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

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

Keywords: biophysics; erythrocyte; human; infectious disease; invasion; malaria; microbiology; plasmodium; structural biology.

Figure 1.. Production of functional recombinant PfRh5.

(A) Purified recombinant PfRh5 was analysed by SDS-PAGE analyses and by erythrocyte binding assays. (B) Formation of the PfRh5–basigin complex was monitored by size-exclusion chromatography. The chromatographic profiles are shown for PfRh5 (panel 1), basigin (panel 2), and the PfRh5-basigin complex (panel 3). The fractions eluted from the column in panel 3 were analysed by SDS-PAGE. (C) The binding affinity of the recombinant PfRh5 to human basigin was measured by SPR on Biacore 3000 with the basigin coupled to a sensor chip. (D) In vitro growth inhibition assays were performed to assess the abilities of the polyclonal antibodies to the recombinant PfRh5 in blocking P. falciparum parasite invasion into erythrocytes. DOI: http://dx.doi.org/10.7554/eLife.04187.003

Figure 1—figure supplement 1.. Production of full-length PfRh5.

(A) SDS-PAGE analyses of the FLAG-tagged recombinant full-length PfRh5 eluted from an anti-FLAG affinity column. (B) Size-exclusion chromatography analyses of the anti-FLAG bead affinity purified PfRh5. The fractions eluted from the affinity column were pooled, concentrated, and stored at 4°C for 2 days before the analyses. (C) Red blood cell binding assay with PfRh5 purified by anti-FLAG bead affinity chromatography. Both full-length protein and the breakdown fragments bound red blood cells with a 48-kDa species having the highest affinity. DOI: http://dx.doi.org/10.7554/eLife.04187.004

Figure 1—figure supplement 2.. 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

Figure 2.. The crystal structure of PfRh5.

(A) Ribbon representation of the PfRh5 structure. Color scheme is rainbow (N-terminus: blue; C-terminus: red) except for the β-hairpin that is colored magenta for clarity. The cysteine residues that form disulfide bridges (Cys345–Cys351 and Cys224–Cys317) are shown as spheres. Helices α4, α5, α6, and α7 assemble as a triplet-helical coiled-coil domain running the length of the long axis of the molecule, helices α1, α2a, and α3b assemble to form a short triplet-helical bundle and helices α2b and α3a assemble to form a short two-helix coiled coil. (B) Ribbon representation of the PfRh5 structure viewed after a 180^o rotation relative to that in (A). The residues between Ser257 and Asp294 are disordered. (C) Ribbon representation of the PfRh5 structure viewed from the side with the C-terminus on the left. The disulfide bridges are indicated with arrows. (D) Ribbon representation of the PfRh5 structure viewed after a 180^o-rotation relative to that in (C). DOI: http://dx.doi.org/10.7554/eLife.04187.006

Figure 2—figure supplement 1.. The secondary structure of PfRh5.

Density for the N-terminal residues Asp127–Leu145, loop residues Glu258–Asn293, and C-terminal residues Met512–Gln526 was not observed. The helices and β-strands are colored red and green respectively. The vertical line at residue 119 and 319 indicates the position of a helical break. DOI: http://dx.doi.org/10.7554/eLife.04187.008

Figure 2—figure supplement 2.. Two free cysteine residues (Cys203 on α2a and Cys329 on α3b) within the crystal structure of PfRh5.

The side chain of Cys329 is completely buried inside the three-dimensional fold of PfRh5 and the side chain of Cys203 is only partly exposed, consistent with the fact that no intermolecular disulfide bond was observed for the native and recombinant PfRh5. DOI: http://dx.doi.org/10.7554/eLife.04187.009

Figure 2—figure supplement 3.. Superimposition of the PfRh5 structure with N-terminal coiled-coil domain of SipB.

The PfRh5 (green) coiled-coil helix bundle formed by helices α5, α6, and α7 has a very similar fold to the N-terminal coiled-coil domain of SipB (magenta). DOI: http://dx.doi.org/10.7554/eLife.04187.010

Figure 2—figure supplement 4.. A unique pocket on the surface of the PfRh5 molecule.

(A) Location of the pocket on the surface formed by the β-hairpin, the triple-helical bundle, and the triple-helical coiled coil. (B) Close-up view of the pocket; key residues lining the pocket are labeled. DOI: http://dx.doi.org/10.7554/eLife.04187.011

Figure 3.. Binding of mutant PfRh5 to basigin.

(A) The binding affinity of the reduced and alkylated PfRh5 to basigin measured by SPR. (B) The binding affinity of the PfRh5 mutant, in which the disordered loop has been deleted, to basigin measured by SPR. DOI: http://dx.doi.org/10.7554/eLife.04187.012