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Research Article

Structural and Functional Insights into the Malaria Parasite Moving Junction Complex

  • Brigitte Vulliez-Le Normand equal contributor,

    equal contributor Contributed equally to this work with: Brigitte Vulliez-Le Normand, Michelle L. Tonkin, Mauld H. Lamarque

    Affiliations: Unité d'Immunologie Structurale, Institut Pasteur, Paris, France, URA 2185 CNRS, Paris, France

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  • Michelle L. Tonkin equal contributor,

    equal contributor Contributed equally to this work with: Brigitte Vulliez-Le Normand, Michelle L. Tonkin, Mauld H. Lamarque

    Affiliation: Department of Biochemistry & Microbiology, University of Victoria, Victoria, British Columbia, Canada

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  • Mauld H. Lamarque equal contributor,

    equal contributor Contributed equally to this work with: Brigitte Vulliez-Le Normand, Michelle L. Tonkin, Mauld H. Lamarque

    Affiliation: UMR 5235 CNRS, Université de Montpellier 2, Montpellier, France

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  • Susann Langer,

    Affiliation: Department of Biochemistry & Microbiology, University of Victoria, Victoria, British Columbia, Canada

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  • Sylviane Hoos,

    Affiliation: Plate-Forme de Biophysique des Macromolécules et de leurs Interactions, Institut Pasteur, Paris, France

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  • Magali Roques,

    Affiliation: UMR 5235 CNRS, Université de Montpellier 2, Montpellier, France

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  • Frederick A. Saul,

    Affiliations: Unité d'Immunologie Structurale, Institut Pasteur, Paris, France, URA 2185 CNRS, Paris, France

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  • Bart W. Faber,

    Affiliation: Department of Parasitology, Biomedical Primate Research Centre, Rijswijk, The Netherlands

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  • Graham A. Bentley mail,

    graham.bentley@pasteur.fr (GAB); mboulang@uvic.ca (MJB); mylebrun@univ-montp2.fr (ML)

    Affiliations: Unité d'Immunologie Structurale, Institut Pasteur, Paris, France, URA 2185 CNRS, Paris, France

    These authors also contributed equally to this work.

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  • Martin J. Boulanger mail,

    graham.bentley@pasteur.fr (GAB); mboulang@uvic.ca (MJB); mylebrun@univ-montp2.fr (ML)

    Affiliation: Department of Biochemistry & Microbiology, University of Victoria, Victoria, British Columbia, Canada

    These authors also contributed equally to this work.

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  • Maryse Lebrun mail

    graham.bentley@pasteur.fr (GAB); mboulang@uvic.ca (MJB); mylebrun@univ-montp2.fr (ML)

    Affiliation: UMR 5235 CNRS, Université de Montpellier 2, Montpellier, France

    These authors also contributed equally to this work.

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  • Published: June 21, 2012
  • DOI: 10.1371/journal.ppat.1002755

Abstract

Members of the phylum Apicomplexa, which include the malaria parasite Plasmodium, share many features in their invasion mechanism in spite of their diverse host cell specificities and life cycle characteristics. The formation of a moving junction (MJ) between the membranes of the invading apicomplexan parasite and the host cell is common to these intracellular pathogens. The MJ contains two key parasite components: the surface protein Apical Membrane Antigen 1 (AMA1) and its receptor, the Rhoptry Neck Protein (RON) complex, which is targeted to the host cell membrane during invasion. In particular, RON2, a transmembrane component of the RON complex, interacts directly with AMA1. Here, we report the crystal structure of AMA1 from Plasmodium falciparum in complex with a peptide derived from the extracellular region of PfRON2, highlighting clear specificities of the P. falciparum RON2-AMA1 interaction. The receptor-binding site of PfAMA1 comprises the hydrophobic groove and a region that becomes exposed by displacement of the flexible Domain II loop. Mutations of key contact residues of PfRON2 and PfAMA1 abrogate binding between the recombinant proteins. Although PfRON2 contacts some polymorphic residues, binding studies with PfAMA1 from different strains show that these have little effect on affinity. Moreover, we demonstrate that the PfRON2 peptide inhibits erythrocyte invasion by P. falciparum merozoites and that this strong inhibitory potency is not affected by AMA1 polymorphisms. In parallel, we have determined the crystal structure of PfAMA1 in complex with the invasion-inhibitory peptide R1 derived by phage display, revealing an unexpected structural mimicry of the PfRON2 peptide. These results identify the key residues governing the interactions between AMA1 and RON2 in P. falciparum and suggest novel approaches to antimalarial therapeutics.

Author Summary

Malaria arises from infection of erythrocytes by single-cell parasites belonging to the genus Plasmodium, the species P. falciparum causing the most severe forms of the disease. The formation of a moving junction (MJ) between the membranes of the parasite and its host cell is essential for invasion. Two important components of the MJ are Apical Membrane Antigen 1 (AMA1) on the parasite surface and the Plasmodium rhoptry neck (RON) protein complex that is translocated to the erythrocyte membrane during invasion. The extra-cellular region of RON2, a component of this complex, interacts with AMA1, providing a bridge between the parasite and its host cell that is crucial for successful invasion. The parasite thus provides its own receptor for AMA1 and accordingly this critical interaction is not subject to evasive adaptations by the host. We present atomic details of the interaction of PfAMA1 with the carboxy-terminal region of RON2 and shed light on structural adaptations by each apicomplexan parasite to maintain an interaction so crucial for invasion. The structure of the RON2 ligand bound to AMA1 thus provides an ideal basis for drug design as such molecules may be refractory to the development of drug resistance in P. falciparum.

