In silico studies on DARC

Abstract

The Duffy Antigen/Receptor for Chemokine (DARC) is a seven segment transmembrane protein. It was firstly discovered as a blood group antigen and was the first specific gene locus assigned to a specific autosome in man. It became more famous as an erythrocyte receptor for malaria parasites (Plasmodium vivax and Plasmodium knowlesi), and finally for chemokines. DARC is an unorthodox chemokine receptor as (i) it binds chemokines of both CC and CXC classes and (ii) it lacks the Asp-Arg-Tyr consensus motif in its second cytoplasmic loop hence cannot couple to G proteins and activate their signaling pathways. DARC had also been associated to cancer progression, numerous inflammatory diseases, and possibly to AIDS. In this review, we will summarize important biological data on DARC. Then we shall focus on recent development of the elaboration and analyzes of structural models of DARC. We underline the difficulty to propose pertinent structural models of transmembrane protein using comparative modeling process, and other dedicated approaches as the Protein Blocks. The chosen structural models encompass most of the biochemical data known to date. Finally, we present recent development of protein-protein docking between DARC structural models and CXCL-8 structures. We propose a hierarchical search based on separated rigid and flexible docking.

Figure 1. The three steps

(1) by comparative modeling, structural models are building; (2) docking approach is performed with the natural ligand structure, i.e., CXC-L8 and (3) with the Duffy Binding Protein (DBP).

Figure 2. Principles of homology modeling for a transmembrane protein.

Figure 3. Phylogenetic analysis of DARC

Different sequences of DARC were aligned using CLUSTALW [91]. The dendogram is plotted with Njplot software [92]. Bootstrap values computed by CLUSTALW are shown at the different nodes. Sequences were extracted from UniProt/UniParc ID [94]: the major 336 aa [93] human (DUFF_HUMAN, Q16570 [95]) was used, gorilla (Gorilla gorilla), AF311914; marmoset (Callithrix jacchus), AF311915; tamarin (Saguinus oedipus), AF311916; night monkey (Aotus trivirgatus), AF311917; squirrel monkey (Samiri boliviensis), AF311918; brown capuchin (Cebus apella), AF311919; chimpanzee (Pan troglodytes), AF311920; rhesus monkey (Macaca mulatta), AF311921; baboon (Papio papio), AF303532; gibbon (Hylobates lar) AF303533 (all the last sequences are from [71]).

Figure 4. Prediction of transmembrane segments of DARC

Are shown the predictions done using HMMTOP [96, 97], TMHMM 2.0 [98], TMAP [113], MEMSAT [117], SOSUI [107], TMpred [104], TSEG [114], TM-FINDER [115], Pred-TMR 2.0 [111], SPLIT [109] and DAS [99]. The final selection corresponds to the positions finally obtained after multiple tests and evaluations. The plot was done using R software [121].

Figure 5. Building structural models of DARC

(a) Prediction of transmembrane helices. (b) Alignment of helical regions with corresponding regions of rhodopsin structure. (c) Potential structural templates for .ECD1 and ICD4 prediction of (d) Addition of these results to the complete alignment for comparative modeling. (e) Structural model generation and refinement of these models. (f) Accessibility computation of amino acids and known to be exposed. (g) In regards to the results, the alignment is modified. (h) At last, some models are selected.

Figure 6. Encoding of the protein structures (3D) in terms of Protein Blocks.

Figure 7. The two DARC selected models

Two views of (a–b) open form structural model and (c–d) closed structural model. Visualization done with PyMol software [160].

Figure 8. Normal Mode Analysis of DARC ‘open’ model

(a) (y-axis) Modes 7 to 9 for (x-axis) the whole sequence. (b) Visualization of the modes 7 to 9 on the ‘open’ model. The cross highlights the hinge region of ECD1. Visualization done with VMD software [162, 163].

Figure 9. Visualization of electrostatics potential on the surface of DARC ‘open’ model

Visualization done with PyMol software [160].

Figure 10. A divide-and-conquer approach

The ECD1 (N terminus of DARC) and the Transmembrane Domain (TMD) are considered separately. (a) A rigid docking is performed on the TMD while (b) a flexible docking is done for ECD1. Then (c) a flexible docking is done to combine both results. (d) the most interesting results are analyzed.

Figure 11. Rigid docking examples

(a – c) Three different examples of rigid docking results between TMD and CXCL8 (monomer form). (d – f) Three different views of a complex obtained by rigid body between TMD and CXCL8 (dimer form).

Figure 12. Selected monomer CXCL8 – DARC TMD docking

Some important residues are highlighted.

Figure 13. Flexible docking

Some snapshots of the flexible search of ECD1 around the rigid structure of CXCL8 monomer are shown.

Figure 14. A good conformation

ECD1, CXCL8 and TD are shown with different grey tones.