Plasmodium falciparum causes malaria in humans with over 450,000 deaths annually. The asexual blood stage involves invasion of erythrocytes by merozoites, in which they grow and divide to release daughter merozoites, which in turn invade new erythrocytes perpetuating the cycle responsible for malaria. A key step in merozoite invasion is the essential binding of PfRh5/CyRPA/PfRipr complex to basigin, a step linked to the formation of a pore between merozoites and erythrocytes. We show CyRPA interacts directly with PfRh5. An invasion inhibitory monoclonal antibody to CyRPA blocks binding of CyRPA to PfRh5 and complex formation thus illuminating the molecular mechanism for inhibition of parasite growth. We determined the crystal structures of CyRPA alone and in complex with an antibody Fab fragment. CyRPA has a six-bladed β-propeller fold, and we identify the region that interacts with PfRh5.

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This functionally conserved epitope is a potential target for vaccines against P. Full-length mature CyRPA (residues 30–362 of the translated protein product) was expressed in insect cells and purified to homogeneity as evidenced by size-exclusion chromatography (SEC) and SDS-PAGE analysis. Whilst CyRPA has been shown to form a co-complex with PfRh5 and PfRipr (; ), it is not known with which of these two proteins it associates. To determine if the recombinant CyRPA can bind to PfRh5 , we combined the two proteins and showed by SEC they are capable of forming a stable 1:1 complex. In these experiments, PfRh5 was incubated with excess CyRPA and the stable CyRPA/PfRh5 complex eluted ahead of free CyRPA. CyRPA/PfRh5 complex formation was also confirmed by incubation of excess CyRPA with FLAG-tagged PfRh5 followed by immunoprecipitation with anti-FLAG antibodies.

In these experiments, CyRPA co-precipitated with PfRh5. Together with the SEC result, this finding suggests an association between CyRPA and PfRh5 within the PfRh5/CyRPA/PfRipr complex during merozoite invasion.

These experiments also indicate that our recombinant CyRPA is correctly folded and also functionally competent. ( A) Purified recombinant CyRPA was analysed by size exclusion chromatography and SDS-PAGE. ( B) Formation of the PfRh5/CyRPA complex was monitored by size exclusion chromatography, CyRPA formed a complex with Rh5 and the complex was eluted in the peak labelled as Rh5/CyRPA.

The chromatographic profiles are shown (top panel) and fractions eluted from the column (lower panel) were analysed by SDS-PAGE. ( C) Immunoprecipitation of FLAG-PfRh5 after incubation with CyRPA. Cy-unb, CyRPA alone; Cy-eluted, elution from anti-FLAG antibody beads of CyRPA alone; Rh5/Cy-unb, FLAG-PfRh5 and excess CyRPA incubated and unbound protein analysed; Rh5/Cy-elu, FLAG-PfRh5 and CyRPA eluted from anti-FLAG antibody beads. CyRPA-specific invasion inhibitory monoclonal antibodies block interaction of CyRPA with PfRh5. Monoclonal antibodies were raised to recombinant CyRPA and their ability to inhibit P. Falciparum growth was determined.

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Three monoclonal antibodies (5B12, 3D1 and 8A7) were identified that inhibited growth to approximately 70% at 2 mg/ml, whereas others (7A6 and 8B9) showed essentially no inhibition. Immunoblot analysis of CyRPA with the monoclonal antibodies showed those that blocked parasite growth reacted with a conformational epitope as they bound to non-reduced protein but not to reduced protein. In contrast, the antibodies that did not inhibit parasite growth reacted with both reduced and non-reduced CyRPA, consistent with them binding to a linear epitope. The three inhibitory anti-CyRPA monoclonal antibodies (5B12, 3D1 and 8A7) have a similar potency as the inhibitory anti-Ripr monoclonal antibody 5G6 in the growth inhibition assays. ( A) In vitro growth inhibition (GIA) assays were performed to assess the abilities of the monoclonal antibodies raised against recombinant CyRPA to block P. Falciparum parasite growth in human erythrocytes.

Assays were performed twice in triplicate and error bars denote SD of the mean of 6 values. Anti-Rh5 mAb 4G4 (not inhibitory) and anti-Ripr mAb 5G6 (inhibitory) are included as GIA controls. ( B) Immunoblot of inhibitory (8A7) and non-inhibitory (7A6) monoclonal antibodies against proteins from CyRPA-HA tagged transgenic P. Falciparum schizonts in reduced (R) and non-reduced (NR) condition. Falciparum parasites expressed haemagglutinin-tagged CyRPA protein. ( C) Monoclonal antibody 8A7 blocked binding of PfRh5 to CyRPA.

PfRh5 and CyRPA were incubated together with 8A7 and the complex formation monitored by size exclusion chromatography and SDS-PAGE analysis under non-reducing conditions. Monoclonal antibody 8A7 bound to CyRPA, preventing the PfRh5/CyRPA complex formation.

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CyRPA was co-eluted with 8A7 in the earlier fractions and the free Rh5 eluted in the later fractions. ( D) Monoclonal antibody 7A6 did not inhibit the formation of the PfRh5/CyRPA complex as monitored by size exclusion chromatography and SDS-PAGE analysis.

