K_D for var2CSA recombinant proteins binding to placental CSPG.

<p>K_D values were determined from the concentration dependence of steady-state SPR response using the Biacore BIAEVALUATION 3.1 software.</p><p>*: from reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020270#pone.0020270-Srivastava1" target="_blank">[34]</a>.</p>#<p>: from reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020270#pone.0020270-Gangnard1" target="_blank">[41]</a>.</p

Cross-reactivity between apical membrane antgen 1 and rhoptry neck protein 2 in <i>P</i>. <i>vivax</i> and <i>P</i>. <i>falciparum</i>: A structural and binding study

<div><p>Malaria, a disease endemic in many tropical and subtropical regions, is caused by infection of the erythrocyte by the apicomplexan parasite <i>Plasmodium</i>. Host-cell invasion is a complex process but two <i>Plasmodium</i> proteins, Apical Membrane Antigen 1 (AMA1) and the Rhoptry Neck protein complex (RON), play a key role. AMA1, present on the surface of the parasite, binds tightly to the RON2 component of the RON protein complex, which is inserted into the erythrocyte membrane during invasion. Blocking the AMA1-RON2 interaction with antibodies or peptides inhibits invasion, underlining its importance in the <i>Plasmodium</i> life cycle and as a target for therapeutic strategies. We describe the crystal structure of the complex formed between AMA1 from <i>P</i>. <i>vivax</i> (PvAMA1) and a peptide derived from the externally exposed region of <i>P</i>. <i>vivax</i> RON2 (PvRON2sp1), and of the heterocomplex formed between <i>P</i>. <i>falciparum</i> AMA1 (PfAMA1) and PvRON2sp1. Binding studies show that the affinity of PvRON2sp1 for PvAMA1 is weaker than that previously reported for the PfRON2sp1-PfAMA1 association. Moreover, while PvRON2sp1 shows strong cross-reactivity with PfAMA1, PfRON2sp1 displays no detectable interaction with PvAMA1. The structures show that the equivalent residues PvRON2-Thr2055 and PfRON2-Arg2041 largely account for this pattern of reactivity.</p></div

Comparison between the homo-complex PvAMA1-PvRON2sp1 and the hetero-complex PfAMA1-PvRON2sp1.

<p>Two orthogonal views of the complexes are shown: (A), (B) The PvAMA1-PvRON2sp1 complex is shown in surface representation with the N-terminal extension in pink (residues 43 to 62), Domain I in light brown (residues 63 to 248), Domain II in mauve (residues 249 to 385) and Domain III in cyan (residues 386 to 474); PvRON2sp1 is shown in stick representation. (C), (D) The PfAMA1-PvRON2sp1 complex is shown with the same relative orientations as in A and B, respectively. PfAMA1 is shown in surface representation with the N-terminal extension in pink (residues 102 to 123), Domain I in light brown (residues 124 to 303), Domain II in mauve (residues 304 to 440); PvRON2sp1 is shown in stick representation.</p

Refinement statistics.

<p>Refinement statistics.</p

Competitive inhibition of recombinant DBL domains binding to decorin using various glycosaminoglycans.

<p>Recombinant proteins (A) 3D7-DBL1X-2X, (B) 3D7-DBL1X-CIDR, (C) 3D7-DBL1X-3X, (D) 3D7-DBL1X-4ε, (E) 3D7-DBL1X-5ε and (E) 3D7-DBL1X-6ε at 1 µg/mL were premixed with increasing amounts of BSA (□) or glycosaminoglycans, 0.156–100 µg/mL of CSA (×), CSC (⋄) or HS (▵) and incubated in plates previously coated with decorin.</p

Binding of recombinant DBL domains from 3D7 and FCR3-DBL1X-6ε to different glycosaminoglycans.

<p>ELISA-based direct binding assay was performed to identify the specificity of (A) 3D7-DBL2X, (B) FCR3-DBL3X, (C) 3D7-DBL5ε, (D) FCR3-DBL3X-4ε, (E) 3D7-DBL1X-2X, (F) 3D7-DBL1X-CIDR, (G) 3D7-DBL1X-3X, (H) 3D7-DBL1X-4ε, (I) 3D7-DBL1X-5ε and (J) 3D7-DBL1X-6ε to different sulfated glycosaminoglycans. Increasing concentrations of recombinant proteins at serial dilutions of 0.31–20 µg/mL were added to wells previously coated with BSA (▵) or different glycosaminoglycans: decorin (○), CSA (□), CSC (⋄), HS (×).</p

Various var2CSA recombinant proteins expressed in HEK293 cells and <i>E. coli</i>.

