Abstract
Invasion of host erythrocytes is essential to the life cycle of Plasmodium parasites and development of the pathology of malaria. The stages of erythrocyte invasion, including initial contact, apical reorientation, junction formation, and active invagination, are directed by coordinated release of specialized apical organelles and their parasite protein contents1. Among these proteins, and central to invasion by all species, are two parasite protein families, the reticulocyte-binding protein homologue (RH) and erythrocyte-binding like proteins, which mediate host–parasite interactions2. RH5 from Plasmodium falciparum (PfRH5) is the only member of either family demonstrated to be necessary for erythrocyte invasion in all tested strains, through its interaction with the erythrocyte surface protein basigin (also known as CD147 and EMMPRIN)3,4. Antibodies targeting PfRH5 or basigin efficiently block parasite invasion in vitro4,5,6,7,8,9, making PfRH5 an excellent vaccine candidate. Here we present crystal structures of PfRH5 in complex with basigin and two distinct inhibitory antibodies. PfRH5 adopts a novel fold in which two three-helical bundles come together in a kite-like architecture, presenting binding sites for basigin and inhibitory antibodies at one tip. This provides the first structural insight into erythrocyte binding by the Plasmodium RH protein family and identifies novel inhibitory epitopes to guide design of a new generation of vaccines against the blood-stage parasite.
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References
Cowman, A. F. & Crabb, B. S. Invasion of red blood cells by malaria parasites. Cell 124, 755–766 (2006)
Tham, W. H., Healer, J. & Cowman, A. F. Erythrocyte and reticulocyte binding-like proteins of Plasmodium falciparum . Trends Parasitol. 28, 23–30 (2012)
Baum, J. et al. Reticulocyte-binding protein homologue 5 - an essential adhesin involved in invasion of human erythrocytes by Plasmodium falciparum . Int. J. Parasitol. 39, 371–380 (2009)
Crosnier, C. et al. Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum . Nature 480, 534–537 (2011)
Douglas, A. D. et al. Neutralization of Plasmodium falciparum merozoites by antibodies against PfRH5. J. Immunol. 192, 245–258 (2014)
Douglas, A. D. et al. The blood-stage malaria antigen PfRH5 is susceptible to vaccine-inducible cross-strain neutralizing antibody. Nature Commun. 2, 601 (2011)
Williams, A. R. et al. Enhancing blockade of Plasmodium falciparum erythrocyte invasion: assessing combinations of antibodies against PfRH5 and other merozoite antigens. PLoS Pathog. 8, e1002991 (2012)
Bustamante, L. Y. et al. A full-length recombinant Plasmodium falciparum PfRH5 protein induces inhibitory antibodies that are effective across common PfRH5 genetic variants. Vaccine 31, 373–379 (2013)
Reddy, K. S. et al. Bacterially expressed full-length recombinant Plasmodium falciparum RH5 protein binds erythrocytes and elicits potent strain-transcending parasite-neutralizing antibodies. Infect. Immun. 82, 152–164 (2014)
Chen, L. et al. An EGF-like protein forms a complex with PfRh5 and is required for invasion of human erythrocytes by Plasmodium falciparum . PLoS Pathog. 7, e1002199 (2011)
Rodriguez, M., Lustigman, S., Montero, E., Oksov, Y. & Lobo, C. A. PfRH5: a novel reticulocyte-binding family homolog of Plasmodium falciparum that binds to the erythrocyte, and an investigation of its receptor. PLoS ONE 3, e3300 (2008)
Manske, M. et al. Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing. Nature 487, 375–379 (2012)
Tran, T. M. et al. Naturally acquired antibodies specific for Plasmodium falciparum reticulocyte-binding protein homologue 5 inhibit parasite growth and predict protection from malaria. J. Infect. Dis. 209, 789–798 (2014)
Tham, W. H. et al. Complement receptor 1 is the host erythrocyte receptor for Plasmodium falciparum PfRh4 invasion ligand. Proc. Natl Acad. Sci. USA 107, 17327–17332 (2010)
Hayton, K. et al. Erythrocyte binding protein PfRH5 polymorphisms determine species-specific pathways of Plasmodium falciparum invasion. Cell Host Microbe 4, 40–51 (2008)
Hayton, K. et al. Various PfRH5 polymorphisms can support Plasmodium falciparum invasion into the erythrocytes of owl monkeys and rats. Mol. Biochem. Parasitol. 187, 103–110 (2013)
Wanaguru, M., Liu, W., Hahn, B. H., Rayner, J. C. & Wright, G. J. RH5-Basigin interaction plays a major role in the host tropism of Plasmodium falciparum . Proc. Natl Acad. Sci. USA 110, 20735–20740 (2013)
Higgins, M. K. & Carrington, M. Sequence variation and structural conservation allows development of novel function and immune evasion in parasite surface protein families. Protein Sci. 23, 354–365 (2014)
