Humoral immune response and protective efficacy induced by <i>Pf</i>EMP1 HABPs derived peptides in <i>Aotus</i> monkeys.

<p><i>Aotus</i> monkeys’ humoral immune responses and protective immunity induced by <i>Pf</i>EMP1-derived peptides, according to our serial numbering system with corresponding amino acid sequence (modifications in bold). Reciprocal IFA antibody titres in bleeding 20 days after second (II₂₀) and third (III₂₀) immunisation and number of protected monkeys in experimental challenge <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088420#pone.0088420-Patarroyo1" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088420#pone.0088420-Curtidor2" target="_blank">[14]</a>.</p

Identification of <i>Pf</i>EMP1 HABPs and variability sequence between <i>Plasmodium falciparum</i> strains.

<p>(<b>A</b>) Dd2 <i>Pf</i>EMP1-based amino-acid sequence synthetic peptides’ RBC and C32 cell binding activity (black bars represent specific binding activity slope); above 2% (dotted line) were considered HABPs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088420#pone.0088420-Rodriguez1" target="_blank">[11]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088420#pone.0088420-Curtidor2" target="_blank">[14]</a>. Blue shows HABPs chosen for immunization and red those containing canonical or homologous (GACxPxRRxxLC) binding motif. Left, schematic representation of <i>Pf</i>EMP1 domains showing H-bonds between HABPs (arrows); head structure recombinant fragments containing NTS and DBL1α (fuchsia), CDR1α (green), DBL3X (orange) and DBL6ε (blue), 3D structure determined by X-ray crystallography. (<b>B</b>) Sequence logos for amino acid conservation in corresponding HABPs according to their frequency in >100 strains; each amino acid height reflects their relative frequency (%) and thus their contribution to conservation.</p

Structural characterization of HABPs present in crystallized Duffy binding like domains (DBL).

<p>DBL domain 3D structure determined by X-ray crystallography A) Head structure: DBL1α (PDB 2XU0) (pink), C) DBL3X (PDB 3CML) (yellow), F) DBL6ε (PDB 2WAU) (pale blue). ¹H-NMR-determined structure localisation, displaying the perfect fit of HABP 6505 (yellow) superimposed onto DBL1α, 6583 (dark blue) and 6584 (purple) onto DBL3X and 6622 (grey) onto DBL6ε. B, D, G). H-bonds between HABP residues and their corresponding sequence on top, displaying relevant residues in binding to A blood group trisaccharides and CSPG (asterisk and black dot, respectively). E) Superimposed conserved binding motif fragments from 6510 and 6573. H) CD spectra for corresponding HABPs.</p

<i>In silico</i> and <i>in vitro</i> analysis of boAP3d1 protein interaction with bovine leukaemia virus gp51 - Fig 1

<p><b><a href="http://web.expasy.org/protscale/pscale/Hphob.Doolittle.html" target="_blank">Kyte & Doolittle</a> hydrophobicity for BLV Env (A) and boAP3D1 (B) proteins.</b> Despite differences regarding the amount of aa in both proteins, their physicochemical characteristics were comparable.</p

boAP3D1 and gp51 docking.

<p>5A. Overview. 5B. Asn170 (gp51) and Lys925 (boAP3D1). 5C. Trp127 (gp51) and Asp807 (boAP3D1). 5D. His115 (gp51) and Asp695 (boAP3D1), hydrogen bonds, non-bonded contacts. 5E. Ala97, Ser98, Glu128 (gp51) and Arg800 (boAP3D1), hydrogen bonds, non-bonded contacts and salt bridges.</p

gp51 recombinant fragment purification.

<p>Lanes 1, 3 and 5 show Western blot detection with an anti-his monoclonal antibody. Lanes 2, 4 and 6 show Coomassie blue stained purified recombinant proteins. The proteins’ molecular weight marker is indicated in the first lane (molecular masses for the three recombinants agreed with expected ones: 30, 16 and 14 kDa for rgp51, rNgp51 and rCgp51, respectively).</p

boAP3D1, BLV Env and recombinant proteins physicochemical properties.

<p>boAP3D1, BLV Env and recombinant proteins physicochemical properties.</p

Binding free energy results for gp51 and boAP3D1 interaction.

<p>Binding free energy results for gp51 and boAP3D1 interaction.</p

gp51 recombinant protein binding assays.

<p>8A. rgp51 MDBK cell binding. Low rgp51 protein concentration (black bars) gave greater MDBK cell binding; however, rgp51 and rCgp51 binding did not change when protein concentration was duplicated (blue bars), whilst rNgp51 increased. 8B. MDBK cell interaction with rNgp51. The black bars represent enzymatically-treated MDBK and rNgp51 binding to the proposed receptor (AP3D1), followed by trypsin and chymotrypsin treatment, resulting in reduced in rNgp51 (98%) and MDBK binding (80%).</p

ClustalOmega sequence alignment of boAP3D1 and huAP3D1.

<p>“-” represents identical aa, the red line represents the BLVR domain.</p