Introduction

Plasmodium spp., and P. falciparum in particular, are devastating global pathogens that place nearly half the human population at risk to malaria, leading to more than 250 million cases yearly and over one million deaths [1]. The success of the malaria parasite can be attributed to its intracellular lifestyle, invading host cells both in liver and blood stages. Invasion of red blood cells is an active process involving a moving junction (MJ), which is formed by intimate contact between erythrocyte and parasite membranes and is thought to be coupled to the parasite's actin-myosin motor [2], [3]. A number of merozoite antigens, either exposed on the surface or stored in secretory organelles, play a role in the invasion process [4]. One of these is Apical Membrane Antigen 1 (AMA1), a type-one transmembrane protein secreted from the micronemes to the merozoite surface and present at the MJ [5], [6]. AMA1 is highly conserved in the Plasmodium genus [6] and, moreover, in the Apicomplexa phylum to which Plasmodium belongs [7], [8], suggesting a common functional role in diverse host cell invasion scenarios. In the apicomplexan organism Toxoplasma gondii, the receptor for AMA1 was shown to be Rhoptry Neck Protein 2 (RON2), a component of the parasite-derived RON protein complex that is secreted into the host cell during invasion and integrated into the host cell membrane [9], [10]. This interaction was subsequently confirmed in P. falciparum as well [11], [12]. Apicomplexans thus provide both receptor and ligand to drive active invasion.

In many malaria-endemic regions, P. falciparum has become resistant to classic drugs, such as chloroquine, and is rapidly developing resistance to recently introduced drugs. Since both AMA1 and RON2 are specific to Apicomplexa and essential for invasion, interruption of the AMA1-RON2 interaction presents an ideal new target for the design and development of inhibitors. This is supported by the recent observation that the invasion-inhibitory peptide R1 [13], [14] blocks interaction between AMA1 and the RON complex in P. falciparum [15], but due to the polymorphism of AMA1, the effectiveness of this peptide inhibitor is limited to a subset of parasite isolates. Interestingly, R1 does not prevent apical contact but no formation of a functional MJ ensues from this event [15].

Crystal structures of PfAMA1 in complex with invasion-inhibitory antibodies [16], [17] have implicated a hydrophobic groove on Domain I (DI) of PfAMA1 as being critical for function. The topological nature of the PfAMA1 groove [18] is conserved in P. vivax AMA1 [19] and T. gondii AMA1 [20], and contains a number of residues that are conserved or semi-conserved across Plasmodium species, as well as other members of Apicomplexa [21], suggesting that it contributes to the receptor-binding site of AMA1. This was recently confirmed by the crystal structure of TgAMA1 in complex with a synthetic peptide, TgRON2sp, which inserts in the groove of TgAMA1 [22].

Here, we report the crystal structure of the complex formed between PfAMA1 and peptide segments of PfRON2, which, together with our previous structural results on the TgAMA1-TgRON2 co-structure [22], highlights a conserved, crucial interaction in apicomplexan host cell invasion. Functional characterization of hot-spot residues driving AMA1-RON2 complex formation leads to a deeper understanding of key interactions occurring at the MJ of P. falciparum and reveals the molecular basis of cross-strain reactivity while preserving specificity for the species. We also describe the crystal structure of PfAMA1 in complex with the invasion-inhibitory peptide R1 [14], and show that this peptide presents an intriguing structural mimicry of PfRON2. Collectively, our results provide an important structural basis for designing cross-strain reactive molecules that inhibit invasion by P. falciparum.

Results

PfRON2sp specifically binds to PfAMA1

From the 67-residue construct, PfRON2-5, that we previously showed to have affinity for PfAMA1 [11], and guided by the TgAMA1-TgRON2sp structure [22], we synthesized two analogous PfRON2 peptides: PfRON2sp1 (residues 2021–2059; numbering from the initiation methionine in PF14_0495), and PfRON2sp2 (residues 2027–2055). Significantly, there is no polymorphism in this sequence among P. falciparum isolates. Both constructs incorporate a disulfide-bound β-hairpin loop proposed to be critical in complex formation [22] while PfRON2sp2 is truncated at both the N- and C-termini (Fig. 1A). Since the extracellular region of PfRON2 is non-polymorphic, we determined the affinity of both peptides for PfAMA1 by Surface Plasmon Resonance (SPR) measurements using the 3D7, CAMP, FVO and HB3 proteins to explore the possible effects of AMA1 polymorphisms. The affinity of PfAMA1 from 3D7 for PfRON2sp1 is 25-fold higher than for PfRON2sp2 (Fig. 1B to E, Table 1), highlighting a moderate, yet influential, role for the N- and C-terminal tails. Interestingly, KD values for the PfRON2sp peptides showed no significant variation in binding to PfAMA1 from the four strains.

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Figure 1. Surface Plasmon Resonance studies of peptides PfRON2sp1 and PfRON2sp2 binding to recombinant PfAMA1 from multiple strains reveal that PfRON2sp1 has a consistently higher affinity.

(A) PfRON2sp1 (orange) and PfRON2sp2 (grey) represent peptides of PfRON2 (green). SP, signal peptide. TMD, putative transmembrane domain. (B). Sensorgrams showing PfRON2sp1 (analyte) binding to PfAMA1 3D7 (immobilized). The PfRON2sp1 concentrations are indicated for each curve (nM). (C). Sensorgrams showing PfRON2sp2 (analyte) binding to PfAMA1 CAMP (immobilized), with PfRON2sp2 concentrations indicated. (D, E). Variation percentage of bound sites (deduced from the steady-state response) with respect to analyte concentration (D, PfRON2sp1; E, PfRON2sp2) obtained from binding to immobilized recombinant PfAMA1 from strains 3D7 (shown in B), CAMP (shown in C), FVO and HB3. The derived apparent equilibrium dissociation constants KD are given in Table 1.

doi:10.1371/journal.ppat.1002755.g001
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Table 1. Apparent equilibrium dissociation constants KD (nM) for the binding of peptides PfRON2sp1 and PfRON2sp2 to AMA1 from different strains of P. falciparum.

doi:10.1371/journal.ppat.1002755.t001

PfRON2sp1 and PfRON2sp2 were co-crystallized with the first two ectoplasmic domains (DI, DII) of recombinant PfAMA1 3D7 or CAMP strains, respectively. The co-structure of PfAMA1 3D7 PfRON2sp1 (PDB entry code 3ZWZ) was refined to 2.2 Å resolution, while PfAMA1 CAMP PfRON2sp2 (PDB entry code 3SRI) was refined to 1.6 Å resolution (Tables 2, 3). The two co-structures overlay with a root mean square deviation (rmsd) of 0.81 Å in 304 Cα positions, and the two peptides alone overlay with a rmsd of 0.34 Å over the complete length of the modeled PfRON2sp2 (25 Cα) (Fig. 2A). These data confirm that the reduced affinity of PfRON2sp2 is due to the truncated N- and C-termini. Since PfRON2sp1 is more biologically relevant than its truncated counterpart, it is used for the following analyses unless otherwise noted.

