Using mutagenesis and structural biology to map the binding site for the Plasmodium falciparum merozoite protein PfRh4 on the human immune adherence receptor

Abstract

To survive and replicate within the human host, malaria parasites must invade erythrocytes. Invasion can be mediated by the P. falciparum reticulocyte-binding homologue protein 4 (PfRh4) on the merozoite surface interacting with complement receptor type 1 (CR1, CD35) on the erythrocyte membrane. The PfRh4 attachment site lies within the three N-terminal complement control protein modules (CCPs 1-3) of CR1, which intriguingly also accommodate binding and regulatory sites for the key complement activation-specific proteolytic products, C3b and C4b. One of these regulatory activities is decay-accelerating activity. Although PfRh4 does not impact C3b/C4b binding, it does inhibit this convertase disassociating capability. Here, we have employed ELISA, co-immunoprecipitation, and surface plasmon resonance to demonstrate that CCP 1 contains all the critical residues for PfRh4 interaction. We fine mapped by homologous substitution mutagenesis the PfRh4-binding site on CCP 1 and visualized it with a solution structure of CCPs 1-3 derived by NMR and small angle x-ray scattering. We cross-validated these results by creating an artificial PfRh4-binding site through substitution of putative PfRh4-interacting residues from CCP 1 into their homologous positions within CCP 8; strikingly, this engineered binding site had an ∼30-fold higher affinity for PfRh4 than the native one in CCP 1. These experiments define a candidate site on CR1 by which P. falciparum merozoites gain access to human erythrocytes in a non-sialic acid-dependent pathway of merozoite invasion.

Keywords: Cell Surface Receptor; Complement System; Convertases; Malaria; NMR; Plasmodium; Protein Structure; Reticulocyte-binding Homologue Proteins; Surface Plasmon Resonance (SPR).

FIGURE 1.

Schematic representation of CR1. The most common allelic form of CR1 contains 30 extracellular CCPs followed by a transmembrane domain (TM) of 28 amino acid residues and a cytoplasmic tail (CYT) of 43 residues. Based on homology, the first 28 CCPs are further grouped into LHRs A, B, C, and D. LHR A (CCPs 1–7) contains a C4b-binding site and possesses decay-accelerating activity (site 1, CCPs 1–3 in yellow), whereas LHRs B (CCPs 8–14) and C (CCPs 15–21) each contain site 2 (duplicated functional units CCPs 8–10 and 15–17 in purple) that are 99% identical, bind C3b and C4b, and have factor I cofactor activity. The binding site for PfRh4 was previously mapped to CCPs 1–3 in LHR A (yellow ovals) (21). LHR D⁺ refers to LHR D plus CCPs 29 and 30.

FIGURE 2.

CCP 1 in LHR A is essential for PfRh4 binding. Microtiter plates were coated with PfRh4 (0.5 μg/well). The interaction between CR1 protein constructs in the supernatants of transfected HEK 293T cells and PfRh4 was detected by a rabbit anti-CR1 polyclonal Ab. A, only LHR A shows binding. B, in LHR A, deletion of CCP 1 abrogates PfRh4 binding, whereas deletion of CCP 2 or 3 has no effect. Displayed values are the means ± S.E. of at least three independent experiments in this figure and in Figs. 3 and 4.

FIGURE 3.

Identification of amino acid residues in CCP 1 important for PfRh4 binding. A, the 27 amino acid residues that differ between CCP 1 and CCP 8 are marked with braces. Because LHR B does not bind to PfRh4, amino acid residues in CCP 1 were substituted with their equivalents in CCP 8. Numbers following the “m” (for mutant) denote the position of substituted amino acids; i.e., m1,3 has both the Q1H and N3Q mutations in LHR A. B, m18–19 and m20–21 show negligible binding to PfRh4, whereas m6–9 and m35 display a >50% reduction in binding. All others are not significantly different from LHR A. C, amino acid residues Asp¹⁸ and Phe²⁰ are essential for PfRh4 binding. Single amino acid substitutions were prepared in the LHR A template, and the proteins were tested for alterations in binding to PfRh4. Experimental design as in Fig. 2 including the use of supernatants of transfected HEK 293T cells containing the CR1 proteins.

FIGURE 4.

A PfRh4 binding capability can be conferred upon LHR B. The two amino acid residues in CCP 1 whose replacement had been shown to abrogate binding were first substituted into LHR B at their homologous positions (m18,20r) but were not sufficient to confer PfRh4 binding. Other mutations that decreased binding of LHR A were also substituted into m18,20r. m7–9,18–20,35r bound 3-fold better than LHR A. m7–9,18–20r bound ∼16-fold better than LHR A. On the other hand, m7–9,35r did not bind. The experimental design for this figure was similar to that in Figs. 2 and 3, except that the CR1 proteins were His-tagged and purified from the supernatants.

FIGURE 5.

LHR A and m7–9,18–20r co-immunoprecipitate with PfRh4. Co-IP experiments were performed by mixing a CR1 variant in the supernatant of HEK 293T transfectants with PfRh4 and then incubating with protein A/G-agarose beads bearing either anti-CR1 monoclonal Ab J3B11 or anti-PfRh4 monoclonal Ab 10C9. A, Western blot analysis of eluants from IP with 10C9 using polyclonal anti-CR1 Ab. The first four lanes (controls) show LHR A, LHR B, LHR A Δ1, and m7–9,18–20r, whereas the last four lanes contain the eluants of IP with 10C9. LHR A (lane 5) and m7–9,18–20r (lane 8) co-IP with PfRh4, whereas LHR B (lane 6) and LHR A Δ1 (lane 7) do not. B, Western blot analysis of eluants from IP blotted with J3B11 shows that PfRh4 co-IP with LHR A (lane 2) and m7–9,18–20r (lane 5) but not with LHR B (lane 3) or LHR A Δ1 (lane 4).

