Molecular modeling of DARC

1. Molecular modeling of DARC A structural model of a seven transmembrane helices receptor: The Duffy Antigen / Receptor for Chemokine (DARC). Alexandre G. de Brevern Equipe de Bioinformatique Génomique et Moléculaire (EBGM) INSERM U726 / Université Paris VII 75251 PARIS Cedex 05 – FRANCE

2. Molecular modeling of DARC • DARC : Duffy Antigen / Receptor for Chemokine • 1 – History • 2- Biological • 3- Principle • 4- Comparative modelling • 5- Analysis • 6- Conclusion

3. Molecular modeling of DARC 1-History 1950/1951: The Duffy protein was first recognized as a blood group antigen, responsible for the expression of the alloantigens Fya and Fyb in humans [1, 2]. 1968: It was the first specific gene locus assigned to a specific autosome in man [3]. [1] M. Cutbush, P.L. Mollison, The Duffy blood group system, Heredity4 (1950) 383-389. [2] E.W. Ikin, A.E. Mourant, H.J. Pettenkofer, G. Blumenthal, Discovery of the expected haemagglutinin, anti-Fyb, Nature168 (1951) 1077-1078. [3] R.P. Donahue, W.B. Bias, J.H. Renwick, V.A. McKusick, Probable assignment of the Duffy blood group locus to chromosome 1 in man, Proc Natl Acad Sci U.S.A. 61 (1968) 949-55

4. Molecular modeling of DARC [3] R.P. Donahue, W.B. Bias, J.H. Renwick, V.A. McKusick, Probable assignment of the Duffy blood group locus to chromosome 1 in man, Proc Natl Acad Sci U.S.A. 61 (1968) 949-55

5. Molecular modeling of DARC Donahue et al. (1968).

6. Molecular modeling of DARC [3] R.P. Donahue, W.B. Bias, J.H. Renwick, V.A. McKusick, Probable assignment of the Duffy blood group locus to chromosome 1 in man, Proc Natl Acad Sci U.S.A. 61 (1968) 949-55

7. Molecular modeling of DARC 2-Biology It was later identified as the erythrocyte receptor for malaria parasites, because erythrocytes of Fy(a–b–) individuals that do not express Duffy cannot be invaded byPlasmodium knowlesi [4] andPlasmodium vivax [5].  American studies  on human  in jail … [4] L.H. Miller, S.J. Mason, J.A. Dvorak, M.H. McGinniss, I.K. Rothman, Erythrocyte receptors for (Plasmodium knowlesi) malaria: Duffy blood group determinants, Science189 (1975) 561-563. [5] L.H. Miller, S.J. Mason, D.F. Clyde, M.H. McGinnis, The resistance factor to Plasmodium vivax in blacks, New England Journal of Medicine, 295 (1976) 302305.

8. Molecular modeling of DARC It was also identified as a chemokine receptor because Fy-positive but Fy-null erythrocytes can bind CXCL-8 (i.e., IL-8) [6, 7]. So, it is involved in classical chemotactism [8]. In human, a single point mutation in an erythroïd regulatory element site of theDARC promoter region is responsible for the disappearance of erythrocyte DARC expression leading to the phenotype Fy(a–b–) [9]. [6] W.C. Darbonne, G.C. et al., Red Blood Cells are a sink for interleukin-8, a leukocyte chemotaxin, J Clin Invest268 (1991) 12247-12249. [7] R. Horuk, C.E. Chitnis, W.C. Darbonne, T.J. Colby, A. Rybicki, T.J. Hadley, L.H. Miller, A receptor for the malarial parasite Plasmodium vivax: the erythrocyte chemokine receptor, Science261 (1993) 1182-1184. [8] C. Murdoch, A. Finn, Chemokine receptors and their role in inflammation and infectious diseases, Blood95 (2000) 3032-3043. [9] C. Tournamille, Y. Colin, J.-P. Cartron, C. Le Van Kim, Disruption of a GATA motif in the Duffy gene promoter abolishes erythroid gene expression in Duffy-negative individuals, Nat Genet10 (1995), 224-228.

9. Molecular modeling of DARC This mutation may have a high selective value in West Africa since 95% of the population in this area exhibits Fy(a–b–) phenotype [9, 10]. In contrast to other chemokine receptors, DARC is a particular promiscuous receptor [11, 12]. [10] R. Horuk, Z.X. Wang, S.C. Peiper, J. Hesselgesser, Identification and characterization of a promiscuous chemokine-binding protein in a human erythroleukemic cell line, J Biol Chem269 (1994) 17730-17733. [11] M.C. Szabo, K.S. Soo, A. Zlotnik, T.J. Schall, Chemokine class differences in binding to the Duffy antigen-erythrocyte chemokine receptor, J Biol Chem270 (1995) 25348-25351. [12] A.B. Lentsch, The Duffy antigen/receptor for chemokines (DARC) and prostate cancer. A role as clear as black and white?, The FASEB Journal16 (2002) 1093-1095.

