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Malaria and toxoplasmosis are infectious diseases caused by the apicomplexan parasites Plasmodium and Toxoplasma gondii , respectively. These parasites have developed an invasion mechanism involving the formation of a moving junction (MJ) that anchors the parasite to the host cell and forms a ring through which the parasite penetrates. The composition and the assembly of the MJ, and in particular the presence of protein AMA1 and its interaction with protein RON2 at the MJ, have been the subject of intense controversy. Here, using reverse genetics, we show that AMA1, a vaccine candidate, interacts with RON2 to maintain the MJ structural integrity in T. gondii and is subsequently required for parasite internalization. Moreover, we show that disruption of the AMA1 gene results in upregulation of AMA1 and RON2 homologues that cooperate to support residual invasion. Our study highlights a considerable complexity and molecular plasticity in the architecture of the MJ. Apicomplexan parasites such as Toxoplasma gondii and Plasmodium form a tight, moving junction with host cells before invading them. Here the authors show that the proteins AMA1 and RON2 of T. gondii cooperate during junction formation and identify additional proteins that have a role in this process.

An essential step in the invasion of red blood cells (RBCs) by Plasmodium falciparum (Pf) merozoites is the binding of rhoptry neck protein 2 (RON2) to the hydrophobic groove of apical membrane antigen 1 (AMA1), triggering junction formation between the apical end of the merozoite and the RBC surface to initiate invasion. Vaccination with AMA1 provided protection against homologous parasites in one of two phase 2 clinical trials; however, despite its ability to induce high-titer invasion-blocking antibodies in a controlled human challenge trial, the vaccine conferred little protection even against the homologous parasite. Here we provide evidence that immunization with an AMA1-RON2 peptide complex, but not with AMA1 alone, provided complete protection against a lethal Plasmodium yoelii challenge in mice. Significantly, IgG from mice immunized with the complex transferred protection. Furthermore, IgG from Pf AMA1-RON2–immunized animals showed enhanced invasion inhibition compared with IgG elicited by AMA1 alone. Interestingly, this qualitative increase in inhibitory activity appears to be related, at least in part, to a switch in the proportion of IgG specific for certain loop regions in AMA1 surrounding the binding site of RON2. Antibodies induced by the complex were not sufficient to block the FVO strain heterologous parasite, however, reinforcing the need to include multiallele AMA1 to cover polymorphisms. Our results suggest that AMA1 subunit vaccines may be highly effective when presented to the immune system as an invasion complex with RON2.

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.

Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa. The interaction of two well-studied proteins, Apical Membrane Antigen 1 (AMA1) and Rhoptry Neck protein 2 (RON2), has been shown to be critical for invasion by the asexual tachyzoite stage. Recently, two paralogues of these proteins, dubbed sporoAMA1 and sporoRON2 (or RON2L2), respectively, have been identified but not further characterized in proteomic and transcriptomic analyses of Toxoplasma sporozoites. Here, we show that sporoAMA1 and sporoRON2 localize to the apical region of sporozoites and that, in vitro, they interact specifically and exclusively, with no detectable interaction of sporoAMA1 with generic RON2 or sporoRON2 with generic AMA1. Structural studies of the interacting domains of sporoRON2 and sporoAMA1 indicate a novel pairing that is similar in overall form but distinct in detail from the previously described interaction of the generic pairing. Most notably, binding of sporoRON2 domain 3 to domains I/II of sporoAMA1 results in major alterations in the latter protein at the site of binding and allosterically in the membrane-proximal domain III of sporoAMA1 suggesting a possible role in signaling. Lastly, pretreatment of sporozoites with domain 3 of sporoRON2 substantially impedes their invasion into host cells while having no effect on tachyzoites, and vice versa for domain 3 of generic RON2 (which inhibits tachyzoite but not sporozoite invasion). These data indicate that sporozoites and tachyzoites each use a distinct pair of paralogous AMA1 and RON2 proteins for invasion into host cells, possibly due to the very different environment in which they each must function.

  [...]pathogens have evolved sophisticated molecular strategies to overcome the mechanical shear forces associated with resisting the flow of biological fluids; in particular, membrane anchored biomolecular complexes enable controlled deceleration, tight adhesion and, in the case of intracellular pathogens, penetration through the host cell membrane (Fig. 1A) [3-7]. Apicomplexans first secrete their own receptor, rhoptry neck protein 2--RON2, into the host cell, which is then engaged by their own surface ligand, apical membrane antigen 1--AMA1 [11-14]. [...]reminiscent of the self-reliant enteropathogenic Escherichia coli intimin-Tir system used to promote colonization of host cell surfaces [15] but unique among characterized eukaryotic invasion mechanisms, the apicomplexans provide both ligand and receptor to promote host cell invasion.

Apicomplexan parasites such as Toxoplasma gondii and Plasmodium species actively invade host cells through a moving junction (MJ) complex assembled at the parasite—host cell interface. MJ assembly is initiated by injection of parasite rhoptry neck proteins (RONs) into the host cell, where RON2 spans the membrane and functions as a receptor for apical membrane antigen 1 (AMA1) on the parasite. We have determined the structure of TgAMA1 complexed with a RON2 peptide at 1.95 angstrom resolution. A stepwise assembly mechanism results in an extensive buried surface area, enabling the MJ complex to resist the mechanical forces encountered during host cell invasion. Besides providing insights into host cell invasion by apicomplexan parasites, the structure offers a basis for designing therapeutics targeting these global pathogens.

