Structure of the DBL3X-DBL4ε region of the VAR2CSA placental malaria vaccine candidate: insight into DBL domain interactions

Abstract

The human malaria parasite, Plasmodium falciparum, is able to evade spleen-mediated clearing from blood stream by sequestering in peripheral organs. This is due to the adhesive properties conferred by the P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) family exported by the parasite to the surface of infected erythrocytes. Expression of the VAR2CSA variant of PfEMP1 leads to pregnancy-associated malaria, which occurs when infected erythrocytes massively sequester in the placenta by binding to low-sulfated Chondroitin Sulfate A (CSA) present in the intervillous spaces. VAR2CSA is a 350 kDa protein that carries six Duffy-Binding Like (DBL) domains, one Cysteine-rich Inter-Domain Regions (CIDR) and several inter-domain regions. In the present paper, we report for the first time the crystal structure at 2.9 Å of a VAR2CSA double domain, DBL3X-DBL4ε, from the FCR3 strain. DBL3X and DBL4ε share a large contact interface formed by residues that are invariant or highly conserved in VAR2CSA variants, which suggests that these two central DBL domains (DBL3X-DBL4ε) contribute significantly to the structuring of the functional VAR2CSA extracellular region. We have also examined the antigenicity of peptides corresponding to exposed loop regions of the DBL4ε structure.

Figure 1. Structure of the FCR3-DBL3X-DBL4ε double domain.

The structure is shown in ribbon representation with DBL3X in yellow and DBL4ε in blue. The interdomain linker between residues K1583 and K1590 inclusive is in cyan.

Figure 2. Interface between the DBL3X and DBL4ε domains.

The domains are shown in surface representation with DBL3X in yellow, DBL4ε in blue and the linker region in cyan. The invariant contacting residues (see Table S1) are in red for DBL3X and orange for DBL4ε; polymorphic contacting residues are green for both domains. The N- and C-termini of the double domain are in magenta and are labelled N and C. (a,b) The DBL3X-DBL4ε double domain viewed edge on from the interdomain interface. The two views are rotated by 180° with respect to each other about the vertical (c) Semi-transparent surface representation of the DBL3X domain viewed from above the interface with the side chains of the contacting residues shown in red. (d) Semi-transparent surface representation of the DBL4ε domain viewed from above the interface with the side chains of the contacting residues shown in orange.

Figure 3. Peptide segments used in antigenicity studies of antibodies raised to the DBL3X-DBL4ε recombinant double domain.

The peptides, chosen from exposed loop regions as described in the main text, are shown in their structural context. Colour code: Pep-1, Ile1640-Lys1651 (IIKNEEGMEKAK), blue; Pep-2, Gln1672-Lys1685 (QYNPTGKGIDDANK), red; Pep-3, Glu1722-Asp1733 (EIFGSSDTNDID), green; Pep-4, Glu1743-ILe1756 (ENETITNGPDRKTI), mauve; Pep-5, Glu1774-Lys1792 (EEKNENFPLSMGVEHIGIAK)*, brown; Pep-6, Asn1864-Asp1890 (NKIYRKSNKESEGGKDYSMIMAPTVID), orange. *Underlined residue in Pep-5 was changed from Cys in the cognate sequence to Ser in order to avoid cross-linking of peptides.

Figure 4. Reactivity of DBL4ε-derived peptides and FCR3-DBL3X-DBL4ε with purified anti- DBL3X-DBL4ε rabbit IgG.

(a) Recognition of peptides (Pep-1 to Pep-6) by anti-DBL3X-DBL4ε purified IgG antibodies. Biotinylated peptides were directly coated in carbonate buffer. ELISA was performed using the pre-Immune IgG (10 μg/mL), the anti-DBL3X-DBL4ε IgG (10 μg/mL) or the anti-biotin Mouse monoclonal IgG (1 μg/mL). (b) Recognition of recombinant FCR3-DBL3X-DBL4ε by immune IgG purified on peptides, named Anti-pep1 to 6. The 3D7-DBL1X-DBL2X double domain and Bovine Serum Albumin (BSA) were used as negative controls. The optical density was measured at 655 nm. All proteins were coated at 1 μg/mL and purified rabbit IgG was used at a dilution of 1:100 in the blocking buffer.