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Figure 2. Structure of PfAMA1 complexed with PfRON2-derived peptides.

(A) Top - Co-crystal structures of PfAMA1 (blue surface) with PfRON2sp1 (orange) and PfRON2sp2 (grey), show a disulfide-anchored U-shaped conformation in the apical groove of PfAMA1. Bottom - Electron density map (orange) for PfRON2sp1 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 PfAMA1 between the apo structure (green; PDB ID 1Z40) and the PfAMA1-PfRON2sp1 co-structure (blue-orange) as observed from a side view. Box 1 - The DII loop of apo PfAMA1 is ejected from the apical groove during binding to PfRON2sp1, leaving room for the PfRON2sp1 N-terminal helix to occupy the space vacated by the DII loop helix. Box 2 - The β-strands of the PfRON2sp1 cystine loop order a PfAMA1 surface loop, generating a contiguous three-stranded β-sheet. (C) In the region of the PfRON2sp1 N-terminal helix, there is notable structural mimicry to the PfAMA1 apo DII loop, including several conserved residues, and a conserved hydrogen bonding network incorporating three buried water molecules. (D) Arg2041, specific to P. falciparum, fits snugly into a deep pocket in the surface of PfAMA1 and is stabilized through a complex network of seven hydrogen bonds.

doi:10.1371/journal.ppat.1002755.g002
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Table 2. Crystallographic parameters, data collection statistics and refinement summary.

doi:10.1371/journal.ppat.1002755.t002
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Table 3. Refinement statistics.

doi:10.1371/journal.ppat.1002755.t003

PfRON2sp1, traced from Thr2023 to Leu2058, includes a disulfide bridge between Cys2037 and Cys2049 and makes several direct contacts with PfAMA1 (Fig. S1), resulting in a total buried surface area of 3154 Å2 (1441 Å2 for PfAMA1 and 1713 Å2 for PfRON2sp1). Overall, the binding paradigm established by TgAMA1-TgRON2sp [22] is maintained, with an N-terminal helix seated at one end of the AMA1 receptor-binding groove and extended through an ordered coil to a disulfide-closed β-hairpin loop, generating a U-shaped conformation (Fig. 2A). Similarly, exposing a functional receptor-binding groove on AMA1 requires displacement of the extended non-polymorphic DII loop, which adopts a disordered state (not modeled between Lys351 to Ala387); this region is stabilized by DI in apo PfAMA1 (Fig. 2B). Intriguingly, the backbone of the N-terminal helix and additional coil of PfRON2sp1 (2024-QQAKDIGAG-2032) overlays remarkably well with a section of the apo PfAMA1 DII loop (360-YEKIKEGFK-368) (rmsd<0.4 Å), which also includes a helical region (Fig. 2B - box 1). Three water molecules buried by the DII loop in the apo form are retained in the receptor-bound state and facilitate a network of hydrogen bonds that bridge PfAMA1 DI to either the DII loop or PfRON2sp in apo PfAMA1 or the receptor complex, respectively (Fig. 2C). The majority of intermolecular contacts are formed by the segment Lys2027-Met2042 of PfRON2sp1. An influential residue on PfRON2 appears to be Arg2041, a residue specific to the P. falciparum species, located at the tip of the β-hairpin with its guanidyl group fitting snugly into a preformed pocket of PfAMA1 (Fig. 2D).

R1 occupies the PfRON2sp-binding site on PfAMA1

The invasion-inhibitory peptide R1, comprising 20 residues (VFAEFLPLFSKFGSRMHILK) [14], has been shown by nuclear magnetic resonance (NMR) to bind to the PfAMA1 hydrophobic groove, but this study gave little structural detail of the interaction [15]. We therefore crystallized PfAMA1 3D7 (DI and II) with R1 to compare with the PfRON2 complex. Surprisingly, two molecules of R1 are bound to PfAMA1, which we denote respectively as the major peptide (R1-major), lying deeply in the binding groove, and the minor peptide (R1-minor), lying above R1-major and making fewer contacts with PfAMA1 (Fig. 3 and Table S1). Several solvent molecules bridge directly between PfAMA1 and R1-major. As in the PfAMA1-PfRON2sp complexes, the N-terminus of R1-major binds to a region of PfAMA1 that becomes exposed after displacement of the DII loop.

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Figure 3. Structure of PfAMA1 complexed with R1 peptide.

(A). The co-crystal structure of PfAMA1 (blue surface) with R1 reveals two bound peptides, R1 major (yellow) and R1 minor (purple). (B). Detailed analysis of interactions at the PfAMA1–R1-major, PfAMA1–R1-minor, and R1-major–R1-minor interfaces. Surface representation of PfAMA1 (blue), with R1-major (yellow) and R1-minor (purple) shown as cartoons. Box 1 – R1-major anchors its N-terminus to PfAMA1 through 3 backbone hydrogen bonds. Box 2 – the central region of the PfAMA1 apical groove is occupied by R1-major through both hydrophobic and polar interactions. Box 3 – R1-minor forms most of its anchor points to PfAMA1 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 PfAMA1 groove through 3 hydrogen bonds. Panel 5 – R1-major integrates into PfAMA1 with the use of an arginine knob-in-hole interaction stabilized by 6 hydrogen bonds, which is also exploited byPfRON2sp.

doi:10.1371/journal.ppat.1002755.g003

R1-major makes several direct contacts with PfAMA1 (113 interatomic distances<3.8 Å), including 19 hydrogen bonds and a salt bridge between the amino group of Lys-P11 (R1 peptide residues numbers are prefixed by P) and the Asp227 carboxylate group of PfAMA1 (Table S1A). Contacts made by R1-minor to PfAMA1 are fewer (26 contacts<3.8 Å) and include only five hydrogen bonds (Table S1B). Interactions between R1-major and R1-minor are maintained by a total of 24 interatomic contacts, including three hydrogen bonds (Table S1C). In total, 3025 Å2 of molecular surface is buried between PfAMA1 and the two peptides, with R1-major contributing about 75% to this area. The buried surface between R1-major and R1-minor is 563 Å2, reflecting the smaller number of close interatomic contacts between these two components.

