Structure of malaria invasion protein RH5 with erythrocyte basigin and blocking antibodies (original) (raw)

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors are deposited at the Protein Data Bank with accession codes 4U0Q, 4U0R and 4U1G.

References

  1. Cowman, A. F. & Crabb, B. S. Invasion of red blood cells by malaria parasites. Cell 124, 755–766 (2006)
    CAS PubMed Google Scholar
  2. Tham, W. H., Healer, J. & Cowman, A. F. Erythrocyte and reticulocyte binding-like proteins of Plasmodium falciparum . Trends Parasitol. 28, 23–30 (2012)
    CAS PubMed Google Scholar
  3. 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)
    CAS PubMed Google Scholar
  4. Crosnier, C. et al. Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum . Nature 480, 534–537 (2011)
    ADS CAS PubMed PubMed Central Google Scholar
  5. Douglas, A. D. et al. Neutralization of Plasmodium falciparum merozoites by antibodies against PfRH5. J. Immunol. 192, 245–258 (2014)
    CAS PubMed Google Scholar
  6. 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)
    ADS Google Scholar
  7. 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)
    CAS PubMed PubMed Central Google Scholar
  8. 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)
    CAS PubMed PubMed Central Google Scholar
  9. 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)
    PubMed PubMed Central Google Scholar
  10. 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)
    CAS PubMed PubMed Central Google Scholar
  11. 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)
    ADS PubMed PubMed Central Google Scholar
  12. Manske, M. et al. Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing. Nature 487, 375–379 (2012)
    ADS CAS PubMed PubMed Central Google Scholar
  13. 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)
    CAS PubMed Google Scholar
  14. 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)
    ADS CAS PubMed PubMed Central Google Scholar
  15. Hayton, K. et al. Erythrocyte binding protein PfRH5 polymorphisms determine species-specific pathways of Plasmodium falciparum invasion. Cell Host Microbe 4, 40–51 (2008)
    CAS PubMed PubMed Central Google Scholar
  16. 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)
    CAS PubMed PubMed Central Google Scholar
  17. 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)
    ADS CAS PubMed PubMed Central Google Scholar
  18. 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)
    CAS PubMed PubMed Central Google Scholar
  19. 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)
    CAS PubMed Google Scholar
  20. 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)
    CAS PubMed PubMed Central Google Scholar
  21. 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)
    CAS PubMed PubMed Central Google Scholar
  22. 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)
    CAS PubMed PubMed Central Google Scholar
  23. Leslie, A. G. The integration of macromolecular diffraction data. Acta Crystallogr. D 62, 48–57 (2006)
    PubMed Google Scholar
  24. Evans, P. R. In Proceedings of the CCP4 Study Weekend (eds Sawyer, L., Isaacs, N. & Bailey, S. ) 114–122 (Daresbury Laboratory, Warrington, UK, 1993)
    Google Scholar
  25. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011)
    CAS PubMed PubMed Central Google Scholar
  26. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)
    CAS PubMed PubMed Central Google Scholar
  27. Zhang, K. Y., Cowtan, K. & Main, P. Combining constraints for electron-density modification. Methods Enzymol. 277, 53–64 (1997)
    CAS PubMed Google Scholar
  28. Cowtan, K. The Buccaneer software for automated model building. Acta Crystallogr. D 62, 1002–1011 (2006)
    PubMed Google Scholar
  29. Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D 67, 355–367 (2011)
    CAS PubMed PubMed Central Google Scholar
  30. Bricogne, G. et al. BUSTER version 2.10.0. (Global Phasing Ltd., Cambridge, UK, 2011)
  31. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)
    CAS PubMed PubMed Central Google Scholar
  32. 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)
    CAS Google Scholar
  33. Sievers, F. et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011)
    PubMed PubMed Central Google Scholar
  34. Gouet, P., Courcelle, E., Stuart, D. I. & Metoz, F. ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15, 305–308 (1999)
    CAS PubMed Google Scholar
  35. 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)
    CAS PubMed Google Scholar
  36. 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)
    CAS Google Scholar
  37. 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)
    CAS Google Scholar
  38. Guinier, A. & Fournet, G. Small-Angle Scattering of X-rays (Wiley, 1955)
    MATH Google Scholar
  39. Porod, G. General Theory Small Angle X-ray Scattering (Academic Press, 1982)
    Google Scholar
  40. 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)
    CAS PubMed PubMed Central Google Scholar
  41. 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)
    CAS Google Scholar
  42. Kozin, M. B. & Svergun, D. I. Automated matching of high- and low-resolution structural models. J. Appl. Crystallogr. 34, 33–41 (2001)
    CAS Google Scholar
  43. 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)
    CAS PubMed Google Scholar
  44. Schuck, P. Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling. Biophys. J. 78, 1606–1619 (2000)
    ADS CAS PubMed PubMed Central Google Scholar
  45. 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)
    CAS PubMed PubMed Central Google Scholar
  46. 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)
    Google Scholar
  47. 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)
    CAS PubMed Google Scholar

Download references

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).

Author information

Authors and Affiliations

  1. Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK,
    Katherine E. Wright, Jonathan Bartlett & Matthew K. Higgins
  2. Jenner Institute, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, UK ,
    Kathryn A. Hjerrild, Alexander D. Douglas, Jing Jin, Rebecca E. Brown, Joseph J. Illingworth, Rebecca Ashfield & Simon J. Draper
  3. ExpreS2ion Biotechnologies, SCION-DTU Science Park, Agern Allé 1, DK-2970 Horsholm, Denmark ,
    Stine B. Clemmensen & Willem A. de Jongh

Authors

  1. Katherine E. Wright
  2. Kathryn A. Hjerrild
  3. Jonathan Bartlett
  4. Alexander D. Douglas
  5. Jing Jin
  6. Rebecca E. Brown
  7. Joseph J. Illingworth
  8. Rebecca Ashfield
  9. Stine B. Clemmensen
  10. Willem A. de Jongh
  11. Simon J. Draper
  12. Matthew K. Higgins

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.

Corresponding authors

Correspondence toSimon J. Draper or Matthew K. Higgins.

Ethics declarations

Competing interests

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 (_R_g) 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 (_R_g) was determined from the Guinier plot using AutoRg37, and the maximum particle dimension (_D_max) 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 Table 1 Crystallographic data collection and refinement statistics

Full size table

Extended Data Table 2 Description of interactions between PfRH5 with basigin or monoclonal antibodies QA1 and 9AD4

Full size table

PowerPoint slides

Rights and permissions

About this article

Cite this article

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

Download citation