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Crystal structure of Plasmodium knowlesi apical membrane antigen 1 and its complex with an invasion-inhibitory monoclonal antibody.

Vulliez-Le Normand B, Faber BW, Saul FA, van der Eijk M, Thomas AW, Singh B, Kocken CH, Bentley GA - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Unité d'Immunologie Structurale, Département de Biologie Structurale et Chimie, Paris, France; CNRS URA 2185, Paris, France.

ABSTRACT
The malaria parasite Plasmodium knowlesi, 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.

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Mechanism of receptor-binding inhibition by monoclonal antibody R31C2.(A) R31C2 blocks interaction of the RON2 receptor by occupying the hydrophobic groove and preventing movement of the D2 loop. PkAMA1 is shown in surface representation with Domain 1 residues lining the hydrophobic groove that are invariant or well conserved across species [18] shown in cyan and the D2 loop shown in red. The CDR residues of R31C2 are shown in ribbon representation; those of VH are blue and those of VL are yellow. (B) Structure of PfAMA1 complexed with the PfRON2 peptide (PDB entry 3ZWZ) [28]. PfAMA1 is shown in surface representation and in the same orientation as PkAMA1 in (A). Species-conserved residues of Domain 1 that line the hydrophobic groove are shown in cyan. The displaced D2 loop is not visible in this structure, probably due to high mobility. Binding of the PfRON2 peptide, shown here in orange as ribbon representation, requires displacement of the D2 loop to expose the complete receptor-bing site.
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pone.0123567.g006: Mechanism of receptor-binding inhibition by monoclonal antibody R31C2.(A) R31C2 blocks interaction of the RON2 receptor by occupying the hydrophobic groove and preventing movement of the D2 loop. PkAMA1 is shown in surface representation with Domain 1 residues lining the hydrophobic groove that are invariant or well conserved across species [18] shown in cyan and the D2 loop shown in red. The CDR residues of R31C2 are shown in ribbon representation; those of VH are blue and those of VL are yellow. (B) Structure of PfAMA1 complexed with the PfRON2 peptide (PDB entry 3ZWZ) [28]. PfAMA1 is shown in surface representation and in the same orientation as PkAMA1 in (A). Species-conserved residues of Domain 1 that line the hydrophobic groove are shown in cyan. The displaced D2 loop is not visible in this structure, probably due to high mobility. Binding of the PfRON2 peptide, shown here in orange as ribbon representation, requires displacement of the D2 loop to expose the complete receptor-bing site.

Mentions: The RON2-binding site on AMA1 comprises the hydrophobic groove and an adjacent region that becomes exposed after displacement of the D2 loop by the receptor [27, 28]. In the crystal structure of the PkAMA1-Fab R31C2 complex, the antibody binds not only to the hydrophobic groove but also to the D2 loop (Fig 6A). The D2 loop makes significant contacts with R31C2 (52 out of 142 interatomic contacts <3.8 Å, including nine hydrogen bonds); it nonetheless preserves the same conformation as found in the unbound PkAMA1. The RON2-binding site is thus inaccessible to the receptor in the presence of R31C2 because interactions with the mAb keep the D2 loop firmly in place as well as blocking access to a large fraction of the hydrophobic groove (Fig 6B). Structural analyses of two other complexes of AMA1 with invasion-inhibitory anti-PfAMA1 mAbs, 1F9 [21] and the IgNAR 14I [22], have shown that these also bind to the hydrophobic groove but their epitopes are displaced towards one end of the groove and do not include the D2 loop. By contrast, epitope mapping of the anti-PfAMA1 mAb 4G2, another extensively studied invasion-inhibitory antibody, shows that this antibody does not bind to the hydrophobic groove but rather to the base of the D2 loop [17, 49]. The mechanism of inhibition in this case is thus most likely indirect by preventing displacement of the D2 loop to fully expose the RON2-binding site. By targeting both the hydrophobic groove and the D2 loop, R31C2 is thus a very effective inhibitor of RBC invasion.