Hirose, S., Shimizu, K., Kanai, S., Kuroda, Y. & Noguchi, T. POODLE-L: a two-level SVM prediction system for reliably predicting long disordered regions. Bioinformatics 23, 2046–2053 (2007)
Miura, K. et al. Anti-apical-membrane-antigen-1 antibody is more effective than anti-42-kilodalton-merozoite-surface-protein-1 antibody in inhibiting plasmodium falciparum growth, as determined by the in vitro growth inhibition assay. Clin. Vaccine Immunol. 16, 963–968 (2009)
Sheehy, S. H. et al. Phase Ia clinical evaluation of the Plasmodium falciparum blood-stage antigen MSP1 in ChAd63 and MVA vaccine vectors. Mol. Ther. 19, 2269–2276 (2011)
Battye, T. G., Kontogiannis, L., Johnson, O., Powell, H. R. & Leslie, A. G. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr. D 67, 271–281 (2011)
Leslie, A. G. The integration of macromolecular diffraction data. Acta Crystallogr. D 62, 48–57 (2006)
Evans, P. R. In Proceedings of the CCP4 Study Weekend (eds Sawyer, L., Isaacs, N. & Bailey, S. ) 114–122 (Daresbury Laboratory, Warrington, UK, 1993)
Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011)
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)
Zhang, K. Y., Cowtan, K. & Main, P. Combining constraints for electron-density modification. Methods Enzymol. 277, 53–64 (1997)
Cowtan, K. The Buccaneer software for automated model building. Acta Crystallogr. D 62, 1002–1011 (2006)
Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D 67, 355–367 (2011)
Bricogne, G. et al. BUSTER version 2.10.0. (Global Phasing Ltd., Cambridge, UK, 2011)
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)
Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. PROCHECK - a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993)
Sievers, F. et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011)
Gouet, P., Courcelle, E., Stuart, D. I. & Metoz, F. ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15, 305–308 (1999)
Kelley, L. A. & Sternberg, M. J. E. Protein structure prediction on the Web: a case study using the Phyre server. Nature Protocols 4, 363–371 (2009)
Konarev, P. V., Volkov, V. V., Sokolova, A. V., Koch, M. H. J. & Svergun, D. I. PRIMUS: a Windows PC-based system for small-angle scattering data analysis. J. Appl. Crystallogr. 36, 1277–1282 (2003)
Petoukhov, M. V., Konarev, P. V., Kikhney, A. G. & Svergun, D. I. ATSAS 2.1 - towards automated and web-supported small-angle scattering data analysis. J. Appl. Crystallogr. 40, s223–s228 (2007)
Guinier, A. & Fournet, G. Small-Angle Scattering of X-rays (Wiley, 1955)
Porod, G. General Theory Small Angle X-ray Scattering (Academic Press, 1982)
Franke, D. & Svergun, D. I. DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering. J. Appl. Crystallogr. 42, 342–346 (2009)
Konarev, P. V., Petoukhov, M. V., Volkov, V. V. & Svergun, D. I. ATSAS 2.1, a program package for small-angle scattering data analysis. J. Appl. Crystallogr. 39, 277–286 (2006)
Kozin, M. B. & Svergun, D. I. Automated matching of high- and low-resolution structural models. J. Appl. Crystallogr. 34, 33–41 (2001)
Birmanns, S., Rusu, M. & Wriggers, W. Using Sculptor and Situs for simultaneous assembly of atomic components into low-resolution shapes. J. Struct. Biol. 173, 428–435 (2011)
Schuck, P. Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling. Biophys. J. 78, 1606–1619 (2000)
Schuck, P., Perugini, M. A., Gonzales, N. R., Howlett, G. J. & Schubert, D. Size-distribution analysis of proteins by analytical ultracentrifugation: Strategies and application to model systems. Biophys. J. 82, 1096–1111 (2002)
Laue, T. M., Shah, B. D., Ridgeway, T. M. & Pelletier, S. L. in Analytical Ultracentrifugation in Biochemistry and Polymer Science (eds Harding, S. & Rowe, A. ) 90–125 (Royal Soc. Chemistry, Cambridge, UK, 1992)
Vistica, J. et al. Sedimentation equilibrium analysis of protein interactions with global implicit mass conservation constraints and systematic noise decomposition. Anal. Biochem. 326, 234–256 (2004)
Acknowledgements
M.K.H. is a Wellcome Trust Investigator (101020/Z/13/Z). K.E.W. is funded by a Wellcome Trust PhD studentship. S.J.D. holds a UK Medical Research Council (MRC) Career Development Fellowship (G1000527), and is a Jenner Investigator and Lister Institute Research Prize Fellow. The project was also funded by the European Vaccine Initiative (EVI) (InnoMalVac); the UK MRC (MR/K025554/1); the European Community’s Seventh Framework Programme (FP7/2007-2013, grant agreement number 242095 – EVIMalaR); and a Wellcome Trust Training Fellowship (089455/2/09/z to ADD). We thank J. Furze and D. Alanine; D. Staunton and E. Lowe; A. Round (ESRF); and R. Flaig and J. Brandao-Neto (Diamond Light Source).
