Plasmodium falciparum causes the severe form of malaria that has high levels of mortality in humans. Blood-stage merozoites of P. falciparum invade erythrocytes, and this requires interactions between multiple ligands from the parasite and receptors in hosts. These interactions include the binding of the Rh5–CyRPA–Ripr complex with the erythrocyte receptor basigin1,2, which is an essential step for entry into human erythrocytes. Here we show that the Rh5–CyRPA–Ripr complex binds the erythrocyte cell line JK-1 significantly better than does Rh5 alone, and that this binding occurs through the insertion of Rh5 and Ripr into host membranes as a complex with high molecular weight. We report a cryo-electron microscopy structure of the Rh5–CyRPA–Ripr complex at subnanometre resolution, which reveals the organization of this essential invasion complex and the mode of interactions between members of the complex, and shows that CyRPA is a critical mediator of complex assembly. Our structure identifies blades 4–6 of the β-propeller of CyRPA as contact sites for Rh5 and Ripr. The limited contacts between Rh5–CyRPA and CyRPA–Ripr are consistent with the dissociation of Rh5 and Ripr from CyRPA for membrane insertion. A comparision of the crystal structure of Rh5–basigin with the cryo-electron microscopy structure of Rh5–CyRPA–Ripr suggests that Rh5 and Ripr are positioned parallel to the erythrocyte membrane before membrane insertion. This provides information on the function of this complex, and thereby provides insights into invasion by P. falciparum.
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We thank L. Chen, J. Thompson, E. Hanssen, A. Leis and P. de Fonseca for experimental assistance, and the Victorian Red Cross Blood Bank for blood. The data presented here were made possible through Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS. The research was directly supported by a National Health and Medical Research Council of Australia (NHMRC).
Nature thanks L. Miller, S. Scheres, A. Sharma and the other anonymous reviewer(s) for their contribution to the peer review of this work.
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Extended data figures and tables
Extended Data Fig. 1 Stoichiometry of the Rh5–CyRPA–Ripr complex, interaction with soluble or full-length basigin and JK-1 cells.
a, Chemical cross-linking of Rh5–CyRPA–Ripr complex by disuccinimidyl suberate (DSS), analysed on an SDS–PAGE. b, Negative-stain electron microscopy of purified Rh5–CyRPA–Ripr ternary complex, showing the elongated shape of the complex. c, Size-exclusion chromotography analysis of a mixture containing recombinant Rh5–CyRPA–Ripr complex and soluble basigin. Soluble basigin was eluted separately from the ternary complex, indicating no binding to basigin. d, Immuno-precipitation of Rh5–CyRPA (tagged with haemagglutinin (HA))–Ripr from parasite schizont-stage extract using anti-HA resins could not pull down soluble basigin. e, Native PAGE analysis of the size-exclusion chromotography (left) and anion-exchange chromatography (right) eluted fractions, showing the migration of the quaternary complex comprised Rh5–CyRPA–Ripr–full-length basigin, close to ~ 600 kDa. f, Western blot analysis of the ion-exchange chromatography elutions indicated the presence of full-length basigin in the complex. g, Size-exclusion chromotography analysis showed that Rh5 and Ripr are eluted separately from each other, which indicates that no complex is formed in the absence of CyRPA. h, Labelling of JK-1 and JK-1ΔBSG cells with anti-CD147 (basigin) and analysis using flow cytometry. i, Analysis of Rh5 (blue line), Rh5–CyRPA (red line) and Rh5–CyRPA–Ripr (green line) binding to JK-1 and JK-1ΔBSG (ΔBSG) cells. j, Differential solubilization showing that peripheral membrane protein (spectrin), integral membrane protein (glycophorin) and detergent-resistant membrane protein flotillin were localized in the sodium-bicarbonate-soluble, TX100-soluble and TX100-insoluble fractions, respectively. Experiments in a–j were repeated three times with biologically independent samples, and were reproducible.
a, Representative flow cytometry plots of unstained JK-1 cells and cells stained with anti-CD147-APC. Populations of cells were gated using forward and side scatter (top). Doublet exclusion was performed using FSC-A and FSC-H (middle). The voltage used for the APC channel (anti-CD147) was set using unstained cells, where the negative population was positioned < 103 (bottom). Experiments were repeated three times independently and were reproducible. b, Representative flow cytometry plots of JK-1 and JK-1ΔBSG cells in the presence or absence of recombinant proteins. Cells were incubated with no protein, single recombinant protein, the binary Rh5–CyRPA complex or the ternary Rh5–CyRPA–Ripr complex, followed by the subsequent incubation of the respective primary and secondary antibodies as indicated. Experiments were repeated three times with biologically independent samples, and were reproducible.
