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Crystal structure of PfRh5, an essential P. falciparum ligand for invasion of human erythrocytes.

Chen L, Xu Y, Healer J, Thompson JK, Smith BJ, Lawrence MC, Cowman AF - Elife (2014)

View Article: PubMed Central - PubMed

Affiliation: Division of Infection and Immunity, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia.

ABSTRACT

Plasmodium falciparum causes the most severe form of malaria in humans and is responsible for over 700,000 deaths annually. It is an obligate intracellular parasite and invades erythrocytes where it grows in a relatively protected niche. Invasion of erythrocytes is essential for parasite survival and this involves interplay of multiple protein–protein interactions. One of the most important interactions is binding of parasite invasion ligand families EBLs and PfRhs to host receptors on the surface of erythrocytes. PfRh5 is the only essential invasion ligand within the PfRh family and is an important vaccine candidate. PfRh5 binds the host receptor basigin. In this study, we have determined the crystal structure of PfRh5 using diffraction data to 2.18 Å resolution. PfRh5 exhibits a novel fold, comprising nine mostly anti-parallel α-helices encasing an N-terminal β-hairpin, with the overall shape being an elliptical disk. This is the first three-dimensional structure determined for the PfRh family of proteins.

Doi:: http://dx.doi.org/10.7554/eLife.04187.001

No MeSH data available.


PfRh5 and human basigin form a 1:1 complex.PfRh5-C and basigin were cross-linked with EDC in the presence of NHS. The mixture was then analysed on a SDS-PAGE gel and the band of the crosslinked complex was excised for trypsin/chymotrypsin digestion followed by mass spectrometric analysis.DOI:http://dx.doi.org/10.7554/eLife.04187.005
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fig1s2: PfRh5 and human basigin form a 1:1 complex.PfRh5-C and basigin were cross-linked with EDC in the presence of NHS. The mixture was then analysed on a SDS-PAGE gel and the band of the crosslinked complex was excised for trypsin/chymotrypsin digestion followed by mass spectrometric analysis.DOI:http://dx.doi.org/10.7554/eLife.04187.005

Mentions: To show that PfRh5-C is functional, we demonstrated that it could bind red blood cells and the receptor human basigin, which is also produced in insect cells (Figure 1A). PfRh5-C formed a stable complex with basigin as evidenced by size-exclusion chromatographic analysis. In these experiments, PfRh5-C was incubated with excess basigin and the stable PfRh5-basign complex eluted ahead of free PfRh5 and basigin (Figure 1B). The stoichiometry of the complex was shown to be 1:1 by chemical cross-linking (Figure 1—figure supplement 2). The binding affinity of the PfRh5-C–basigin interaction was determined by surface plasmon resonance (SPR) to be KD = 43.4 nM (Figure 1C). This KD value is higher than that previously reported (Crosnier et al., 2011). We note that while the PfRh5 sample used for the SPR measurement was prepared in monomeric form by gel-filtration chromatography, it is possible that a dynamic equilibrium with oligomeric forms within the sample has contributed to the higher affinity measurement. Antibodies to the recombinant PfRh5-C block growth of 3D7 and W2mef strains of P. falciparum at levels comparable to previous studies (Douglas et al., 2011; Bustamante et al., 2013; Douglas et al., 2014; Reddy et al., 2014) (Figure 1D). Taken together, these data imply that PfRh5-C is functionally competent.


Crystal structure of PfRh5, an essential P. falciparum ligand for invasion of human erythrocytes.

Chen L, Xu Y, Healer J, Thompson JK, Smith BJ, Lawrence MC, Cowman AF - Elife (2014)

PfRh5 and human basigin form a 1:1 complex.PfRh5-C and basigin were cross-linked with EDC in the presence of NHS. The mixture was then analysed on a SDS-PAGE gel and the band of the crosslinked complex was excised for trypsin/chymotrypsin digestion followed by mass spectrometric analysis.DOI:http://dx.doi.org/10.7554/eLife.04187.005
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356141&req=5

fig1s2: PfRh5 and human basigin form a 1:1 complex.PfRh5-C and basigin were cross-linked with EDC in the presence of NHS. The mixture was then analysed on a SDS-PAGE gel and the band of the crosslinked complex was excised for trypsin/chymotrypsin digestion followed by mass spectrometric analysis.DOI:http://dx.doi.org/10.7554/eLife.04187.005
Mentions: To show that PfRh5-C is functional, we demonstrated that it could bind red blood cells and the receptor human basigin, which is also produced in insect cells (Figure 1A). PfRh5-C formed a stable complex with basigin as evidenced by size-exclusion chromatographic analysis. In these experiments, PfRh5-C was incubated with excess basigin and the stable PfRh5-basign complex eluted ahead of free PfRh5 and basigin (Figure 1B). The stoichiometry of the complex was shown to be 1:1 by chemical cross-linking (Figure 1—figure supplement 2). The binding affinity of the PfRh5-C–basigin interaction was determined by surface plasmon resonance (SPR) to be KD = 43.4 nM (Figure 1C). This KD value is higher than that previously reported (Crosnier et al., 2011). We note that while the PfRh5 sample used for the SPR measurement was prepared in monomeric form by gel-filtration chromatography, it is possible that a dynamic equilibrium with oligomeric forms within the sample has contributed to the higher affinity measurement. Antibodies to the recombinant PfRh5-C block growth of 3D7 and W2mef strains of P. falciparum at levels comparable to previous studies (Douglas et al., 2011; Bustamante et al., 2013; Douglas et al., 2014; Reddy et al., 2014) (Figure 1D). Taken together, these data imply that PfRh5-C is functionally competent.

View Article: PubMed Central - PubMed

Affiliation: Division of Infection and Immunity, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia.

ABSTRACT

Plasmodium falciparum causes the most severe form of malaria in humans and is responsible for over 700,000 deaths annually. It is an obligate intracellular parasite and invades erythrocytes where it grows in a relatively protected niche. Invasion of erythrocytes is essential for parasite survival and this involves interplay of multiple protein–protein interactions. One of the most important interactions is binding of parasite invasion ligand families EBLs and PfRhs to host receptors on the surface of erythrocytes. PfRh5 is the only essential invasion ligand within the PfRh family and is an important vaccine candidate. PfRh5 binds the host receptor basigin. In this study, we have determined the crystal structure of PfRh5 using diffraction data to 2.18 Å resolution. PfRh5 exhibits a novel fold, comprising nine mostly anti-parallel α-helices encasing an N-terminal β-hairpin, with the overall shape being an elliptical disk. This is the first three-dimensional structure determined for the PfRh family of proteins.

Doi:: http://dx.doi.org/10.7554/eLife.04187.001

No MeSH data available.