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Contributions
K.E.W. purified and crystallized the proteins, collected and analysed SAXS data, and performed surface plasmon resonance and analytical ultracentrifugation analysis. M.K.H. and K.E.W. prepared crystals for data collection and solved the structures. W.A.J. and S.B.C. made S2 cell lines, and K.A.H. and J.J.I. purified proteins. A.D.D. and J.B. provided hybridomas. J.J., R.E.B. and R.A. designed and performed parasite assays and ELISAs. K.E.W., M.K.H. and S.J.D. designed the project, analysed the data, and wrote the paper.
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A.D.D., J.J.I., K.E.W., M.K.H. and S.J.D. are named on patent applications relating to PfRH5 and/or other malaria vaccines. W.A.J. is named on patents relating to Drosophila S2 protein expression and is co-founder of ExpreS2ion Biotechnologies.
Extended data figures and tables
Extended Data Figure 1 PfRH5 disorder predictions and structural alignment.
a, Long-range disorder was predicted by POODLE-L19 and was used to determine domain boundaries for the PfRH5ΔNL crystallization construct. The disorder predictions are shown above the sequence of PfRH5, with values of >0.5 indicative of disorder. The residues visible in the PfRH5ΔNL crystal structure are shown below the PfRH5 sequence as secondary structure elements (sheets as blue arrows, and helices as tubes in rainbow colouring) linked by blue lines. The missing loop (248–296) is shown as a break in the blue line. The secretion signal sequence is indicated (black underline). b, Two copies of PfRH5 from the PfRH5–basigin structure (red and orange), two copies from the PfRH5–QA1 structure (blue and cyan), and one copy from PfRH5–9AD4 (green) structure were aligned using Coot31, giving an r.m.s.d. of 1.7 Å. The C terminus and the loop between helices 4 and 5 were the only regions showing significant differences. For the remaining 95% of PfRH5, the r.m.s.d. is 0.9 Å.
Extended Data Figure 2 Investigation of the interaction of PfRH5ΔNL with a panel of mouse monoclonal antibodies using ELISA.
Five monoclonal antibodies that bind to PfRH5 were coated on an ELISA plate and probed using PfRH5ΔNL at concentrations of 12.5, 50, 200 or 800 ng ml−1. Antibodies 9AD4, QA1 and QA5 interacted with PfRH5ΔNL while RB3 and 4BA7 did not. Indeed, RB3 and 4BA7 bind to the flexible N terminus and the truncated loop, respectively, both features lacking in PfRH5ΔNL5. The error bars are standard error of mean (n = 3).
Extended Data Figure 3 SAXS analysis of the PfRH5–basigin complex.
a, The theoretical scattering calculated from the average of 20 ab initio reconstructions (continuous lines, with PfRH5 in orange and PfRH5–basigin in blue) plotted with the experimental scattering intensity curves (diamonds). The data are presented as the natural logarithm of the intensity. Guinier plots are displayed in the inset. b, The distance distribution function, P(r), of PfRH5 (orange) and PfRH5–basigin (blue). c, To the left, the crystal structure of PfRH5ΔNL (yellow) was docked into the average ab initio SAXS envelope of full-length PfRH5 (grey). Extra density corresponding to some or all of the truncated regions is visible at the bottom of the kite-like structure, near the C terminus. To the right, the crystal structure of PfRH5ΔNL–basigin is docked into the average ab initio SAXS envelope of full-length PfRH5–basigin (grey). PfRH5ΔNL is yellow. In dark blue and cyan are basigin molecules from the two PfRH5ΔNL–basigin complexes in the asymmetric unit, superimposed based on the structure of PfRH5ΔNL. d, Summary of SAXS parameters. The radius of gyration (Rg) was determined from the Guinier plot using AutoRg37, and the maximum particle dimension (Dmax) and the Porod volume39 were calculated using GNOM37. An estimate of the molecular weight was obtained by dividing the Porod volume by 1.7. Ab initio modelling was used to generate 20 shape reconstructions from the data. The normalized spatial discrepancy parameter (NSD) diagnoses the similarity of these models42. The models were averaged and the fit of the average model to the experimental data are indicated by the χ value.
