Molecular basis for sialic acid-dependent receptor recognition by the Plasmodium falciparum invasion protein erythrocyte-binding antigen-140/BAEBL

Abstract

Plasmodium falciparum erythrocyte invasion is dependent on high affinity recognition of sialic acid on cell surface receptors. The erythrocyte binding-like (EBL) family of invasion ligands mediates recognition of sialic acid on erythrocyte glycoproteins. Erythrocyte-binding antigen-140 (PfEBA-140/BAEBL) is a critical EBL ligand that binds sialic acid on its receptor glycophorin C. We present here the crystal structure of the two-domain receptor-binding region of PfEBA-140 in complex with a glycan containing sialic acid. The structure identifies two glycan-binding pockets unique to PfEBA-140 and not shared by other EBL ligands. Specific molecular interactions that enable receptor engagement are identified and reveal that the glycan binding mode is distinct from that of apicomplexan and viral cell surface recognition ligands as well as host immune factors that bind sialic acid. Erythrocyte binding experiments elucidated essential glycan contact residues and identified divergent functional roles for each receptor-binding site. One of four polymorphisms proposed to affect receptor binding was localized to a glycan-binding site, providing a structural basis for altered erythrocyte engagement. The studies described here provide the first full description of sialic acid-dependent molecular interactions at the P. falciparum erythrocyte invasion interface and define a framework for development of PfEBA-140-based therapeutics, vaccines, and diagnostics assessing vaccine efficacy and natural immunity to infection.

FIGURE 1.

The crystal structure of RII PfEBA-140 bound to sialyllactose reveals two receptor glycan-binding sites. A, RII PfEBA-140 bound to two sialyllactose molecules in the crystal structure. The F1 and F2 DBL domains each form a contact surface with one sialyllactose molecule. The F1 domain is shown in orange, and the F2 domain is in blue. A short linker connecting the two DBL domains is shown in gray. The modeled sialic acid molecules in each glycan contact site are shown in green and boxed in black. B, the bound sialic acid inserts into the F1- and F2-binding pockets, whereas the galactose extends away from the pocket surface. The sialic acid molecules and corresponding electron density observed in the F1 domain (left, orange) and F2 domain (right, blue) are displayed within their binding sites. The sialic acid is shown in red for clarity. Density for the galactose is shown to illustrate the location of this sugar moiety relative to the sialic acid-binding site. The F_o − F_c map prior to sialic acid modeling is shown in green and contoured at 3.0σ. The 2F_o − F_c map obtained following modeling of the sialic acid into the binding pocket is shown in blue and contoured at 1.0σ. C, close-up view of the electron density observed for the sialic acid and galactose of the bound sialyllactose in the F1 (left) and F2 (right) domains. The density maps are contoured and colored as in B. D, the two glycan-binding sites are located in a region of concentrated positively charged residues that may function as a high affinity interface for GPC engagement. Surface charge potential is colored from +3.5 eV (blue) to −3.5 eV (red). E, the sialic acid-binding pockets are in structurally similar locations on the F1 and F2 DBL domains. The glycan bound to the F1 domain is shown in green, and the glycan bound to the F2 domain is shown in yellow. F, the individual binding sites in each DBL domain are structurally similar. Specifically, the sialic acid inserts into a valley at the interface of a helix and an extended loop present on both domains. The glycan coloring is the same as in E.

FIGURE 2.