Since the structure of the PfAMA1 3D7-R1 complex revealed two bound peptide molecules, binding measurements of R1 to PfAMA1 3D7 were made by isothermal titration calorimetry (ITC) to examine the stoichiometry (Fig. S2). The measured KD of 145 nM is comparable with previous measurements by SPR [13] and the deduced stoichiometry was 1:1 over the peptide concentrations used. This implies that the second binding site in the crystal structure (R-minor) has an affinity that could not be determined under the experimental conditions used for ITC but can be estimated to be at least 10-fold weaker than the major site.

R1 mimicry of PfRON2

While R1-major follows the general contour of the receptor-binding groove, it does so in a linear rather than the U-shaped conformation adopted by PfRON2sp1 (Fig. 4A). R1-minor occupies a similar region in space as the second strand of the PfRON2sp β-hairpin, contacting the same DI loop of PfAMA1 but running in the opposite direction to form a parallel two-stranded β-sheet with the major peptide (Fig. 4A). Portions of R1-major exhibit structural similarity to PfRON2, displaying a 1.2 Å rmsd in the twelve Cα positions (PfRON2sp1, Ala2031 to Met2042; R1-major, Phe-P5 to Met-P16) (Fig. 4A). Moreover, sequence alignment based on the structural superposition reveals a remarkable similarity between the central regions of the two ligands; the segments Ala2031-Met2042 of PfRON2 and Phe-P5–Met-P16 of R1 have five identical amino acids and two conservative differences (Fig. 4B). R1-major residue Arg-P15 contributes the most contacts to PfAMA1 and is positioned within the same pocket of PfAMA1 as PfRON2 Arg2041 (Fig. 4A - box 3) where it maintains six of the seven hydrogen bonds observed for PfAMA1-PfRON2sp. Interestingly, while PfRON2 mimicry is observed in the cystine loop-binding region (Phe2038/Phe-P12 to Arg2041/Arg-P15), R1-major establishes clear anchor points in the hydrophobic groove different from PfRON2; Phe-P2 and Phe-P5 brace the peptide N-terminus in the region exposed by displacement of the DII loop, with Phe-P5 occupying the pocket left vacant by Phe367 of PfAMA1 (Fig. 4A - box 1).

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Figure 4. Structural mimicry of PfRON2 by peptide R1 in binding to PfAMA1.

(A) Top (left) and end-on (right) views of PfAMA1-PfRON2sp1 (orange cartoon) overlayed on PfAMA1-R1-major (yellow)/R1-minor (purple), show that the PfAMA1 groove is capable of accepting only PfRON2sp1 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, PfRON2sp1 in orange and apo PfAMA1 in green. (B). Comparison of the R1 and PfRON2sp1 sequences reveals five identical (red) and two similar (blue) residues.

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PfAMA1 Polymorphisms at positions 175 and 225 are determinant for the 3D7 specificity of R1

R1 is strain specific, binding to PfAMA1 from the 3D7 (cognate antigen) and D10 strains, but with much reduced affinity to the HB3 or W2mef proteins, as determined by ELISA [14] or SPR [13] measurements (recapitulated in Table S2). In contrast, PfRON2sp1 bound to all the PfAMA1 proteins tested (Table 1) with a higher affinity than for R1 peptide. Consistent with these values, PfRON2sp1 displayed a higher capacity to inhibit red cell invasion by P. falciparum 3D7 than the R1 peptide (Fig. 5). Moreover, PfRON2sp1 shows cross-strain inhibition of invasion as expected from its biological function (Table 1), contrasting with the more restricted strain specificity of R1 (Fig. 5, Table S2) [14].

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Figure 5. Highly potent cross-strain inhibition of red blood cell invasion of PfRON2sp1.

Comparison of PfRON2sp1 and R1 peptides (concentrations 0.2 to 20 µM) in inhibiting red blood cell invasion by P. falciparum 3D7 or HB3 highlights the higher inhibitory efficiency and cross-strain reactivity of PfRON2sp1. 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.

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The PfAMA1 3D7-R1 crystal structure shows that three polymorphic residues (175, 224 and 225) contact R1-major (Table S2). The 224 polymorphism, Met/Leu, is conservative and since contacts are formed by the main chain only, this should not affect R1 specificity. The 3D7 and D10 antigens both carry Tyr175 and Ile225; for the W2mef and HB3 antigens, residue 175 is Tyr and Asp, respectively, and residue 225 is Asn in both. Thus, polymorphisms at positions 225 and possibly 175 appear to be determinant for the 3D7 specificity of R1 at the major peptide-binding site (Table S2A). R1-minor contacts polymorphic residue 230, which is Lys in all strains studied (Table S2B). As our data suggest a weak affinity for this binding site, however, it is unlikely that this polymorphism has a significant effect on the specificity for R1. We examined these polymorphisms further using the mutant PfAMA1 Dico3 [23], which differs only at residue 175 for the 3D7-contacting residues (Table S2A), and a 3D7 mutant with the substitution Ile225Asp, which we call 3D7mut. The equilibrium KD, determined from the SPR steady-state responses to R1 binding, was 15.2±1.9 µM for 3D7mut and 22.3±3.3 µM for Dico3, showing a reduction in affinity of over 200-fold with respect to the native 3D7 antigen (Fig. 6, Table S2C). This affinity is comparable to that observed for HB3 and W2mef [13] (recapitulated in Table S2), and confirms that both Tyr175 and Ile225 are important for the strain-specific recognition of R1. Tyr175, located at the tip of a flexible DI loop that is solvent-exposed in the apo antigen [18], becomes buried by R1-major and forms a hydrogen bond to this ligand via the phenol group. Ile225 is also buried by R1-major, forming a pair of hydrogen bond via its main chain to the R1-major main chain.

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Figure 6. Surface Plasmon Resonance studies of peptide R1 binding to PfAMA1 mutants 3D7mut and Dico3.

