.1517/13543770902755129

    Article  CAS  Google Scholar 

  20. Baba M, Nishimura O, Kanzaki N, Okamoto M, Sawada H, Iizawa Y, Shiraish M, Aramaki Y, Okonogi K, Ogawa Y, Meguro K, Fujino MA (1992) Small molecule non-peptide CCR5 antagonist with highly potent and selective anti-HIV activity. Proc Natl Acad Sci USA 96: 5698–5703. doi:10.1073/pnas.96.10.5698

    Article  Google Scholar 

  21. Kobilka BK, Deupi X (2007) Conformational complexity of G-protein-coupled receptors. Trends Pharmacol Sci 28: 397–406. doi:10.1016/j.tips.2007.06.003

    Article  PubMed  CAS  Google Scholar 

  22. Qin L, Cai S, Zhu Y, Inouye M (2003) Cysteine-scanning analysis of the dimerization domain of EnvZ, an osmosensing histidine kinase. J Bacterial 185: 3429–3435. doi:10.1128/JB.185.11.3429-3435.2003

    Article  CAS  Google Scholar 

  23. Kouadio JLK, Horn JR, Pal G, Kossiakoff AA (2005) Shotgun alanine scanning shows that growth hormone can bind productively to its receptor through a drastically minimized interface. J Biol Chem 280: 25524–25535. doi:10.1074/jbc.M502167200

    Article  PubMed  CAS  Google Scholar 

  24. Bromberg Y, Rost B (2008) Comprehensive in silico mutagenesis highlights functionally important residues in proteins. Bioinformatics 24: 207–212. doi:10.1093/bioinformatics/btn268

    Article  Google Scholar 

  25. Kortemme T, Baker DA (2002) simple physical model for binding energy hot spots in protein–protein complexes. Proc Natl Acad Sci USA 99: 14116–14121. doi:10.1073/pnas.202485799

    Article  PubMed  CAS  Google Scholar 

  26. Wang J, Morin P, Wang W, Kollman PA (2001) Use of MM-PBSA in reproducing the binding free energies to HIV-1 RT of TIBO derivatives and predicting the binding mode to HIV-1 RT of efavirenz by docking and MM-PBSA. J Am Chem Soc 123: 5221–5230. doi:10.1021/ja003834q

    Article  PubMed  CAS  Google Scholar 

  27. Rao SN, Singh UC, Bash PA, Kollman PA (1987) Free energy perturbation calculations on binding and catalysis after mutating Asn 155 in subtilisin. Nature 328: 551–554. doi:10.1038/328551a0

    Article  PubMed  CAS  Google Scholar 

  28. Hansson T, Marelius J, Åqvist J (1998) Ligand binding affinity prediction by linear interaction energy methods. J Comput Aid Mol Des 12: 27–35. doi:10.1023/A:1007930623000

    Article  CAS  Google Scholar 

  29. Cherezov V, Rosenbaum DM, Hanson MA, Rasmussen SGF, Thian FS, Kobilka TS, Choi HJ, Kuhn P, Weis WI, Kobilka BK (2007) High-resolution crystal structure of an engineered human β 2-Adrenergic G protein-coupled receptor. Science 318: 1258–1265. doi:10.1126/science.1150577

    Article  PubMed  CAS  Google Scholar 

  30. Fiser A, Do RK, Sali A (2000) Modeling of loops in protein structures. Protein Sci 9: 1753–1773. doi:10.1110/ps.9.9.1753

    Article  PubMed  CAS  Google Scholar 

  31. Molecular Operating Environment (MOE), 2011.10 (2011) Chemical Computing Group, Inc., Montreal, QC, Canada

  32. Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26: 283–291. doi:10.1107/S0021889892009944

    Article  CAS  Google Scholar 

  33. Singh R, Sobhia ME (2010) Homology modeling of human CCR2 receptor. Med Chem Res 20: 1704–1712. doi:10.1007/s00044-010-9497-9

    Article  Google Scholar 

  34. Friesner RA, Banks JL, Murphy RB, Halgren TA, Klicic JJ, Mainz DT, Repasky MP, Knoll EH, Shelley M, Perry JK (2004) Glide: a new approach for rapid, accurate docking and scoring. 1. method and assessment of docking accuracy. J Med Chem 47: 1739–1749. doi:10.1021/jm030644s

    Article  PubMed  CAS  Google Scholar 

  35. Mirzadegan T, Diehl F, Ebi B, Bhakta S, Polsky I, McCarley D, Mulkins M, Weatherhead GS, Lapierre J, Dankwardt J, Morgans D, Wilhelm R Jr, Jarnagin K (2000) Identification of the binding site for a novel class of CCR2b chemokine receptor antagonists binding to a common chemokine receptor motif within the helical bundle. J Biol Chem 275: 25562–25571. doi:10.1074/jbc.M000692200

