Crystal Structure of Plasmodium knowlesi Apical Membrane Antigen 1 and Its Complex with an Invasion-Inhibitory Monoclonal Antibody

The malaria parasite Plasmodiumknowlesi, previously associated only with infection of macaques, is now known to infect humans as well and has become a significant public health problem in Southeast Asia. This species should therefore be targeted in vaccine and therapeutic strategies against human malaria. Apical Membrane Antigen 1 (AMA1), which plays a role in Plasmodium merozoite invasion of the erythrocyte, is currently being pursued in human vaccine trials against P. falciparum. Recent vaccine trials in macaques using the P. knowlesi orthologue PkAMA1 have shown that it protects against infection by this parasite species and thus should be developed for human vaccination as well. Here, we present the crystal structure of Domains 1 and 2 of the PkAMA1 ectodomain, and of its complex with the invasion-inhibitory monoclonal antibody R31C2. The Domain 2 (D2) loop, which is displaced upon binding the Rhoptry Neck Protein 2 (RON2) receptor, makes significant contacts with the antibody. R31C2 inhibits binding of the Rhoptry Neck Protein 2 (RON2) receptor by steric blocking of the hydrophobic groove and by preventing the displacement of the D2 loop which is essential for exposing the complete binding site on AMA1. R31C2 recognizes a non-polymorphic epitope and should thus be cross-strain reactive. PkAMA1 is much less polymorphic than the P. falciparum and P. vivax orthologues. Unlike these two latter species, there are no polymorphic sites close to the RON2-binding site of PkAMA1, suggesting that P. knowlesi has not developed a mechanism of immune escape from the host’s humoral response to AMA1

Structural and Functional Insights into the Malaria Parasite Moving Junction Complex

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

Les épitopes de l'enveloppe du virus de l'hépatite B: approche-structurale.

International audienceHepatitis B is a major public health problem. More than 300 million people are chronically infected by the virus. During infection very large quantities of complete virions and empty envelopes, consisting of spherical or filamentous lipoprotein particles, are present in the blood. DNA genome coding for envelopes is divided into three domains, preS1, preS2 and S. All available data suggest that the preS1 and preS2 products are exposed at the surface of the virions. These proteins are more immunogenic than S in terms of in vivo antibody response and the number of epitopes identified. The three dimensional mapping of antigenic sites of the HBV will provide important strategic information for vaccine development and identification of targets for immunorecognition or immunoregulation of the disease

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

Refinement statistics.

<p>Refinement statistics.</p

Refinement statistics.

<p>Refinement statistics.</p

Comparison of contacts between AMA1 and the RON2 peptide in the homo- and hetero-complexes.

<p>The number of interatomic contacts < 3.8 Å (ordinate) is given as a function of the RON2 sequence (abscissa; PvRON2/PfRON2). In the histogram, the PvAMA1-PvRON2sp1 complex is shown in blue, the PfAMA1(FVO)-PvRON2sp1 complex is in red and the PfAMA1(3D7)-PfRON2sp1 complex [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183198#pone.0183198.ref010" target="_blank">10</a>] is in black.</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

Crystallographic parameters and data collection statistics.

<p>Crystallographic parameters and data collection statistics.</p