FIGURE 6.

PfRh4 binds to immobilized CR1 LHR A. A, PfRh4 was injected at 5.5, 2.8, 1.4, 0.7, 0.35, and 0.18 μm over parallel flow cells of nickel-purified, surface plasmon resonance chip immobilized LHR A (3,000 RU), LHR B (3,600 RU), and LHR D (3,800 RU). The reference-subtracted sensorgrams demonstrate concentration-dependent binding to LHR A. No binding of PfRh4 to LHR B or LHR D was detected. B, the PfRh4:LHR A binding data conform to a 1:1 Langmuir interaction model. A nonlinear regression of response units versus PfRh4 concentrations demonstrate a K_D (indicated by a dotted line) of 490 ± 60 nm.

FIGURE 7.

Mutations in CCP 1 of CR1 abrogate PfRh4 binding. A, supernatants containing His-tagged CR1 recombinant constructs were injected at 220 nm over immobilized PfRh4 (3,300 RU). Reference subtracted sensorgrams demonstrate that LHR A Δ1, m18, and m20 behave like LHR B and do not bind to immobilized PfRh4. A parallel flow cell bearing 4,000 RU immobilized mouse IgG was employed as the reference. B, duplicate injections of supernatants containing His-tagged constructs passed over immobilized PfRh4 demonstrate that m7–9,18–20r carrying six single amino acid substitutions from LHR A confers a PfRh4-binding site if placed in homologous positions of LHR B. Consistent with ELISA and IP results, m7–9,18–20 protein has a many-fold greater affinity for PfRh4 than LHR A.

FIGURE 8.

PfRh4 interactions with sCR1, LHR A, and m7–9,18–20r. CR1 constructs were injected at multiple concentrations over surface plasmon resonance chip immobilized PfRh4 (850 RU). A parallel flow path bearing 630 RU of immobilized mouse IgG was used as a reference. Dissociation constants were calculated from R_eq values derived from reference-subtracted curves and fitted to a Langmuir 1:1 interaction model. A, sCR1 was injected at 10, 6.7, 3.3, 2.2, 0.7, 0.2, and 0.08 μm. The K_D (indicated by a dotted line) was calculated to be 8 ± 1 μm. B, supernatants from transfected 293T containing LHR A were injected at 5, 1.7, 0.6, 0.2, and 0.1 μm. K_D = 2 ± 1 μm. C, supernatants from transfected 293T cells containing the m7–9,18–20r protein were injected in duplicate at 250, 128, 64, 32, and 17 nm. K_D = 61 ± 9 nm, ∼30-fold greater than that observed for PfRh4:LHR A.

FIGURE 9.

CR1 interaction with PfRh4 is ionic strength-dependent. Supernatants containing sCR1, LHR A, and m7–9,18–20r were injected over immobilized PfRh4 in 75, 150, or 300 mm NaCl buffer. A–C, representative reference-subtracted sensorgrams are presented for each protein: sCR1 (A), LHR A (B), and m7–9,18–20r (C).

FIGURE 10.

Summary of NMR/SAXS-derived structures. Ensembles (for the 20 lowest energy structures) are shown as overlaid backbone traces; the root mean square deviation (heavy backbone atoms) value is shown for each overlay. Closest to mean structures are drawn as cartoons (PyMOL) based on β-strands determined by STRIDE (65), and cysteine sulfur atoms are drawn as spheres. A, ensemble for CR1 1–2. Upper left, overlay on module 1; upper right, overlay on module 2; lower left, overlay on both modules; lower right, closest to mean structure. B, ensemble for CR1 2–3. Upper left, overlay on module 2; upper right, overlay on module 3; lower left, overlay on both modules; lower right, closest to mean structure. C, ensemble for CR1 1–3. Top, from left to right, overlays on modules 1, 2, and 3; bottom left, overlay on all modules; bottom right, closest to mean structure.

FIGURE 11.

Identification of the PfRh4-binding site in CCP 1 of CR1. In the top left are two views of the surface of the (lowest energy) NMR-derived solution structure of CR1 1–3. Residues of CCP 1 that are conserved in CCP 8 are in white; nonconserved residues that may be substituted by equivalent CCP 8 residues without any measurable effect on PfRh4 binding are colored pink; residues substituted in the m6–9 mutant of LHR A (cyan) and in the m18,19 and m20,21 mutants (yellow) are also highlighted. Gly³⁵ and other residues previously identified as important for C4b binding by CR1 1–3 are colored orange. Residues 6–9 and 18–21 are also shown by stick representation in the expansion (right), with carbons colored cyan and yellow (respectively), oxygens in red, and nitrogens in blue. Asp¹⁸ and Phe²⁰ (blue labels) are essential for PfRh4 binding; substitution of Glu⁶ (red label) by Asp increases binding 3-fold. In the bottom right are shown (as sticks) the residues of CCP 8 (modeled on structure of CCP 15 that is nearly identical) equivalent to CCP 1 residues 6–9 and 18–21. Replacement of these (in LHR B) by the equivalent CCP 1 residues creates a novel, strong PfRh4-binding site in LHR B. These studies led to the proposal of the binding site shown in the bottom left. The surface here is cut away to reveal atom representations of Trp⁷ as well as Phe²⁰ and the surrounding negatively charged side chains of Glu⁶, Asp¹⁸, Glu¹⁹, and Glu²¹ (colored as in the stick representation).