Crystal structure of Plasmodium knowlesi apical membrane antigen 1 and its complex with an invasion-inhibitory monoclonal antibody.

Vulliez-Le Normand B, Faber BW, Saul FA, van der Eijk M, Thomas AW, Singh B, Kocken CH, Bentley GA - PLoS ONE (2015)

Mechanism of receptor-binding inhibition by monoclonal antibody R31C2.(A) R31C2 blocks interaction of the RON2 receptor by occupying the hydrophobic groove and preventing movement of the D2 loop. PkAMA1 is shown in surface representation with Domain 1 residues lining the hydrophobic groove that are invariant or well conserved across species [18] shown in cyan and the D2 loop shown in red. The CDR residues of R31C2 are shown in ribbon representation; those of VH are blue and those of VL are yellow. (B) Structure of PfAMA1 complexed with the PfRON2 peptide (PDB entry 3ZWZ) [28]. PfAMA1 is shown in surface representation and in the same orientation as PkAMA1 in (A). Species-conserved residues of Domain 1 that line the hydrophobic groove are shown in cyan. The displaced D2 loop is not visible in this structure, probably due to high mobility. Binding of the PfRON2 peptide, shown here in orange as ribbon representation, requires displacement of the D2 loop to expose the complete receptor-bing site.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4401722&req=5

pone.0123567.g006: Mechanism of receptor-binding inhibition by monoclonal antibody R31C2.(A) R31C2 blocks interaction of the RON2 receptor by occupying the hydrophobic groove and preventing movement of the D2 loop. PkAMA1 is shown in surface representation with Domain 1 residues lining the hydrophobic groove that are invariant or well conserved across species [18] shown in cyan and the D2 loop shown in red. The CDR residues of R31C2 are shown in ribbon representation; those of VH are blue and those of VL are yellow. (B) Structure of PfAMA1 complexed with the PfRON2 peptide (PDB entry 3ZWZ) [28]. PfAMA1 is shown in surface representation and in the same orientation as PkAMA1 in (A). Species-conserved residues of Domain 1 that line the hydrophobic groove are shown in cyan. The displaced D2 loop is not visible in this structure, probably due to high mobility. Binding of the PfRON2 peptide, shown here in orange as ribbon representation, requires displacement of the D2 loop to expose the complete receptor-bing site.
Mentions: The RON2-binding site on AMA1 comprises the hydrophobic groove and an adjacent region that becomes exposed after displacement of the D2 loop by the receptor [27, 28]. In the crystal structure of the PkAMA1-Fab R31C2 complex, the antibody binds not only to the hydrophobic groove but also to the D2 loop (Fig 6A). The D2 loop makes significant contacts with R31C2 (52 out of 142 interatomic contacts <3.8 Å, including nine hydrogen bonds); it nonetheless preserves the same conformation as found in the unbound PkAMA1. The RON2-binding site is thus inaccessible to the receptor in the presence of R31C2 because interactions with the mAb keep the D2 loop firmly in place as well as blocking access to a large fraction of the hydrophobic groove (Fig 6B). Structural analyses of two other complexes of AMA1 with invasion-inhibitory anti-PfAMA1 mAbs, 1F9 [21] and the IgNAR 14I [22], have shown that these also bind to the hydrophobic groove but their epitopes are displaced towards one end of the groove and do not include the D2 loop. By contrast, epitope mapping of the anti-PfAMA1 mAb 4G2, another extensively studied invasion-inhibitory antibody, shows that this antibody does not bind to the hydrophobic groove but rather to the base of the D2 loop [17, 49]. The mechanism of inhibition in this case is thus most likely indirect by preventing displacement of the D2 loop to fully expose the RON2-binding site. By targeting both the hydrophobic groove and the D2 loop, R31C2 is thus a very effective inhibitor of RBC invasion.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Unité d'Immunologie Structurale, Département de Biologie Structurale et Chimie, Paris, France; CNRS URA 2185, Paris, France.

ABSTRACT
The malaria parasite Plasmodium knowlesi, 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.

Show MeSH
Related in: MedlinePlus