Sialic acid modeling and erythrocyte binding studies provide a detailed molecular description of the RII PfEBA-140 erythrocyte invasion interface. A, close-up view of the F1-binding pocket. Four residues directly contact the sialic acid. Hydrogen bonds are designated with black lines. B, close-up view of the F2-binding pocket. Four residues directly contact the sialic acid. The single hydrogen bond interaction is shown with a black line. C, erythrocyte binding by RII PfEBA-140 expressed on the surface of HEK-293 cells is sensitive to trypsin and neuraminidase treatment of red blood cells, but chymotrypsin-resistant. Erythrocytes were treated with 0.1 mg/ml trypsin, 0.1 mg/ml chymotrypsin, or 5 milliunits of V. cholerae neuraminidase at 37 °C prior to the rosetting assay. Bound erythrocytes appear black around the transfected mammalian cells. The images are displayed at 20× magnification. D and E, RII PfEBA-140 mutants were expressed on the surface of mammalian cells and tested for erythrocyte binding. GFP was used to assess proper expression of each construct. Wild type RII PfEBA-140 extensively binds erythrocytes, and a construct containing GFP alone is not capable of binding. To identify critical binding interactions, individual glycan contact residues in the F1- and F2-binding pockets were mutated to alanine or glutamate and tested for erythrocyte binding by rosetting assay. Bound erythrocytes appear black around the transfected mammalian cells. The images are displayed at 20× magnification. F and G, the percentage of cells expressing point mutants of RII PfEBA-140 that bind erythrocytes relative to wild type RII. The binding phenotypes are representative of the binding percentage quantified for 30 fields of view per mutation. Lys-347, mutated to alanine as a surface control residue, displays wild type binding. The black bars indicate wild type RII PfEBA-140 binding. The white bars display binding percentages for single alanine mutants. The gray bar represents the surface control Lys-347. The hatched bars represent binding percentages for glutamate mutants in the F2-binding pocket. Error bars indicate S.E.

FIGURE 3.

RII PfEBA-140 glycan-binding sites are distinct from other classes of sialic acid-binding proteins. A–D, the PfEBA-140 F1 (orange) and F2 domains (blue) are shown overlaid with the TgMIC1 (light blue) (A), hemagglutinin (red) (B), selectin (purple) (C), and Siglec (light green) (D) sialic acid-binding sites. The sialic acid molecule bound to PfEBA-140 is shown in green, and the sialic acid bound to the protein of comparison is shown in gray. Arrows highlight structural differences in each case.

FIGURE 4.

The F1-binding pocket surface is altered when Ile-185 is mutated to valine in silico. A, the glycan-binding surface containing Ile-185 (the residue present in the construct used for crystallization) exhibits strong shape complimentary with the sialic acid. The overall surface is shown in gray on the top, and the electrostatic surface is shown on the bottom (surface charge potential is colored from +3.5 eV (blue) to −3.5 eV (red)). The bound sialic acid is shown in green. B, the binding pocket surface is altered following in silico mutation of residue 185 to valine. The top displays the overall surface of the pocket in gray, and the bottom displays the electrostatic surface (surface charge potential is colored as in A). The altered surface cavity is identified with an arrow, and the sialic acid is shown in green.

FIGURE 5.

The sialyllactose-bound structure illuminates a glycan-induced structural change and allows for localization of putative sulfate-binding sites. A, overlay of the sialyllactose bound (F1 domain in orange, F2 domain in blue) and unbound (F1 domain in light orange, F2 domain in light blue) structures of RII PfEBA-140. The bound sialic acid molecules are shown in green. The glycan-induced structural change is outlined in black. B, close-up view of residues Thr-493, Tyr-494, and Leu-495, which show a pronounced movement following glycan binding. C, the helix shift in the F2 domain observed upon glycan binding is propagated by sialic acid interference with main chain water molecule interactions. Glycan binding precludes access of two water molecules to the cavity indicated by the black arrow (the waters are present in the apo structure). In the absence of contacts mediated by these water molecules, the helix is destabilized, allowing the movement observed in the bound crystal structure. The water molecules are shown in cyan, and the sialic acid is in green. The bound F2 domain is shown in dark blue, and the unbound F2 domain is in light blue. D, three sulfate-binding motifs in RII PfEBA-140 are adjacent to the sialic acid-binding sites. Two other putative sulfate-binding motifs are distal to the binding sites. The putative sulfate-binding sites are shown in purple and identified with an arrow.

FIGURE 6.

PfEBA-140 sialic acid recognition is distinct from that of PfEBA-175. Shown here is an overlay of the RII PfEBA-140 monomer with the dimer of RII PfEBA-175. The PfEBA-140 F1 domain is shown in orange, and the F2 domain is shown in blue. The RII PfEBA-175 monomer overlaid with RII PfEBA-140 is shown in light green. The second monomer of RII PfEBA-175 is shown in light purple. Residues of PfEBA-140 involved in sialic acid binding are shown in red. The sialic acid molecules bound to RII PfEBA-140 are shown in black for clarity. Residues involved in PfEBA-175 sialic acid binding are shown in purple.