Extended Data Figure 4 A conserved PfRH5-like fold in other Plasmodium RH proteins.
a, P. falciparum RH1, RH4, RH2b, RH2a and RH3 (a pseudogene); P. vivax RBP-1 and RBP-2; P. reichenowi RH5; and P. yoelii Py01365 were aligned using Clustal Omega33 and were threaded using the Phyre2 server35, giving more than 98% confidence of fold conservation over >260 residues in each case. The secondary structure of PfRH5 is shown below the sequence in a rainbow colour scheme as in Fig. 1a. Residues from PfRH5 that interact with basigin, QA1 and 9AD4 are indicated above the sequence by blue, red or green stars, respectively. Cysteine residues that make disulphide bonds are indicated by pink numbers, with residues sharing the same number forming a disulphide bond. b, PfRH5 is shown in yellow, with residues similar among RH proteins (from the alignment in a) highlighted as pink sticks. The majority of the similar residues appear to play a structural role stabilizing the architecture of the domain.
Extended Data Figure 5 Location of PfRH5 polymorphisms, and residues of PfRH5 and basigin implicated in host tropism.
a, b, Indicated are the locations of PfRH5 SNPs that are common (10% frequency or greater; red sticks) or uncommon (blue sticks) among 227 field isolates7,8,12, as well as additional SNPs observed in lab strains (green sticks)15,16. b, Basigin (blue) is shown in addition to PfRH5 (yellow). SNPs Y203, I204, N347, Y358 and E362 are localized in or near the PfRH5–basigin interface. Not visible in this orientation is lab strain polymorphism K429. c, Highlighted are basigin residues F27, Q100 and H102 (orange sticks). Mutation of F27 or Q100, or an insertion adjacent to H102, all change the affinity for PfRH517. Also shown are two SNPs of PfRH5, namely N347 and I204 (pink sticks), found in the PfRH5–basigin binding interface and linked to the strain’s ability to invade Aotus monkey erythrocytes15.
Extended Data Figure 6 Arrangement of two PfRH5–basigin complexes in the asymmetric unit of the crystal.
One complex, shown in yellow (PfRH5) and blue (basigin), interacts with the second, shown in silver (PfRH5) and cyan (basigin), primarily through packing between the two C-terminal domains of basigin. The two C termini of basigin are in close proximity (top view).
Extended Data Figure 7 Analysis of the PfRH5–basigin complex using analytical ultracentrifugation.
a, b, Sedimentation velocity analysis. The continuous sedimentation coefficient distributions that best fit the data are shown for basigin (top), full-length PfRH5 (middle), and a gel-filtered PfRH5–basigin complex (bottom). The inset shows the fitting residuals. c, d, Sedimentation equilibrium analysis. PfRH5ΔN (residues 140–526), basigin, and a gel-filtered PfRH5ΔN–basigin complex were analysed. The runs lasted 20 h at different speeds, as indicated in the inset legends. Ultraviolet absorbance was monitored at 280 nm. The residuals are shown below fitted data. The calculated molecular weights are consistent with the formation of a 1:1 complex between PfRH5ΔN and basigin.
Extended Data Figure 8 SAXS of PfRH5 in complex with growth-inhibitory Fab fragments.
a, The theoretical scattering calculated from the average of 20 ab initio reconstructions (continuous lines, with PfRH5 in orange, PfRH5–9AD4 in green, PfRH5–QA1 in red, and PfRH5–QA5 in blue) plotted with the experimental scattering intensity curves (black diamonds). The data are presented as the natural logarithm of the intensity. The Guinier plots are displayed in the inset. b, The distance distribution function, P(r), with colours as in a. c, The crystal structures of PfRH5ΔNL–QA1(left) and PfRH5ΔNL–9AD4 (middle) were docked into the corresponding full-length PfRH5–Fab envelopes (grey). PfRH5ΔNL is shown in yellow, QA1 in red, and 9AD4 in green. PfRH5ΔNL and a Fab fragment (cyan) were docked into the PfRH5–QA5 SAXS envelope to generate a model of the PfRH5–QA5 structure (right). d, A summary of SAXS parameters. The radius of gyration (Rg) was determined from the Guinier plot using AutoRg37, and the maximum particle dimension (Dmax) and the Porod volume39 were calculated using GNOM37. An estimate of the molecular weight was obtained by dividing the Porod volume by 1.7. Ab initio modelling was used to generate 20 shape reconstructions from the data. The normalized spatial discrepancy parameter (NSD) diagnoses the similarity of these models42. The models were averaged and the fit of the average model to the experimental data are indicated by the χ value.
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Wright, K., Hjerrild, K., Bartlett, J. et al. Structure of malaria invasion protein RH5 with erythrocyte basigin and blocking antibodies. Nature 515, 427–430 (2014). https://doi.org/10.1038/nature13715
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DOI: https://doi.org/10.1038/nature13715
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