(A). Left - sensorgrams, showing R1 (analyte) binding to PfAMA1 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 KD derived from the steady state binding curves is 15.2 µM for 3D7mut and 22.3 µM for Dico3.

doi:10.1371/journal.ppat.1002755.g006

Hot spots driving specific PfAMA1-PfRON2 complex formation

Guided by the similarities between the PfRON2sp and R1 co-structures, and the conservation of key contact residues (Fig. 7A), we probed the functional importance of a subset of PfRON2 residues by testing the binding to BHK-21 cells expressing PfAMA1 of GST-PfRON2-5 fusion proteins carrying single alanine mutations at: Pro2033 (aligns structurally with Pro7 of peptide R1, which was shown to be critical for binding [24]), Phe2038 (interacts with invariant residue Phe183 in the hydrophobic groove and aligns structurally with Phe12 of R1), Arg2041 (extensive contacts with PfAMA1 and structurally equivalent to Arg-P15 of R1) and Pro2044 (the peptide bond Ser2043-Pro2044 is cis and is thus important for the β-hairpin conformation). Consistent with the structure, mutation of Arg2041 to Ala abrogated binding to PfAMA1 (Fig. 7B). Similar effects were observed with Pro2044, Phe2038 and Pro2033 mutations, the latter also shown to be a key residue in the TgAMA1-TgRON2 interaction [22].

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Figure 7. Mutations of PfAMA1 and PfRON2-5 reveal residues critical for high affinity interaction.

(A) Interface between PfAMA1 and PfRON2sp1 shown in open-book presentation. Residues of both components that were mutated are labeled. (B). Binding characteristics of recombinant GST-PfRON2-5 mutants to dissect hot-spot residues in PfRON2. PfAMA1-expressing BHK-21 cells were incubated with 10 µg/ml of PfRON2 or mutated proteins (GST-fusion proteins), washed and the binding of recombinant PfRON2 fragment was revealed with anti-GST antibody. PfAMA1 was detected with mAb F8.12.19, which recognizes extracellular Domain III. (C). Binding consequences of PfAMA1 mutations. Mutated versions of PfAMA1 were expressed on the surface of BHK-21 cells and incubated with wild-type PfRON2 recombinant proteins at 10 and 1 µg/ml.

doi:10.1371/journal.ppat.1002755.g007

Similarly, a subset of key PfAMA1 residues was also chosen for mutation: Phe183 (an invariant residue that contributes to the hydrophobic groove and that interacts with Phe2038 of PfRON2 via aromatic interactions), Asn223 (which makes important polar interactions with PfRON2), residue 225 (a polymorphic residue that contributes many contacts to PfRON2 in the structure both the CAMP (Asn225) and 3D7 (Ile225) complexes), Tyr234 (which makes polar contacts to Arg2041 of PfRON2) and Tyr251 (which has been suggested by previous studies to be important [12], [25]). A clear role for Phe183 in the PfAMA1-PfRON2 complex formation was evident when expressed on the surface of BHK-21 cells and tested for their ability to bind GST-PfRON2-5 fusion protein (Fig. 7C). A less pronounced role of Tyr234 was observed and none for the remaining residues, including Tyr251. Although these conclusions differ from those of others [12], [25], these results are consistent with the limited contacts shown by this residue in the structures and with our earlier findings on the TgAMA1-TgRON2 interaction, where the equivalent TgAMA1 residue, Tyr230, had a minimal effect on the binding.

Discussion

The structure of PfAMA1 in complex with the extracellular region of its receptor PfRON2 and the accompanying functional analysis reveal atomic details of the interaction between two key partners at the MJ. The binding site on PfAMA1 includes the hydrophobic groove and a region that becomes exposed by displacement of the flexible DII loop from its apo conformation. Comparison of residues from both components at the PfAMA1-PfRON2 interface with those of other apicomplexan homologs underscores the separate co-evolution of the receptor-ligand pair in members of the phylum.

The DII loop displays a strong propensity for mobility in P. falciparum [16], [18] and P. vivax AMA1 structures [19], particularly at its N- and C-terminal extremities (weak or absent electron density); the central region of the DII loop is more structured and stabilized by contacts with DI, and is better defined in some of these AMA1 structures. Here, we show that the DII loop is displaced by PfRON2sp, as well as by the R1 peptide. In T. gondii, the DII loop is 14 residues shorter than in the Plasmodium orthologs and appears less mobile [20] but nonetheless is readily displaced by TgRON2sp [22]. Flexibility may therefore have an important functional role: it protects a significant portion of the binding site in apo AMA1 against the host's immune response but can be readily displaced to extend the hydrophobic groove for effective binding to RON2. The anti-PfAMA1 invasion-inhibitory monoclonal antibody 4G2, which binds to the N- and C-termini of the DII loop [19], probably prevents its displacement for effective binding to PfRON2. The absence of polymorphisms in the DII loop in spite of immune targeting of this region underlines its important functional role [21].

We have previously demonstrated an evolutionary constraint on the AMA1–RON2 interaction within apicomplexan parasites [11]. Our functional analysis of the TgAMA1-TgRON2sp co-structure suggested that the cystine loop initially anchors the receptor to the hydrophobic groove, causing expulsion of the DII loop to promote interaction throughout the entire binding site [22]. Comparison of the TgAMA1-TgRON2sp and PfAMA1-PfRON2sp co-structures reveals that the cystine loop, while conserved across the two genera, is the most divergent region within the RON2 (Fig. 8). The separate co-evolution of the AMA1-RON2 pair in Apicomplexa is clearly illustrated by the difference between the cystine loop conformations of PfRON2sp and TgRON2sp. In particular, this allows Arg2041 to access the specific PfAMA1 pocket (Fig. 8), where it participates in an intricate network of polar interactions. From mutagenesis, we have demonstrated a crucial role of Arg2041 in complex formation (Fig. 7B). Moreover, this region of the cystine loop also appears to play an influential role in species selectivity as superposition of PvAMA1 structure [19] onto PfAMA1-PfRON2sp shows that Arg2041 would be sterically hindered at the interface but Thr, the equivalent residue in PvRON2 from P. vivax, can be accommodated (Fig. 9A). This accounts for our prior observation that the original 67-residue segment of PfRON2 does not bind to PvAMA1 [11].

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Figure 8. The RON2 cystine loop governs specificity.

