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. 2015 Apr 17;10(4):e0123567.
doi: 10.1371/journal.pone.0123567. eCollection 2015.

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

Affiliations

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

Brigitte Vulliez-Le Normand et al. PLoS One. .

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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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Structure of domains 1 and 2 of PkAMA1 in the non-complexed form.
The structure of PkAMA1 (molecule B) is shown in ribbon representation with Domain 1 in green and Domain 2 in light brown. The Domain 2 loop is shown in red and the c-myc tail is shown in mauve. The N- and C-termini are indicated by N and C, respectively. A gap occurs within the D2 loop since the protein could not be traced from residues Gly328 to Ser332.
Fig 2
Fig 2. Electron density of the D2 loop.
The D2 loop is shown in stereo. (A) free PkAMA1 (molecule A). (B) PkAMA1-Fab R31C2 complex. The contour level is drawn at the r.m.s. of the weighted Fobs electron density.
Fig 3
Fig 3. Structure of the PkAMA1-Fab-R31C2 complex.
The structure of the complex is shown in ribbon representation. Domain 1 of PkAMA1 is shown in green and Domain 2 in light brown, with the Domain 2 loop in red. The light chain of Fab-R31C2 is shown in yellow (variable and constant domains labeled VL and CL, respectively) and the heavy chain is shown in light blue (variable and constant domains labeled VH and CH1, respectively).
Fig 4
Fig 4. The PkAMA1 epitope recognized by R31C2.
PkAMA1 is shown in surface representation with the epitope residues on Domain 1 in green and the epitope residues on the D2 loop in red. In addition, residues of both PkAMA1 and R31C2 that are involved in polar interactions are drawn in stick representation with hydrogen bonds indicated by dotted lines. The stick models of the PkAMA1 residues and their labels are green for Domain 1 and red for the D2 loop. Stick models for R31C2 are blue for VH residues and yellow for VL residues with residue labels in black. (The complete list of PkAMA1 and R31C2 residues in contact is given in Table 3, Table 4).
Fig 5
Fig 5. Germline sequences and somatic mutations of the R31C2 variable domains.
(A) Amino acid and nucleotide sequences of the R31C2 VL domain compared with the Vκ and Jκ germline genes. (B) Amino acid and nucleotide sequences of the R31C2 VH domain compared with the VH, D and JH germline genes.
Fig 6
Fig 6. 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.
Fig 7
Fig 7. Comparison of the D2 loop of PkAMA1 and PfAMA1.
(A) PkAMA1. (B) PfAMA1 (PDB entry 2Z8V). The two structures are viewed from equivalent orientations and are shown in surface representation with Domain 1 in green, Domain 2 in light brown, and the D2 loop in red. The D2 loop conformation of both orthologues is shown in ribbon representation and the N- and C-terminal residues are labelled.

References

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