<p>(A). Schematic view of the var2CSA domain organization and sequence limits of the recombinant domains studied (3D7-DBL1X, 3D7-DBL2X, FCR3-DBL3X, 3D7-DBL5ε, 3D7-DBL1X-2X, FCR3-DBL3X-4ε, 3D7-DBL1X-CIDR, 3D7-DBL1X-3X, 3D7-DBL1X-4ε, 3D7-DBL1X-5ε and 3D7-DBL1X-6ε). Var2CSA comprises six DBL domains (DBL1X to DBL6ε), a CIDRpam domain and three inter-domain regions (INT1-3) in the extracellular region, together with a trans-membrane segment and acidic C terminus sequence (ATS). DBL1X, DBL2X, DBL3X, DBL4ε, DBL5ε and DBL6ε are shown in green; CIDR in orange; N-terminal sequence (NTS) and inter-domain regions (INT) in grey; the trans-membrane and ATS regions in blue. The length of each bar corresponds to the domain size. (B). Purification of var2CSA derived proteins expressed in HEK293 and <i>E. coli</i>. Nu-SDS-PAGE Precast 4–12% Bis-Tris gel under nonreducing (B) and reducing (C) conditions was loaded with purified recombinant proteins. Lane 1: Marker, lane 2: 3D7-DBL1X, lane 3: 3D7-DBL2X, lane 4: FCR3-DBL3X, lane 5: 3D7-DBL5ε, lane 6: 3D7-DBL1X-2X, lane 7: FCR3-DBL3X-4ε, lane 8: 3D7-DBL1X-CIDR, lane 9: 3D7-DBL1X-3X, lane 10: 3D7-DBL1X-4ε, lane 11: 3D7-DBL1X-5ε and lane 12: 3D7-DBL1X-6ε. Proteins were visualized with Coomassie blue.</p

Comparison of the PvRON2sp1 peptides in the homo- and hetero-complexes, and PfRON2sp1 in the homo-complex PfAMA1-PfRON2sp1.

<p>PvRON2sp1 is shown in cyan for the homo-complex and in orange for the hetero-complex; PfRON2sp1 from the PfAMA1-PfRON2sp1 homo-complex (PDB entry 3ZWZ) is shown in magenta. (A) The three ligands, shown in ribbon representation, were superimposed using the main-chain coordinates of the disulphide-bridged β-hairpin (Cys2051-Cys2063 in PvRON2sp1, Cys2037-Cys2049 in PfRON2sp1). The side chains of Arg2041 of PfRON2sp1, which makes critical contacts with PfAMA1 in the <i>P</i>. <i>falciparum</i> homo-complex, and of the equivalent residue in PvRON2sp1, Thr2055, are also shown. The superimposed ligands are oriented such that the N-terminal helix of PvRON2sp1 in the homo-complex (cyan) is vertical. (B) View of the superimposed ligands rotated by 90° about the horizontal axis.</p

Depletion of purified adhesion-inhibitory rabbit IgGs (anti 3D7-DBL1X-6ε) by various recombinant proteins.

<p>Immunization of rabbit with 3D7-DBL1X-6ε induces adhesion-blocking antibodies. Purified IgG against 3D7-DBL1X-6ε (Anti-DBL1X-6ε was depleted on 3D7-DBL1X, 3D7-DBL2X, FCR3-DBL3X, 3D7-DBL5ε, 3D7-DBL6ε, 3D7-DBL1X-2X, 3D7-DBL1X-CIDR, FCR3-DBL3X-4ε, 3D7-DBL1X-3X, 3D7-DBL1X-4ε, 3D7-DBL1X-5ε and 3D7-DBL1X-6ε immobilized on tosylactivated beads. Unbound (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020270#pone-0020270-g005" target="_blank">Fig. 5A</a>) and bound (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020270#pone-0020270-g005" target="_blank">Fig. 5B</a>) fractions were tested for inhibition of binding of 3D7-DBL1X-6ε to decorin. Data are express as % of inhibition compare to control.</p

Binding measurements of PvRON2sp1 to PvAMA1 and PfAMA1 using surface plasmon resonance.

<p>Sensograms for the binding of PvRON2sp1 to (A) PvAMA1, (B) PfAMA1 (FVO strain), and (C) PfAMA1 (3D7 strain). (D) Steady state binding curves showing the fraction of bound sites as a function of PvRON2sp1 concentration. The following peptide concentrations were injected: 10, 39, 156, 313, 625, 1250, 2500 nM for PvAMA1, and 12.5, 25, 50, 100, 200, 400, 600, 800 and 1000 nM for 3D7 and FVO.</p