(A). Alignment of RON2 sequences truncated to correlate PfRON2sp1 with RON2 sequences from the following accession numbers: TgRON2 - TGME49_100100, NcRON2 - NCLIV_064620, PfRON2 - PF14_0495, PvRON2 - PVX_117880, PyRON2 – PY_06813, BbRON2 (BBOV_I001630). (B). Overlay of TgRON2sp (green; PDB ID 2Y8T) onto PfAMA1-PfRON2sp (blue-orange) shows that both peptides adopt a helix/coil/cystine loop/coil architecture in the AMA1 groove, with the highest divergence localized to the cystine loop (black arrow). (C). Electrostatic surface renderings of PfAMA1 (left) and TgAMA1 (right), with the secondary structure of the RON2 binding partner and residues defining the base of cystine loop shown, illustrates that both interactions are highly complementary, but highly genus specific.

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Figure 9. The Arg knob-in-hole interaction is critical for species selectivity and interaction with invasion inhibitory antibodies and peptides.

(A). Left - A cut-away surface of PfAMA1 (blue), reveals that Arg2041 of PfRON2sp1 (orange) integrates deeply into a well-defined pocket. Right - However, no analogous pocket is observed in PvAMA1 (grey; PDB ID 1W8K). (B). Peptides and antibodies known to be invasion inhibitory for P. falciparum occupy the key Arg binding site, as shown by orthogonal views of the PfAMA1-PfRON2sp1 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).

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An additional feature of the PfRON2sp cystine loop region is the presence of a cis peptide bond between Ser2043 and Pro2044; the Ser-Pro-Pro segment contributes negligible buried surface area but is important for maintaining the β-hairpin conformation for efficient complex formation. Sequence alignment reveals that the Pro duo (Pro2044–Pro2045) is preserved in all analyzed Plasmodium species (Fig. 8A) and is thus likely important for specific recognition of AMA1. We propose that it provides necessary internal structure at the tip of the cystine loop and places the disulfide bond in the proper orientation to brace the AMA1-RON2 interaction. The influential role of Pro2044 is confirmed by mutagenesis where substitution with Ala, which would disfavor the cis peptide bond, abrogates PfAMA1-PfRON2 binding (Fig. 7B). While T. gondii does not share the conserved proline pair, its cystine loop is two residues shorter (Fig. 8A), which mirrors the narrower groove of TgAMA1. Altogether, the overall U-shape architecture of RON2 in complex with AMA1 appears to be remarkably well maintained within apicomplexan parasites but specific features are clearly visible in the cystine loop of PfRON2 and TgRON2, highlighting how a receptor-ligand complex has evolved to maintain a common and crucial event in the biology of these parasites.

Although the PfAMA1-PfRON2 interface is highly conserved, five polymorphic residues of PfAMA1 contact the non-polymorphic PfRON2sp [26]. Of these, however, only residue 225 (Asn/Ile) varies significantly. The remaining polymorphisms should not affect binding as they involve main chain contacts only (residues 172, 174, 187 and 224). Our study allows a detailed structural assessment of polymorphism at residue 225 since complexes with PfAMA1 from the 3D7 (Ile225) and CAMP (Asn225) strains were determined. The 3D7 and CAMP orthologs both maintain two hydrogen bonds between the main chain of residue 225 and PfRON2 Thr2039. However, Ile225 presents a deep pocket to Arg2041 with apolar contacts formed between the aliphatic regions of these two side chains, while Asn225 presents a shallower pocket to Arg2041 with the Asn225 amide group stacking against the guanidyl group. Nonetheless, our binding studies by SPR show no significant difference in the affinity of these two PfAMA1 homologs for PfRON2sp2. Sequence variations at PfRON2-interacting positions, 172(Glu/Gly), 187(Glu/Asn) and 225 (Ile/Asn) are represented by the strains 3D7, CAMP, FVO and HB3 that we have analyzed by SPR; the very similar KD constants, ranging from approximately 10 to 20 nM, confirm that these exert little effect in the strength of the interaction.

Peptide R1 shows a more restricted specificity as it binds strongly to the cognate 3D7 and closely related D10 antigens but only weakly to orthologs that do not carry the same polymorphic amino acids at position 175 or 225 (Table S2). Tyr175 in PfAMA1 3D7 makes a hydrogen bond to the main chain of R1-major but, as this residue is located in a flexible loop with some freedom to adapt to the PfAMA1-R1 interface, it is unclear why the Asp175 polymorphism leads to reduced affinity. In the case of Ile225 of PfAMA1 3D7, the main chain forms two hydrogen bonds to the main chain of R1-major but the preference of R1 for the Ile225 polymorphism remains unexplained as it contrasts with PfRON2sp where main chain hydrogen bonds are also formed by both Ile225 (3D7) and Asn225 (CAMP) to the main chain of PfRON2. This emphasizes that specificity differences may present subtleties that are difficult to decipher. Here, the crystal structure of R1 in complex with the 3D7mut (Ile225Asn) and Dico3 (Tyr175Asp) mutants of PfAMA1 would provide invaluable insights into this question. Taken together, these results highlight that unlike the natural ligand PfRON2, R1, which was selected by phage display, is highly susceptible to polymorphisms.

R1 exhibits a close structural similarity to PfRON2, with the major/minor peptide pair displaying a similar boomerang form as PfRON2, binding to the same region of PfAMA1 and following the same general contour of the binding-site groove. Our structural data show that binding of R1-minor is dependent upon prior binding of R1-major as it lies above the latter in the binding groove and makes fewer contacts to PfAMA1. This, indeed, is consistent with the ITC measurements that show a stoichiometry of 1:1, indicating a weaker affinity for the minor peptide-binding site. R1-major is thus favored as the principle inhibitor of the interaction with PfRON2, but this does not preclude a contribution by the minor peptide-binding site at high peptide concentrations.

