Fig 1.
RH5 from an ancient inter-clade introgression in Laverania parasites binds both human and gorilla basigin.
(A) Phylogenetic relationships and host tropisms of the parasites within the Laverania subgenus. The inter-clade introgression event encoding rh5 and cyrpa is marked with a yellow arrow. Hosts are indicated as chimpanzee (blue), gorilla (black), and human (red) icons. (B) Organization of the RH5 invasion complex. RH5 is tethered through its N terminus (RH5Nt) to the parasite surface via P113 and binds host basigin on the erythrocyte surface; CyRPA directly interacts with RH5, thereby recruiting RIPR to the complex. (C) Ancestral introgressed RH5 binds to gorilla and human, but not chimpanzee, basigin. Serial dilutions of the indicated concentrations of purified introgressed RH5 were injected over gorilla, human, and chimpanzee basigin immobilized on a sensor chip. Raw surface plasmon resonance (SPR) traces are shown in black and fitted to a simple 1:1 Langmuir binding isotherm (red) to derive the equilibrium dissociation constants (KDs). Underlying numerical data can be found in S1 Data. A representative experiment of three is shown. BSG, basigin; cyrpa, cysteine-rich protective antigen; RH5, reticulocyte-binding protein homologue 5; RH5Ct, RH5 C-terminal domain; RH5Nt, RH5 N-terminal domain; RIPR, RH5-interacting protein; SPR, surface plasmon resonance.
Fig 2.
P. praefalciparum and clade A Laverania RH5 does not exhibit strict host basigin binding specificity.
(A) The binding of Laverania RH5 proteins to human, gorilla, and chimpanzee basigin was quantified by SPR. Sensorgram data from serial dilutions of the RH5 proteins are shown in black and were fitted to a simple 1:1 Langmuir binding isotherm (red). Background shading of the graphs indicates where binding was expected based on the known parasite tropism (beige) or were unexpected (gray). (B) Binding specificities of Laverania parasite RH5 proteins with human basigin were confirmed using an RH5 cell binding assay. Avid RH5 binding probes were presented to basigin-expressing HEL cells, and binding specificity was demonstrated by showing that all RH5 probe binding was abolished if the cells were preincubated with an anti-basigin monoclonal antibody (mAb) that blocks RH5 binding (Ab1—blue histograms) compared with a cell-binding isotype-matched anti-CD59 mAb (red). Control is streptavidin-PE alone (gray). Summary numerical data are provided in S1 Data; gating strategy and original .fcs files in S2 Data. Representative results of at least three independent experiments are shown. Ab1, anti-basigin mAb; HEL, human erythroid-like; IntRH5, introgressed ancestral RH5; mAb, monoclonal antibody; PE, phycoerythrin; RH5, reticulocyte-binding protein homologue 5; SPR, surface plasmon resonance.
Fig 3.
RH5 complex interactions are conserved across the Laverania, including the introgressed RH5.
(A) Interactions between the introgressed (Int) and ancestral (Anc) Laverania RH5 complex components are conserved. Introgressed RH5 binds introgressed CyRPA and ancestral P113, and introgressed CyRPA binds ancestral RIPR, as shown by the AVEXIS assay using the named bait and prey proteins. Bars represent means ± SEM; n = 3. (B) Summary of the interactions within the Laverania RH5 complex. The orthologues of RH5, P113, and CyRPA from the named Laverania species were synthesized, expressed, and systematically tested for binding using the AVEXIS assay. The RH5-CyRPA (upper panel) and RH5-P113 (lower panel) interactions were conserved within (indicated by red boxes) and across all tested species, with the exception of P. billcollinsi RH5, which did not interact with P. adleri CyRPA. The scale graphically represents the normalized quantitative readout of the AVEXIS assay but is not expected to provide relative measures of interaction affinity; values above 0.4 are considered positive. Underlying numerical data can be found in S1 Data. A single representative of three experiments is shown. Anc, ancestral; AVEXIS, avidity-based extracellular interaction screening; CyRPA, cysteine-rich protective antigen; HsBSG, human basigin; Int, introgressed; RH5, reticulocyte-binding protein homologue 5; RIPR, RH5-interacting protein; -ve, negative control.
Fig 4.
A single amino acid in RH5 is responsible for human basigin binding specialization.
(A) Structural representation of the RH5–basigin complex, with five of the six residues that differ between the introgressed RH5 and P. falciparum RH5 highlighted in yellow. Note that residue 148 was not resolved in the RH5 structure. (B) Proteins corresponding to IntRH5 and the six mutations where the amino acids have been mutated to the corresponding residue in the P. falciparum RH5 sequence were expressed and purified and the binding to either human or gorilla basigin determined by SPR. Sensorgrams of four 2-fold serial dilutions (600 to 75 nM) are shown, with the raw data shown in black and the fits to a simple 1:1 binding model in red. Representative sensorgrams from two experiments are shown. All mutants bound human basigin with comparable affinity (top row), and one (H200Y) showed no detectable binding to gorilla basigin. (C) Calculated interaction half-lives (t1/2) of all mutants to human and gorilla basigin. Bars represent means ± SEM from at least three different analyte concentrations. Underlying numerical data can be found in S1 Data. IntRH5, introgressed ancestral RH5; RH5, reticulocyte-binding protein homologue 5; SPR, surface plasmon resonance.