10. Molecular modeling of DARC • Indeed, chemokines are small proteins sharing a high structural similarity and grouped into two major distinct classes: the CC and the CXC chemokines. • Most of chemokine receptors are selective of only one of the two classes, but the DARC binds chemokines of both CC and CXC classes [7, 11, 12]. • CC chemokines monocyte chemotatic protein-1 (MCP-1, CCL-2) and regulated upon activation normal Texpressed and secreted (RANTES, CCL-5),  CXC chemokines growth related gene alpha (GRO-α, CXCL-1), and neutrophil activating peptide-2 (NAP-2, CXCL-7). • [12] A. Chaudhuri, V. Zbrzezna, J. Polyakova, A.O. Pogo, J. Hesselgesser, R. Horuk, Expression of the Duffy antigen in K562 cells. Evidence that it is the human erythrocyte chemokine receptor, J Biol Chem269 (1994) 7835-7838.

11. Molecular modeling of DARC Similar to other chemokine receptors, DARC is probably a seven-transmembrane receptor. However, unlike other chemokine receptors, ligands bound by DARC do not induce G-protein coupled signal transduction nor a Ca2+-flux [13]. Indeed, DARC lacks a highly conserved DRY motif in the second intracellular loop of the protein that is known to be associated with G-protein signaling [14]. DARC could so be considered as a “silent” chemokine receptor. In fact, evidence is accumulating that ligand-induced signaling may not be required for “silent” receptors to exert major role in chemokine biology [15]. [13] K. Neote, et al., Functional and biochemical analysis of the cloned Duffy antigen: identity with the red blood cell chemokine receptor, Blood84 (1994) 44-52. [14] T.J. Hadley, S.C. Peiper, From malaria to chemokine receptor: the emerging physiologic role of the Duffy blood group antigen, Blood89 (1997) 3077-3091. [15] R. Nibbs, G. Graham, A. Rot, Chemokines on the move: control by the chemokine “interceptors” Duffy blood group antigen and D6, Seminars in Immunology15 (2003) 287-294.

12. Chemokines [CXC & CC] (CXCL-8, RANTES) 1 chemotactism 1 DARC Molecular modeling of DARC Physiological condition extracellular Membrane intracellular

13. Plasmodium vivax (human), Plasmodium knowlesi (simian) 2 Duffy Binding Protein (DBP) DARC 2 Parasites invasion Molecular modeling of DARC Sexual cycle of parasite paludic infection physiological condition extracellular Membrane intracellular

14. Molecular modeling of DARC Goal 1 2 3

15. Molecular modeling of DARC DARC DBP DARC can be divided into three distinct regions: (1) the transmembrane part with the 7 putative a-helices and the 6 connecting loops, (2) the long ECD1 (60 residues) and (3) a short Cter. (20 aa), i.e., ICD4. (P1) The structure of CXCL-8 is known. (P2) The DBP (200 amino acids) is part of a protein (1500 aa) anchored to the membrane with a single a-helix domain. P2 P1 2 ECD1 Protein (transmembrane helices and connecting loops) 1 3 ICD4

16. Molecular modeling of DARC Biological data [16] C. Tournamille, A. Filipe, K. Wasniowska, G. Kazimiera, P. Gane, E. Lisowska, J.-P. Cartron, Y. Colin, C. Le Van Kim, Structurefunction analysis of the extracellular domains of the Duffy antigen/receptor for chemokines: characterization of antibody and chemokine binding sites, British Journal of Haematology 122 (2003), 1014-1023.

17. Localization of helical regions 1 Molecular modeling of DARC ECD1 ICD4 DARC (sequence)

18. Molecular modeling of DARC Secondary structure prediction

19. Localization of helical regions 1 3 2 rhodopsin (3D) Alignment 4 Molecular modeling of DARC ECD1 ICD4 DARC (sequence)

20. Localization of helical regions 1 ECD1 and ICD4 predictions 5 Molecular modeling of DARC ECD1 ICD4 DARC (sequence)

21. Molecular modeling of DARC Structural information ECD1 ICD4 Structural homologues 0 0 Secondary structure prediction not good not good Threading some some Ab initio / de novo done done Protein Blocks done done

22. Molecular modeling of DARC Threading results [17] J. Shi, T. L. Blundell, K. Mizuguchi, FUGUE: sequence-structure homology recognition using environment-specific substitution tables and structure- dependent gap penalties, J Mol Biol310 (2001) 243-257.

