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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

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slide1

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

slide2

Molecular modeling of DARC

  • DARC : Duffy Antigen / Receptor for Chemokine
    • 1 – History
    • 2- Biological
    • 3- Principle
    • 4- Comparative modelling
    • 5- Analysis
    • 6- Conclusion
slide3

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

slide4

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

slide5

Molecular modeling of DARC

Donahue et al. (1968).

slide6

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

slide7

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.

slide8

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.

slide9

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.

slide10

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.
slide11

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.

slide12

Chemokines [CXC & CC]

(CXCL-8, RANTES)

1

chemotactism

1

DARC

Molecular modeling of DARC

Physiological condition

extracellular

Membrane

intracellular

slide13

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

slide15

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

slide16

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.

slide17

Localization of helical regions

1

Molecular modeling of DARC

ECD1

ICD4

DARC (sequence)

slide18

Molecular modeling of DARC

Secondary structure prediction

slide19

Localization of helical regions

1

3

2

rhodopsin (3D)

Alignment

4

Molecular modeling of DARC

ECD1

ICD4

DARC (sequence)

slide20

Localization of helical regions

1

ECD1 and ICD4

predictions

5

Molecular modeling of DARC

ECD1

ICD4

DARC (sequence)

slide21

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

slide22

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.

slide24

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.

slide25

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

slide26

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

slide27

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.

slide31

Molecular modeling of DARC

Accessibility of model 1

Not accessible … because

slide32

C

C

ECD2

ECD1

C

C

ECD3

ECD4

Molecular modeling of DARC

Simulated annealing

Principle

slide33

Molecular modeling of DARC

Simulated annealing

ECD1

ECD2

ECD3

ECD4

slide35

Molecular modeling of DARC

Simulated annealing

Movie 3

slide36

Molecular modeling of DARC

Simulated annealing

ECD1

slide37

Molecular modeling of DARC

Simulated annealing

ECD4

slide38

Molecular modeling of DARC

Electrostatics

DARC

CXCL-8

slide39

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.

slide40

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.

slide41

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
slide42

Molecular modeling of DARC

Docking (preliminary results)

slide43

Molecular modeling of DARC

Docking (preliminary results)

Movie 4