Extended Data Fig. 3 The Rh5–CyRPA–Ripr complex does not stimulate Ca2+ flux across the erythrocyte membrane.
a, FACS kinetic plot of online stimulation of Fluo-4-loaded erythrocytes, stimulated with a dilution series of the Ca2+ ionophore A23187 (1 μM–0.031 μM). An equivolume of a twofold concentration of A23187 was added to erythrocytes loaded with Fluo-4 at 10 s, and fluorescence was monitored continuously for 1 min 30 s. An equivolume of a 2× concentration of Rh5 only or preassembled Rh5–CyRPA–Ripr complex was also added at 10 s after the start of acquisition. Rh5- and Rh5–CyRPA–Ripr complex-bound erythrocytes were then re-measured at 4 min and 6 min. At 7 min post-addition of Rh5–CyRPA–Ripr, Fluo-4-loaded erythrocytes were re-challenged with 1 μM of A23187. b, Kinetic plot of samples to which Rh5 alone or Rh5–CyRPA–Ripr complex was added, plotted only with stimulation with 0.031 μM A23187. c, Representation of mean fluorescence intensity values at 80 s, as above. Experiments were repeated three times with biologically independent samples, and were reproducible.
a, A micrograph, after drift correction and dose-weighting. b, Reference-free 2D class averages. c, 3D classification resulted in separation of the binary CyRPA–Ripr complex (left) and the ternary Rh5–CyRPA–Ripr complex (right). d, FSC curves indicating the overall resolutions of the ternary Rh5–CyRPA–Ripr (blue) and binary CyRPA–Ripr (red) reconstructions. e, FSC curves between the final refined Rh5–CyRPA–Ripr ternary model and full map, excluding an unbuilt region of Ripr density (black); between the model refined in half map 1 and the reconstruction from that same half (FSCwork, blue); and between the model refined in half map 1 and the reconstruction from half map 2 (FSCtest, red) for the Rh5–CyRPA–Ripr ternary complex. f, g, Local-resolution estimation colour spectrum of the ternary Rh5–CyRPA–Ripr map (f) and the binary CyRPA–Ripr map (g).
Extended Data Fig. 5 Cryo-EM densities of CyRPA in the binary complex and Rh5 in the ternary complex
a, Electron microscopy density showing the top view of the CyRPA β-propeller (left), and a cross-section of the same region showing the resolution of the 6-bladed β-sheets of CyRPA (right). b, Density of β-strands resolved in blades 1, 3 and 6 of the CyRPA β-propeller. c, Electron microscopy densities showing the individual α-helices (α2–α7) of Rh5. d, Model showing α7 helix of Rh5 inserted into the central cavity of CyRPA. e, Hydrophobic residues (L393, L397, F494 and I498) form a groove of Rh5, in contact with aromatic residues (Y185, F187 and F226) presented by B4 and B4–B5 loops of CyRPA. f, Models showing the hydrophobic groove of Rh5 and the binding aromatic residues of CyRPA (Y185, F187 and F226), shown in orange. g, Density maps and the refined atomic models showing the disordered B5 loop in CyRPA upon binding of Rh5 (left), the corresponding region showing the ordered B5 loop in CyRPA in the absence of Rh5 (middle) and the two superimposed blade 5 β-sheets of CyRPA in the absence (blue) and presence (red) of Rh5 (right). h, Tandem mass spectra of DSS cross-linked peptides identified from tryptic digestion of gel-purified Rh5–CyRPA–Ripr complex. High-resolution spectra from Q-Exactive mass spectrometer for two cross-linked peptides between Rh5(520–526) and CyRPA(37–50). Experiments were repeated three times with biologically independent samples, and were reproducible.
Extended Data Fig. 6 Electron microscopy density map of Ripr in the ternary complex, and orientation of the Rh5–CyRPA–Ripr complex on the erythrocyte membrane.
a, Density map corresponding to Ripr (yellow) and CyRPA (red), showing several β-sheets in Ripr. b, Secondary structure prediction using the Phyre2 server indicated that two putative high-confidence α-helices (in dashed rectangles) reside in the N terminus of Ripr23. c, The cryo-EM structure of Rh5–CyRPA–Ripr overlaid with the crystal structure of the Rh5–basigin (BSG) complex. The overlaid structures suggest the Rh5–CyRPA–Ripr complex is positioned parallel to the erythrocyte membrane before insertion. The crystal structures of CyRPA–C12, CyRPA–8A7 and Rh5–9AD4 antigen–antibodies complexes were also overlaid with the Rh5–CyRPA–Ripr cryo-EM structure. The overlaid structures suggest these monoclonal antibodies function to inhibit the docking of the invasion complex to the erythrocyte membrane. d, The crystal structure of the N-terminal domain of SipB is superimposed with the C-terminal helical bundles of Rh5. Over 144 residues of SipB were aligned with Rh5 C terminus, with a root mean square deviation of 3.4 Å. e, The Rh5 C-terminal helical bundle containing the α4–α7 helices is shown in cartoon and surface representation. Hydrophobic residues lining one side of the helical bundle are coloured in red, whereas hydrophilic residues lining the opposite side of the helical bundle are coloured in blue.
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Wong, W., Huang, R., Menant, S. et al. Structure of Plasmodium falciparum Rh5–CyRPA–Ripr invasion complex. Nature 565, 118–121 (2019). https://doi.org/10.1038/s41586-018-0779-6
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