Structure of <i>Pf</i>AMA1 complexed with R1 peptide.

<p>(A). The co-crystal structure of <i>Pf</i>AMA1 (blue surface) with R1 reveals two bound peptides, R1 major (yellow) and R1 minor (purple). (B). Detailed analysis of interactions at the <i>Pf</i>AMA1–R1-major, <i>Pf</i>AMA1–R1-minor, and R1-major–R1-minor interfaces. Surface representation of <i>Pf</i>AMA1 (blue), with R1-major (yellow) and R1-minor (purple) shown as cartoons. Box 1 – R1-major anchors its N-terminus to <i>Pf</i>AMA1 through 3 backbone hydrogen bonds. Box 2 – the central region of the <i>Pf</i>AMA1 apical groove is occupied by R1-major through both hydrophobic and polar interactions. Box 3 – R1-minor forms most of its anchor points to <i>Pf</i>AMA1 through the apical loops and does not contact the base of the groove, which is occupied by R1-major. Panel 4 – Backbone hydrogen bonds between R1-minor and R1-major generate a β-sheet, while R1-major is further pinned to the <i>Pf</i>AMA1 groove through 3 hydrogen bonds. Panel 5 – R1-major integrates into <i>Pf</i>AMA1 with the use of an arginine knob-in-hole interaction stabilized by 6 hydrogen bonds, which is also exploited by<i>Pf</i>RON2sp.</p

Refinement statistics.

<p>Refinement statistics.</p

Surface Plasmon Resonance studies of peptides <i>Pf</i>RON2sp1 and <i>Pf</i>RON2sp2 binding to recombinant <i>Pf</i>AMA1 from multiple strains reveal that <i>Pf</i>RON2sp1 has a consistently higher affinity.

<p>(A) <i>Pf</i>RON2sp1 (orange) and <i>Pf</i>RON2sp2 (grey) represent peptides of <i>Pf</i>RON2 (green). SP, signal peptide. TMD, putative transmembrane domain. (B). Sensorgrams showing <i>Pf</i>RON2sp1 (analyte) binding to <i>Pf</i>AMA1 3D7 (immobilized). The <i>Pf</i>RON2sp1 concentrations are indicated for each curve (nM). (C). Sensorgrams showing <i>Pf</i>RON2sp2 (analyte) binding to <i>Pf</i>AMA1 CAMP (immobilized), with <i>Pf</i>RON2sp2 concentrations indicated. (D, E). Variation percentage of bound sites (deduced from the steady-state response) with respect to analyte concentration (D, <i>Pf</i>RON2sp1; E, <i>Pf</i>RON2sp2) obtained from binding to immobilized recombinant <i>Pf</i>AMA1 from strains 3D7 (shown in B), CAMP (shown in C), FVO and HB3. The derived apparent equilibrium dissociation constants K_D are given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002755#ppat-1002755-t001" target="_blank">Table 1</a>.</p

Mutations of <i>Pf</i>AMA1 and <i>Pf</i>RON2-5 reveal residues critical for high affinity interaction.

<p>(A) Interface between <i>Pf</i>AMA1 and <i>Pf</i>RON2sp1 shown in open-book presentation. Residues of both components that were mutated are labeled. (B). Binding characteristics of recombinant GST-<i>Pf</i>RON2-5 mutants to dissect hot-spot residues in <i>Pf</i>RON2. <i>Pf</i>AMA1-expressing BHK-21 cells were incubated with 10 µg/ml of <i>Pf</i>RON2 or mutated proteins (GST-fusion proteins), washed and the binding of recombinant <i>Pf</i>RON2 fragment was revealed with anti-GST antibody. <i>Pf</i>AMA1 was detected with mAb F8.12.19, which recognizes extracellular Domain III. (C). Binding consequences of <i>Pf</i>AMA1 mutations. Mutated versions of <i>Pf</i>AMA1 were expressed on the surface of BHK-21 cells and incubated with wild-type <i>Pf</i>RON2 recombinant proteins at 10 and 1 µg/ml.</p

Crystallographic parameters, data collection statistics and refinement summary.

<p>Values in parenthesis are for the last resolution shell.</p

Highly potent cross-strain inhibition of red blood cell invasion of <i>Pf</i>RON2sp1.

<p>Comparison of <i>Pf</i>RON2sp1 and R1 peptides (concentrations 0.2 to 20 µM) in inhibiting red blood cell invasion by <i>P. falciparum</i> 3D7 or HB3 highlights the higher inhibitory efficiency and cross-strain reactivity of <i>Pf</i>RON2sp1. Parasitemia of control infected red blood cells (IRBC) 16 hours post-invasion was used as the 100% invasion reference. Means (± SD for N = 3) are shown.</p

The Arg knob-in-hole interaction is critical for species selectivity and interaction with invasion inhibitory antibodies and peptides.