Therapeutic strategies aimed at inhibiting the interaction between PfAMA1 and PfRON2 should be very effective in treating malaria as they address a critical phase in the life cycle of the parasite and, importantly, should not be compromised by polymorphism since the PfAMA1-PfRON2 interface is highly conserved. Our results provide a structural basis for designing inhibitors against the most virulent malaria parasite. The PfRON2sp1 peptide used in this study has a very high affinity to PfAMA1 and is very efficient at inhibiting invasion. Moreover, in contrast to the less strongly binding peptide R1, PfRON2sp1 is not strain specific. Structural details of the PfAMA1-PfRON2 interaction offer the possibility to design molecules with the desired specific inhibitory properties by in silico screening and structural validation. The binding of PfRON2 Arg2041 to a specific pocket on PfAMA1 could be a critical target region. Indeed, the important role played by Arg-P15 at the PfAMA1-R1 interface closely mirrors the equivalent interaction in the PfAMA1-PfRON2sp complexes and, interestingly, the same pocket is occupied by Arg and Lys in PfAMA1 complexes with the invasion inhibitory antibodies IgNAR [17] and 1F9 [16], respectively (Fig. 9B). Phe2038 (corresponding to Phe-P12 in R1) is also a key residue, as its substitution by Ala affected binding. The importance of this sub-site is further highlighted by the concomitant loss in affinity when Phe183 (with which it interacts) was mutated in PfAMA1. Collectively, these data provide a firm basis for designing molecules with optimal inhibitory properties to treat malarial infection.

Materials and Methods

Recombinant protein production

(i) Baculovirus insect cell expression: A synthetic codon-optimized gene encoding DI and DII of PfAMA1 3D7 [27] (residues 104–438; numbering based on the initiation methionine, PF11_0344) (GenScript) was subcloned into a modified pAcGP67B vector (Pharmingen) for expression in insect cells using established protocols [20]. Final yield of recombinant protein was approximately 3 mg per L of culture.

(ii) P. pastoris expression: Synthetic genes were optimized for PfAMA1 coding of residues 97–442, from strains 3D7 (Genbank accession number U33274), CAMP (accession number M34552) and HB3 (accession number U33277). Potential N-glycosylation sites were mutated and genes were cloned EcoRI-KpnI in the pPicZalpha A vector (Invitrogen), resulting in an 11-residues sequence extension followed by myc-epitope and hexa-His tags at the C-terminus), expressed in P. pastoris, and purified as described [28]. Yield after purification was approximately 20 mg per L of culture. PfAMA1 FVO (residues 25–545, no tags, accession number AJ277646) was produced as described before [29]. The DiCo3 protein was modified compared to the published protein [23]; it includes the PfAMA1 FVO prodomain (amino acids 25–96) and one additional mutation to minimize proteolytic cleavage Lys376–>Arg (B. Faber, unpublished results). The PfAMA1 3D7mut (Ile225–>Asn, residues 25–545, no tags) mutant was generated by site-directed mutagenesis (Genscript) and produced in P. pastoris in a similar fashion to the native protein [29].

Peptide synthesis

A 39-residue peptide corresponding to residues 2021 to 2059 of PfRON2 (PfRON2sp1) was synthesized by Kinexus (Vancouver, Canada) and disulfide cyclized. Lyophilized PfRON2sp1 was solubilized in 100% DMSO and subsequently diluted in HBS (20 mM HEPES pH 7.5, 150 mM NaCl) for use in co-crystallization and functional studies. Peptides PfRON2sp2 (residues 2027 to 2054) and R1 were synthesized by PolyPeptide (Strasbourg, France) and solubilized in 3.5% DMSO for subsequent use.

Crystallization and data collection

Crystals of PfAMA1 3D7 PfRON2sp1 were grown in 30% PEG400, 100 mM Tris-HCl pH 8.5, 200 mM tri-sodium citrate dihydrate and the protein (5 mg/mL final concentration) incubated with PfRON2sp1 (1:2 molar excess). A crystal in cryoprotectant buffer was flash cooled at 100 K and diffraction data were collected on beamline 9-2 at SSRL (Stanford Synchrotron Radiation Laboratory, Stanford, US). Crystals of PfAMA1 CAMP PfRON2sp2 were obtained in 20% PEG 4000, 0.1 M Tris/HCl pH 8.6, 0.1 M sodium acetate and 20% isopropanol and the protein (6.4 mg/mL final concentration) incubated with PfRON2sp2 (1:5 molar excess). Diffraction data were collected from a crystal in cryoprotectant buffer at 100 K on beamline ID29 at European Synchrotron Radiation Facility (Grenoble, France). Crystals of PfAMA1 3D7 R1 were obtained in 15% PEG 4000, 0.1 M Tris/HCl pH 8.5, 0.1 M sodium acetate and 10% isopropanol and the protein (5.4 mg/mL final concentration) incubated with R1 (1:6 molar excess). Diffraction data were collected at 100 K on beamline PROXIMA 1 at SOLEIL (St. Aubin, France).

Data processing, structure solution and refinement

Diffraction data were processed using Imosflm [30] or XDS [31] and Scala [32] in the CCP4 suite of programs [33]. Crystallographic parameters and data collection statistics are given in Table 2. Initial phases were obtained by molecular replacement using PHASER [34] or AMoRe [35] with the unliganded PfAMA1 structure (PDB 1Z40). Tracing of the PfRON2 and R1 peptides, and addition of solvent molecules, was performed manually in COOT [36] and refinement was performed with Refmac5 [37] or autoBUSTER (Global Phasing Ltd, Cambridge, UK). A summary of refinement statistics is given in Table 3. All molecular representation figures were generated in the PyMOL Molecular Graphics System, version 1.2r3pre, Schrödinger, LLC. Coordinates and structure factors have been deposited in the Protein Data Bank with the following entry codes: PfAMA1-PfRON2sp1, 3ZWZ; PfAMA1-PfRON2sp2, 3SRI; PfAMA1-R1, 3SRJ.

Binding studies by SPR

SPR measurements were made with a Biacore 2000 instrument (Biacore AB). AMA1 proteins diluted in 10 mM sodium acetate pH 4.5 for 3D7, CAMP, HB3 and FVO strains, or pH 4.0 for 3D7mut and Dico3, were covalently immobilized by an amine-coupling procedure on CM5 sensor chips (GE Healthcare). The reference flow cell was prepared by the same procedure in absence of protein. Binding assays were performed at 25°C in PBS and 0.005% Tween 20 by injecting a series of peptide (PfRON2sp1 and PfRON2sp2 on 3D7, CAMP, HB3 and FVO, and R1 on 3D7mut and Dico3) concentrations at a constant flow rate of 5 µL/min. A heterologous peptide was used to verify the absence of non-specific binding. Peptide dissociation was realized by injecting the running buffer, and the surface was regenerated by injecting glycine/HCl pH 1.5 followed by SDS 0.05%. Control flow cell sensorgrams were subtracted from the ligand flow cell sensorgrams and averaged buffer injections were subtracted from analyte sensorgrams. For peptide R1, steady-state signals (Req) were obtained directly from the plateau region of the sensorgrams, while for PfRON2sp peptides, estimated values of Req were obtained by extrapolation from the experimental curves since the association phase did not reach a final equilibrium state. All calculations were made using the BIAevaluation 4.2 software (BIAcore AB). The saturation curves obtained by plotting Req versus the peptide concentration were fitted with a steady-state model to obtain the Rmax and the apparent equilibrium dissociation constants, KD. To normalize the response for the different ligands, these curves were reported as the percentage of bound sites (ratio Req/Rmax) versus the analyte concentration..