    Article  PubMed  CAS  Google Scholar 

  36. Berkhout TA, Blaney FE, Bridges AM, Cooper DG, Forbes IT, Gribble AD, Groot PHE, Hardy A, Ife RJ, Kaur R, Moores KW, Shillito H, Willetts J, Witherington J (2003) CCR2: characterization of the antagonist binding site from a combined receptor modeling/mutagenesis approach. J Med Chem 46: 4070–4086. doi:10.1021/jm030862l

    Article  PubMed  CAS  Google Scholar 

  37. Gavrilin MA, Gulinac IV, Kawanoc T, Draganc S, Chakravartic L, Kolattukudyb PE (2005) Site-directed mutagenesis of CCR2 identified amino acid residues in transmembrane helices 1, 2, and 7 important for MCP-1 binding and biological functions. Biochem Biophys Res Commun. 327: 533–540. doi:10.1016/j.bbrc.2004.12.037

    Article  PubMed  CAS  Google Scholar 

  38. Marshall TG, Lee RE, Marshall FE (2006) Common angiotensin receptor blockers may directly modulate the immune system via VDR, PPAR and CCR2b. Theor Biol Med Model 3: 1. doi:10.1186/1742-4682-3-1

    Article  PubMed  Google Scholar 

  39. Hall SE, Mao A, Nicolaidou V, Finelli M, Wise EL, Nedjai B, Kanjanapangka J, Harirchian P, Chen D, Selchau V, Ribeiro S, Schyler S, Pease JE, Horuk R, Vaidehi N (2009) Elucidation of binding sites of dual antagonists in the human chemokine receptors CCR2 and CCR5. Mol Pharmacol 75: 1325–1336. doi:10.1124/mol.108.053470

    Article  PubMed  CAS  Google Scholar 

  40. Ponder JW, Case DA (2003) Force fields protein simulations. Adv Prot Chem 66: 27–86. doi:10.1016/S0065-3233(03)66002-X

    Article  CAS  Google Scholar 

  41. Jakalian A, Jack DB, Bayly CI (2002) Fast, efficient generation of high-quality atomic charges. AM1-BCC Model: II. parameterization and validation. J Comput Chem 23: 1623–1641. doi:10.1002/jcc.10128

    Article  PubMed  CAS  Google Scholar 

  42. Altschul SF, Madden TL, Schfer AA, Zhang JZ, Miller DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389–3402. doi:10.1093/nar/25.17.3389

    Article  PubMed  CAS  Google Scholar 

  43. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res 22(22): 4673–4680. doi:10.1093/nar/22.22.4673

    Article  PubMed  CAS  Google Scholar 

  44. Laskowski RA, Rullmann JAC, MacArthur MW, Kaptein R, Thornton JM (1996) AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR 8: 477–486. doi:10.1007/BF00228148

    Article  PubMed  CAS  Google Scholar 

  45. Case DA, Darden TA, Cheatham TE III, Simmerling CL, Wang J, Duke RE, Luo R, Crowley M, Walker RC, Zhang W (2008) AMBER 10. University of California, San Francisco

    Google Scholar 

  46. Essmann U, Perera L, Berkowitz ML, Darden T, Lee H, Pedersen LG (1995) A smooth particle mesh Ewald method. J Chem Phys 103: 8577–8593. doi:10.1063/1.470117

    Article  CAS  Google Scholar 

  47. Van Gunsteren WF, Berendsen HJC (1977) Algorithms for macromolecular dynamics and constraint dynamics. Mol Phys 34: 1311–1327. doi:10.1080/00268977700102571

    Article  CAS  Google Scholar 

  48. Samson M, LaRosa G, Libert F, Paindavoine P, Detheux M, Vassart G, Parmentier M (1997) The second extracellular loop of CCR5 is the major determinant of ligand specificity. J Biol Chem 272: 24934–24941. doi:10.1074/jbc.272.40.24934

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research, Sector 67, S.A.S. Nagar, Punjab, 160 062, India

    Swapnil Chavan, Shirishkumar Pawar, Rajesh Singh & M. Elizabeth Sobhia

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  1. Swapnil Chavan
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  2. Shirishkumar Pawar
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  3. Rajesh Singh
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  4. M. Elizabeth Sobhia
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Correspondence to M. Elizabeth Sobhia.

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Chavan, S., Pawar, S., Singh, R. et al. Binding site characterization of G protein-coupled receptor by alanine-scanning mutagenesis using molecular dynamics and binding free energy approach: application to C-C chemokine receptor-2 (CCR2). Mol Divers 16, 401–413 (2012). https://doi.org/10.1007/s11030-012-9368-z

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