This antibody did not bind to native CyRPA indicating the linear epitope is not surface exposed. ( E) Non-inhibitory anti-PfRh5 monoclonal antibody 4G4 did not significantly inhibit the formation of the PfRh5/CyRPA complex. The PfRh5/CyRPA complex bound to antibody 4G4 and was eluted together with 4G4 as a tri-molecule complex in the earlier fractions.To determine the growth-inhibitory mechanism of the monoclonal antibody 8A7, we performed experiments to examine if the antibody blocked the interaction of CyRPA with PfRh5. Both proteins were incubated together in the presence of either inhibitory monoclonal antibody 8A7 or non-inhibitory monoclonal antibody 7A6. The mixtures were analysed by SEC and fractions visualized on SDS-PAGE and stained with Coomassie Blue R250. In the presence of inhibitory monoclonal antibody 8A7, the formation of the PfRh5/CyRPA complex is disrupted with PfRh5 eluted alone in the later fractions , and CyRPA eluted as a complex with 8A7 in the earlier fractions. However, in the presence of non-inhibitory monoclonal antibody 7A6, CyRPA remains bound to PfRh5.

The antibody 7A6 also did not bind to native CyRPA consistent with binding to a linear epitope in immunoblots that is not exposed on the surface of the protein. In addition, in the presence of non-inhibitory anti-PfRh5 monoclonal antibody 4G4 , the PfRh5/CyRPA complex remained largely intact and was eluted as a tri-molecular complex in the earlier fractions, with free CyRPA eluted in the later fractions. These results suggested that growth-inhibitory mechanism of the monoclonal antibody 8A7 is likely due to the disruption of PfRh5/CyRPA complex formation.

The crystal structures of CyRPA alone and in complex with the Fab fragment of invasion inhibitory monoclonal antibody 8A7. ( A) Orthogonal view of the ribbon representation of CyRPA, colored in rainbow fashion from the N-terminus ( blue) through to the C- terminus ( red).

( B) Amino acid sequence and the secondary structure of CyRPA, showing the location of the 24 canonical strands of the six-bladed β-propeller labelled ‘β ms n’ where the index n denotes the strand and the index m the sheet ( n = 1.,4; m = 1.,6). ( C) Assembly of the 222 pseudo-symmetric tetramer of CyRPA within the crystallographic asymmetric unit, showing the formation of the extended 5–5 and 6–6 sheets ( orange and red, respectively). Each monomer of CyRPA in the asymmetric unit is labelled a, b, c, d.Statistics for the highest-resolution shell are shown in parentheses.The CyRPA structure displays the canonical six-bladed β-propeller (6BBP) fold. Within the β-propeller structure are four intra-sheet disulfide bonds (Cys19-Cys35, Cys90-Cys92, Cys151-Cys169 and Cys229-Cys239) and one inter-sheet disulfide bond (Cys274-Cys298). The crystal of uncomplexed CyRPA contains four copies of the protein in the crystallographic asymmetric unit, arranged with approximate 222-point group symmetry.

Within this tetramer, a pairwise association of CyRPA molecules is mediated by the formation of two eight-strand anti-parallel β-sheets, one (termed sheet ‘6–6’) comprises two copies of the sixth β-sheet of the CyRPA monomer and the second (termed sheet ‘5–5’) comprises two copies of the (adjacent) fifth β-sheet of the CyRPA monomer. In each case, formation of the eight-stranded sheet is mediated by the respective outermost strands of the two constituent four-stranded sheets. These dimers of CyRPA monomers then associate with each other via the formation of a β-sandwich comprising sheet 6–6 of one CyRPA dimer and sheet 6–6 of the second CyRPA dimer. The monomer-monomer interfaces, although extensive, are most likely an artefact of crystallization, as we find no evidence for higher-order association upon SEC of CyRPA.

However, we cannot rule out such associations occurring within the hetero-complexes formed by CyRPA and its partner proteins on the surface of the merozoite.The 6BBP fold is found in a number of enzyme families, as well as in some attachment proteins. A DALI search reveals that the closest structural homologue to CyRPA is the attachment glycoprotein of a henipah virus isolated from Ghanaian bats (PDB entry 4UF7 ), with the next closest homologues being various sialidases of viral, bacterial and mammalian origin (; ). CyRPA has up to 16% sequence identity with all such structural homologues found by the DALI search, suggesting only distant evolutionary relationships, if any, with other thus-far structurally-characterized 6BBPs. Nevertheless, given that sialidases appear consistently as the highest-ranking homologues, it is instructive to consider whether CyRPA contains any specific sequence motifs that are characteristic of sialidases and, indeed, whether it has any sialidase activity. We compared the sequence of CyRPA with that of Clostridium perfringens NanI sialidase to identify potential conserved residues that might be relevant to sialidase enzyme activity. Localization of these residues on an overlay of structures revealed the absence of any potential active site.

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There are two signature motifs for the active site of all sialidases (; ). The first is a set of three conserved arginine residues that coordinate the binding of the carboxylate moiety of sialic acid (; ), these residues are located respectively in the loop between the first and second β-sheets, in the loop between the third and fourth strand of the fifth β-sheet and in the loop connecting the second and third strand in the fourth β-sheet. Inspection reveals only one possible equivalent, an arginine at residue 21 in the loop between the first and second β-sheets of CyRPA (.

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