Isothermal calorimetry.

ITC measurements were made using a ITC200 calorimeter (MicroCal). PfAMA1 3D7 (P. pastoris) and peptide R1 were diluted in PBS to final concentrations of 0.6 µM and 55 µM, respectively. PfAMA1 3D7 (initial volume 200 µL) was titrated at 25°C by consecutive injections of the peptide R1 (2 µL aliquots at 3 min intervals). Raw data were normalized and corrected for the heat of dilution of R1 in PBS. Binding stoichiometry was determined by fitting the final data to a 1:1 interaction model using the Origin7 software (OriginLab).

P. falciparum cultures and invasion assays

The P. falciparum cell cultures and the invasion assays were performed as described previously [11]. Briefly, highly synchronized P. falciparum 3D7 and HB3 schizonts (1.5% hematocrit, 1.5% parasitemia) were incubated with R1 or PfRON2sp1 peptides. Blood smears were collected 16 hours post-invasion and used for ring-stage parasites counting. The results presented are representative of three independent experiments, each performed in triplicate.

Transient transfection experiments and cell binding assays

Cell binding assays using PfAMA1-expressing BHK-21 cells and recombinant GST-PfRON2-5 fusion proteins were performed as previously described [11]. Although not quantitative, this cell-binding assay truly reflects the interaction between AMA1 and RON2 as we carefully checked all the experimental steps as well as the image recording as described below. Transfections were carried out using Lipofectamine Reagent (Invitrogen) as instructed by the manufacturer with 3×105 BHK-21 cells grown on coverslips for 24 h in 6 well plates. Cells were grown for an additional 24 h post-transfection before subsequent analysis. Expression and correct folding of PfAMA1 (and the mutants) at the host cell surface was verified by IFA performed with or without permeabilisation, using antibodies either specific to the cytoplasmic tail (anti-myc tag) or specific to the extracellular ectodomain of PfAMA1 (mouse mAb F8.12.19 [38]). For binding assays, coverslips from a same transfection experiment were washed in HBSS (Invitrogen) before addition of recombinant PfRON2-5 wild type or mutants diluted in HBSS at 10, 1 or 0.1 µg/ml. Coverslips incubated with GST were systematically used as a control. After five washes in PBS to remove unbound protein, cells were fixed in 4% PAF and further processed for IFA as described above [11]. The binding characteristics of RON2 (anti-GST labelling) on the PfAMA1 mutant were only considered valid when its signal was identical to that of wild type PfAMA1. All other micrographs were obtained with a Zeiss Axiophot microscope equipped for epifluorescence. Adobe photoshop (Adobe Systems, Mountain View, CA) was used for image processing. Matching pairs of images were recorded with the same exposure time and processed identically.

The PfAMA1 and GST-PfRON2 mutated constructs were generated by site directed mutagenesis using Quickchange II XL protocol (Stratagene).

Supporting Information

Figure S1.

Detailed analysis of interactions at the PfAMA1-PfRON2sp1 interface. (A). Open-book surface representation of PfAMA1 (left) and PfRON2sp1 (right) showing the extensive involvement of residues from both molecules in forming a complex interface. Residues involved in hydrogen bonding are coloured blue, while residues contributing significant buried surface area (BSA>20 Å2 for PfAMA1, >5 Å2 for PfRON2sp1) are colored green. (B). Table of residues involved in hydrogen bonding at the PfAMA1- PfRON2sp1 interface (left) and residues contributing significant buried surface area (right), as calculated by PISA (http://www.ebi.ac.uk/msd-srv/prot_int/pi​start.html). Polymorphic residues of PfAMA1 are shown in blue.

doi:10.1371/journal.ppat.1002755.s001

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Figure S2.

Isothermal titration calorimetry of peptide R1 binding to Pf AMA1 3D7.

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Table S1.

Polar interactions and buried surface areas in the PfAMA1-R1 crystal structure. (A). Polar contacts between PfAMA1 3D7 and R1-major (column 1), and buried surface areas of individual residues of PfAMA1 3D7 (column 2) and R1-major (column 3). Salt bridges are indicated in bold. (B). Polar contacts between PfAMA1 3D7 and R1-minor (column 1), and buried surface areas of individual residues of PfAMA1 3D7 (column 2) and R1-minor (column 3). (C). Polar contacts between R1-major and R1-minor (column 1), and buried surface areas of individual residues of R1-major (column 2) and R1-minor (column 3). Polymorphic residues of PfAMA1 are shown in blue.

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Table S2.

Polymorphic residues of PfAMA1 contacting peptide R1. (A). Polymorphic residues contacting R1-major showing the sequence for strains analyzed using ELISA (*) [14], SPR (+) [13] and in this study using SPR (°). (B). Polymorphic residues contacting R1-minor, showing the sequence for strains as presented in (A). (C). Binding to PfAMA1, classified as strong (s) or weak (w) for the studies presented in (A) and (B).

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Table S3.

Primers used in this study.

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Acknowledgments

We thank Roberto Rodriguez-Garcia and Marjolein van der Eijk for technical assistance, Patrick England for helpful discussions and the staff of the European Synchrotron Radiation Facility (Grenoble, France) and SOLEIL (St. Aubin, France), for providing facilities for diffraction measurements and for assistance.

Author Contributions

Conceived and designed the experiments: ML MJB GAB. Performed the experiments: BVLN MLT MHL SL SH MR FAS MJB GAB. Analyzed the data: BVLN ML MHL MLT MJB GAB. Contributed reagents/materials/analysis tools: BWF. Wrote the paper: BVLN ML MLT MJB GAB.

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