<p>(A). Left - A cut-away surface of <i>Pf</i>AMA1 (blue), reveals that Arg2041 of <i>Pf</i>RON2sp1 (orange) integrates deeply into a well-defined pocket. Right - However, no analogous pocket is observed in <i>Pv</i>AMA1 (grey; PDB ID 1W8K). (B). Peptides and antibodies known to be invasion inhibitory for <i>P. falciparum</i> occupy the key Arg binding site, as shown by orthogonal views of the <i>Pf</i>AMA1-<i>Pf</i>RON2sp1 co-structure (blue-orange) overlayed with the mAb 1F9 co-structure (1F9, green; PDB ID 2Q8B), IgNAR14l-1 co-structure (IgNAR, purple; PDB ID 2Z8V), and R1 co-structure (R1, yellow; reported here).</p

Structure of <i>Pf</i>AMA1 complexed with <i>Pf</i>RON2-derived peptides.

<p>(A) Top - Co-crystal structures of <i>Pf</i>AMA1 (blue surface) with <i>Pf</i>RON2sp1 (orange) and <i>Pf</i>RON2sp2 (grey), show a disulfide-anchored U-shaped conformation in the apical groove of <i>Pf</i>AMA1. Bottom - Electron density map (orange) for <i>Pf</i>RON2sp1 contoured at 1.0 σ, highlighting well ordered density from the N-terminal helix, through the cystine loop, to the C-terminal coil. (B) Notable changes in the structure of <i>Pf</i>AMA1 between the apo structure (green; PDB ID 1Z40) and the <i>Pf</i>AMA1-<i>Pf</i>RON2sp1 co-structure (blue-orange) as observed from a side view. Box 1 - The DII loop of apo <i>Pf</i>AMA1 is ejected from the apical groove during binding to <i>Pf</i>RON2sp1, leaving room for the <i>Pf</i>RON2sp1 N-terminal helix to occupy the space vacated by the DII loop helix. Box 2 - The β-strands of the <i>Pf</i>RON2sp1 cystine loop order a <i>Pf</i>AMA1 surface loop, generating a contiguous three-stranded β-sheet. (C) In the region of the <i>Pf</i>RON2sp1 N-terminal helix, there is notable structural mimicry to the <i>Pf</i>AMA1 apo DII loop, including several conserved residues, and a conserved hydrogen bonding network incorporating three buried water molecules. (D) Arg2041, specific to <i>P. falciparum</i>, fits snugly into a deep pocket in the surface of <i>Pf</i>AMA1 and is stabilized through a complex network of seven hydrogen bonds.</p

Surface Plasmon Resonance studies of peptide R1 binding to <i>Pf</i>AMA1 mutants 3D7mut and Dico3.

<p>(A). Left - sensorgrams, showing R1 (analyte) binding to <i>Pf</i>AMA1 3D7mut (immobilized). R1 concentrations are indicated for each curve (µM). Right - the variation in percentage of bound sites (deduced from the steady-state response) with respect to analyte concentration. (B). Left - sensorgrams, showing R1 (analyte) binding to Dico3 (immobilized), with R1 concentrations indicated. Right - the variation in percentage of bound sites (deduced from the steady-state response) with respect to analyte concentration. The equilibrium dissociation constant K_D derived from the steady state binding curves is 15.2 µM for 3D7mut and 22.3 µM for Dico3.</p

Structural mimicry of <i>Pf</i>RON2 by peptide R1 in binding to <i>Pf</i>AMA1.

<p>(A) Top (left) and end-on (right) views of <i>Pf</i>AMA1-<i>Pf</i>RON2sp1 (orange cartoon) overlayed on <i>Pf</i>AMA1-R1-major (yellow)/R1-minor (purple), show that the <i>Pf</i>AMA1 groove is capable of accepting only <i>Pf</i>RON2sp1 or the two R1 peptides at one time. Box 1 shows that Phe-P5 of R1 mimics Phe367 of the DII loop, while boxes 2 and 3 highlight spatial conservation of a phenylalanine anchor at the center of the groove, and a knob-in-hole interaction incorporating the peptide Arg-P15. R1-major is shown in yellow, <i>Pf</i>RON2sp1 in orange and apo PfAMA1 in green. (B). Comparison of the R1 and <i>Pf</i>RON2sp1 sequences reveals five identical (red) and two similar (blue) residues.</p