Effect of arginine mutation on EBA-175 RII binding to human erythrocytes.

a<p>the rosette number was an average counted from two biological repeats in each experiment.</p>b<p>erythrocyte-binding assay was performed with normal erythrocytes in two independent experiments.</p>c<p>erythrocyte-binding assay was performed with neuraminidase-treated erythrocytes Nm, neuraminidase.</p>*<p>Residue 566 of EBA-175 corresponds to residue 422 of rEBA-175 RII.</p

Deduced amino acid sequences of clones enriched for each EBA-175 RII specific mAb.

<p>Deduced amino acid sequences of clones enriched for each EBA-175 RII specific mAb.</p

F2βf peptide competes with mAbs R215 (A) and R217 (B) but not R218 (C) for binding to recombinant EBA-175 RII by Surface Plasmon Resonance studies.

<p>Monoclonal antibody sensograms show binding to immobilized EBA-175 RII with the concentrations of mAb and cyclic F2βf peptide as indicated. The concentrations of cyclic F2βf peptide were added to a fixed concentration of mAb.</p

Mapping of epitope predictions to the rEBA-175 RII crystal structure.

<p>(A) rEBA-175 RII crystal structure as a dimer of two RII molecules (cyan and grey), the region corresponding to the F2βf peptide is highlighted in yellow and R422 is highlighted in magenta. The amino- and carboxy- terminal residues are colored in blue and red respectively. The white box represents the region highlighted in panel B. (B) The F2 domain of one monomer in the rEBA-175 RII crystal structure is shown as a ribbon diagram and residues are colored by the number of times they were predicted by either PepSurf and Mapitope to be part of the epitope for mAbs R215, R217, or R256 from never (cyan) to most often (magenta; see SOM for raw data). Residues discussed in the text are labeled according to rEBA-175 RII numbering (EBA-175 3D7 sequence number –144) and shown in stick representation.</p

Summary of binding properties of EBA-175 RII specific mAbs.

<p>SPR: surface plasmon resonance. ND: not determined.</p>*<p>Does not inhibit parasite growth <i>in vitro</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056326#pone.0056326-Sim2" target="_blank">[21]</a>.</p>**<p>None of the mAbs demonstrated binding to linear F2βf.</p>c<p>These mAbs were shown to compete against each other for binding by competition ELISA.</p

The Epitope of Monoclonal Antibodies Blocking Erythrocyte Invasion by <em>Plasmodium falciparum</em> Map to The Dimerization and Receptor Glycan Binding Sites of EBA-175

<div><p>The malaria parasite, <em>Plasmodium falciparum</em>, and related parasites use a variety of proteins with Duffy-Binding Like (DBL) domains to bind glycoproteins on the surface of host cells. Among these proteins, the 175 kDa erythrocyte binding antigen, EBA-175, specifically binds to glycophorin A on the surface of human erythrocytes during the process of merozoite invasion. The domain responsible for glycophorin A binding was identified as region II (RII) which contains two DBL domains, F1 and F2. The crystal structure of this region revealed a dimer that is presumed to represent the glycophorin A binding conformation as sialic acid binding sites and large cavities are observed at the dimer interface. The dimer interface is largely composed of two loops from within each monomer, identified as the F1 and F2 β-fingers that contact depressions in the opposing monomers in a similar manner. Previous studies have identified a panel of five monoclonal antibodies (mAbs) termed R215 to R218 and R256 that bind to RII and inhibit invasion of erythrocytes to varying extents. In this study, we predict the F2 β-finger region as the conformational epitope for mAbs, R215, R217, and R256, and confirm binding for the most effective blocking mAb R217 and R215 to a synthetic peptide mimic of the F2 β-finger. Localization of the epitope to the dimerization and glycan binding sites of EBA-175 RII and site-directed mutagenesis within the predicted epitope are consistent with R215 and R217 blocking erythrocyte invasion by <em>Plasmodium falciparum</em> by preventing formation of the EBA-175– glycophorin A complex.</p> </div

Additional file 1: of Tagging to endogenous genes of Plasmodium falciparum using CRISPR/Cas9

PCR primers and sgRNAs used for plasmid construction. (PDF 78 kb

Target Elucidation by Cocrystal Structures of NADH-Ubiquinone Oxidoreductase of <i>Plasmodium falciparum</i> (<i>Pf</i>NDH2) with Small Molecule To Eliminate Drug-Resistant Malaria

Drug-resistant malarial strains have been continuously emerging recently, which posts a great challenge for the global health. Therefore, new antimalarial drugs with novel targeting mechanisms are urgently needed for fighting drug-resistant malaria. NADH-ubiquinone oxidoreductase of <i>Plasmodium falciparum</i> (<i>Pf</i>NDH2) represents a viable target for antimalarial drug development. However, the absence of structural information on <i>Pf</i>NDH2 limited rational drug design and further development. Herein, we report high resolution crystal structures of the <i>Pf</i>NDH2 protein for the first time in Apo-, NADH-, and RYL-552 (a new inhibitor)-bound states. The <i>Pf</i>NDH2 inhibitor exhibits excellent potency against both drug-resistant strains in vitro and parasite-infected mice in vivo via a potential allosteric mechanism. Furthermore, it was found that the inhibitor can be used in combination with dihydroartemisinin (DHA) synergistically. These findings not only are important for malarial <i>Pf</i>NDH2 protein-based drug development but could also have broad implications for other NDH2-containing pathogenic microorganisms such as <i>Mycobacterium tuberculosis</i>