23. Molecular modeling of DARC Protein Blocks

24. Molecular modeling of DARC Protein Blocks results [18] A.G. de Brevern, C. Etchebest, S. Hazout, Bayesian probabilistic approach for predicting backbone structures in terms of protein blocks, Proteins41 (2000) 271-287. [19] A.G. de Brevern, C. Benros, R. Gautier, H. Valadié, S. Hazout, C. Etchebest, Local backbone structure prediction of proteins, In silico Biology4 (2004) 31. [20] A.G. de Brevern, New assessment of a structural alphabet, In silico Biology5 (2005) 26.

25. 6 6 Complete alignment Molecular modeling of DARC Molecular modeling of DARC Localization of helical regions 1 ECD1 ICD4 DARC (sequence) 3 2 rhodopsin (3D) 4 Alignment 5 ECD1 and ICD4 predictions

26. Localization of helical regions 1 adjustments 10 Simulated annealing … ECD1 ICD4 9 DARC (sequence) adjustments 3 2 rhodopsin (3D) Minimization, Analysis (accessibility) Alignment 4 8 ECD1 and ICD4 predictions 5 6 6 7 Building the models Complete alignment Molecular modeling of DARC

27. Molecular modeling of DARC Final alignment Mean sequence identity = 22 % in central region (25 % for the helices). [0% -13%] for the loops. Automatic = 12% identity.

28. Molecular modeling of DARC Selected models

29. Molecular modeling of DARC Model 1 Movie 1

30. Molecular modeling of DARC Model 2 Movie 2

31. Molecular modeling of DARC Accessibility of model 1 Not accessible … because

32. C C ECD2 ECD1 C C ECD3 ECD4 Molecular modeling of DARC Simulated annealing Principle

33. Molecular modeling of DARC Simulated annealing ECD1 ECD2 ECD3 ECD4

34. Molecular modeling of DARC Protein Blocks

35. Molecular modeling of DARC Simulated annealing Movie 3

36. Molecular modeling of DARC Simulated annealing ECD1

37. Molecular modeling of DARC Simulated annealing ECD4

38. Molecular modeling of DARC Electrostatics DARC CXCL-8

39. Molecular modeling of DARC Partial conclusions We have described the construction of a model structure for the Duffy Antigen/Receptor for Chemokine (DARC). The final model of DARC contains the seven transmembrane helices, the extra- and intra-cellular loops including the long N-terminal domain (ECD1) and the C-terminal tail (ICD4). Such a model will be extremely valuable to help understanding the structure/function relationship of DARC. Given the limited amount of structural data available for these transmembrane proteins, building a model structure for a receptor of this family is a difficult and challenging task. The model presented has been built carefully, making use of several highly relevant bioinformatics tools. Both bioinformatics analyses and experimental data have been used as guidelines/restraints to build the model. The strategy used here is an illustration of the fact that protein structure prediction is often very demanding, especially in the case of membrane proteins and can not always rely on automated procedures. We have carefully considered and combined all available information, and evaluated thoroughly the models produced. The resulting model complies with experimental data and will surely be useful in the design of new experiments. Additionally, the strategy itself is relevant for other scientists interested in modeling GPCRs and other transmembrane proteins. A.G. de Brevern, H. Wong, C. Tournamille, Y. Colin, C. Le Van Kim & C. Etchebest, A structural model of a seven transmembrane helices receptor: The Duffy Antigen / Receptor for Chemokine (DARC), in revision.

40. Molecular modeling of DARC Partial conclusions DARC seems to be a really hard case. Two of the prediction methods (PRO-TMHMM and S-TMHMM) have predicted only a 6 transmembrane proteins, missing the 7th helix. S-TMHMM predicted the ECD1 as intracellular. Helix 3 is shifted for 8 residues between PRODIV-TMHMM and PRO-TMHMM. Moreover, the confidence given in the predictions is really poor (0.1 for an index that goes from 0 to 1), showing the complexity of the prediction. H. Viklund, A. Elofsson, Best alpha-helical transmembrane protein topology predictions are achieved using hidden Markov models and evolutionary information, Protein Sci13 (2004) 1908-1917.

41. Molecular modeling of DARC Future works • New analyses on the models. • - Normal Mode Analysis / Principal Component Analysis on ECDs • New parameterization of Hex for the docking of DARC / CXCL-8 • Modeling of Duffy Binding Protein (DBP) • - … the other chemokine receptors

42. Molecular modeling of DARC Docking (preliminary results)

43. Molecular modeling of DARC Docking (preliminary results) Movie 4