Invasion of the red blood cell by Plasmodium falciparum parasites requires formation of an electron dense circumferential ring called the Moving Junction (MJ). The MJ is anchored by a high affinity complex of two parasite proteins: Apical Membrane Antigen 1 ( Pf AMA1) displayed on the surface of the parasite and Rhoptry Neck Protein 2 that is discharged from the parasite and imbedded in the membrane of the host cell. Structural studies of Pf AMA1 revealed a conserved hydrophobic groove localized to the apical surface that coordinates RON2 and invasion inhibitory peptides. In the present work, we employed computational and biophysical methods to identify competitive P. falciparum AMA1–RON2 inhibitors with the goal of exploring the ‘druggability’ of this attractive antimalarial target. A virtual screen followed by molecular docking with the Pf AMA1 crystal structure was performed using an eight million compound collection that included commercial molecules, the ChEMBL malaria library and approved drugs. The consensus approach resulted in the selection of inhibitor candidates. We also developed a fluorescence anisotropy assay using a modified inhibitory peptide to experimentally validate the ability of the selected compounds to inhibit the AMA1–RON2 interaction. Among those, we identified one compound that displayed significant inhibition. This study offers interesting clues to improve the throughput and reliability of screening for new drug leads.

Background S100 proteins are a family of small, EF-hand containing calcium-binding signaling proteins that are implicated in many cancers. While the majority of human S100 proteins share 25-65% sequence similarity, S100A7 and its recently identified paralog, S100A15, display 93% sequence identity. Intriguingly, however, S100A7 and S100A15 serve distinct roles in inflammatory skin disease; S100A7 signals through the receptor for advanced glycation products (RAGE) in a zinc-dependent manner, while S100A15 signals through a yet unidentified G-protein coupled receptor in a zinc-independent manner. Of the seven divergent residues that differentiate S100A7 and S100A15, four cluster in a zinc-binding region and the remaining three localize to a predicted receptor-binding surface. Results To investigate the structural and functional consequences of these divergent clusters, we report the X-ray crystal structures of S100A15 and S100A7D24G, a hybrid variant where the zinc ligand Asp24 of S100A7 has been substituted with the glycine of S100A15, to 1.7 Å and 1.6 Å resolution, respectively. Remarkably, despite replacement of the Asp ligand, zinc binding is retained at the S100A15 dimer interface with distorted tetrahedral geometry and a chloride ion serving as an exogenous fourth ligand. Zinc binding was confirmed using anomalous difference maps and solution binding studies that revealed similar affinities of zinc for S100A15 and S100A7. Additionally, the predicted receptor-binding surface on S100A7 is substantially more basic in S100A15 without incurring structural rearrangement. Conclusions Here we demonstrate that S100A15 retains the ability to coordinate zinc through incorporation of an exogenous ligand resulting in a unique zinc-binding site among S100 proteins. The altered surface chemistry between S100A7 and S100A15 that localizes to the predicted receptor binding site is likely responsible for the differential recognition of distinct protein targets. Collectively, these data provide novel insight into the structural and functional consequences of the divergent surfaces between S100A7 and S100A15 that may be exploited for targeted therapies.

[...]pathogens have evolved sophisticated molecular strategies to overcome the mechanical shear forces associated with resisting the flow of biological fluids; in particular, membrane anchored biomolecular complexes enable controlled deceleration, tight adhesion and, in the case of intracellular pathogens, penetration through the host cell membrane (Fig. 1A) [3-7]. Apicomplexans first secrete their own receptor, rhoptry neck protein 2--RON2, into the host cell, which is then engaged by their own surface ligand, apical membrane antigen 1--AMA1 [11-14]. [...]reminiscent of the self-reliant enteropathogenic Escherichia coli intimin-Tir system used to promote colonization of host cell surfaces [15] but unique among characterized eukaryotic invasion mechanisms, the apicomplexans provide both ligand and receptor to promote host cell invasion.

Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa. The interaction of two well-studied proteins, Apical Membrane Antigen 1 (AMA1) and Rhoptry Neck protein 2 (RON2), has been shown to be critical for invasion by the asexual tachyzoite stage. Recently, two paralogues of these proteins, dubbed sporoAMA1 and sporoRON2 (or RON2L2), respectively, have been identified but not further characterized in proteomic and transcriptomic analyses of Toxoplasma sporozoites. Here, we show that sporoAMA1 and sporoRON2 localize to the apical region of sporozoites and that, in vitro, they interact specifically and exclusively, with no detectable interaction of sporoAMA1 with generic RON2 or sporoRON2 with generic AMA1. Structural studies of the interacting domains of sporoRON2 and sporoAMA1 indicate a novel pairing that is similar in overall form but distinct in detail from the previously described interaction of the generic pairing. Most notably, binding of sporoRON2 domain 3 to domains I/II of sporoAMA1 results in major alterations in the latter protein at the site of binding and allosterically in the membrane-proximal domain III of sporoAMA1 suggesting a possible role in signaling. Lastly, pretreatment of sporozoites with domain 3 of sporoRON2 substantially impedes their invasion into host cells while having no effect on tachyzoites, and vice versa for domain 3 of generic RON2 (which inhibits tachyzoite but not sporozoite invasion). These data indicate that sporozoites and tachyzoites each use a distinct pair of paralogous AMA1 and RON2 proteins for invasion into host cells, possibly due to the very different environment in which they each must function.