Function
Pregnancy-associated malaria (PAM) is caused by Plasmodium falciparum-infected erythrocytes (IEs) accumulating in the placenta. The multi-domain antigen VAR2CSA confers specific adhesion of IEs to Chondroitin Sulphate A (CSA) in the placenta. This fixation is the main function of DBL3x domain.
Disease
The interaction between the DBL3x domain and the CSA, on the placenta surface, plays a key role in the development of the Pregnancy-Associated Malaria (PAM). Malaria is a disease infected by a parasite of the genus Plasmodium, transmitted by the bite of the Anopheles mosquito, in many tropical regions. Plasmodium Falciparum is one of the most dangerous parasites. It causes the accumulation of infected red blood cells, leading to placental inflammation and block of blood flow to the developing fetus.
According to last OMS estimations, 212 million cases and 429 000 deaths occured in 2015 in the world.
Each person, suffering from this disease, can experience several symptoms : fever, vomiting, headaches, fatigue etc.
Structural highlights
Overall of DBL3x
The DBL3x structure is composed of (using the nomenclature of ref 1).
The (residues 1220−1292 ; yellow) has not regular secondary structure except for a single turn of helix and is held together by two disulphide bonds between Cys1230-Cys1273 and Cys1251-Cys1264.
The (residues 1293−1444 ; blue) contains four helices (H1-H4) linked by four loops. In helix H4, an unpaired cysteine (Cys1418) reacted with cystamine during refolding, gaining a cysteamine adduct. The C-terminal domain of this subdomain (residues 1424−1444) forms an extended structure that connects to the third subdomain. Cys1437 forms a disulfide bond with Cys1344 on helix H2.
The (residues 1445−1580 ; red) has two long antiparallel helices, H5 (residues 1449−1476) and H6 (residues 1499−1529), that are linked to each other by a large loop (residues 1477−1498) and that make contacts with the first and the second subdomains. The C-terminal domain of this subdomain (residues 1563−1580) forms a flat structure of small helices connected by short linker regions. Subdomain 3 contains four disulfide bonds: Cys1462-Cys1546, Cys1476-Cys1501, Cys1505-Cys1574 and Cys1486-Cys1576. .
Comparaison with known DBL structures
DBL3x is compared to two other proteins that does not belong to PfEMP1 family : the P. falciparum erythrocyte binding antigen (EBA)-175, which has two DBL domains F1 and F2 (PDB 1ZRL), and the Plasmodium knowlesi (Pk)α-DBL protein, which has one DBL domain (PDB 2C6J).
The structure of the DBL3x shows that PfEMP1 domains have the conserved DBL protein fold. Structure-based sequence alignments of DBL3x with EBA-175 and Pkα-DBL show conserved cysteines amid a few other conserved residues. Futhermore, conserved helices have been observed in the absence of substantial sequence identity.
Overlays of the four structures based on the structural superimposition of the long helices in the third subdomain made it clear that each DBL domain has its three subdomains positioned differently.
Location of CSA binding site
A sulfate ion seems to be coordinated by Lys1324, Arg1467 and Lys1504. The lysines of DBL3x are required for binding CSA.
Mechanism
Clusters of several PfEMP1 molecules occur on protuberances, or 'knobs', which are characteristic surface features of P. falciparum–infected erythrocytes. The adhesin comprises a large extracellular N-terminal region with six DBL domains, a single transmembrane segment and a cytoplasmic acid terminal segment (ATS) at the C terminus. The first three DBL domains belong to class X, whereas the last three belong to class epsilon. Individual recombinant DBL domains from VAR2CSA that have been shown to bind CSA are colored in orange. The DBL3x domain, illustrated as an enlargement in ribbon form, is a highly alpha-helical protein (PDB 3CML). The subdomains 1, 2 and 3 are colored yellow, red and green, respectively. VAR2CSA binds to the CSA moiety of placental chondroitin sulfate proteoglycan present on the surface of the syncytiotrophoblast cells in the placenta. The CSA ligand is shown schematically as hexagons (blue), positioned over the region covering subdomains 2 and 3 identified by the two research groups as the CSA binding site.
However, the molecular mechanism underlying the interaction between VAR2CSA and CSA remains unresolved.The main cause are the difficulties in analyzing protein-glycan binding in vitro. It can easily brings to a false positive identification of nonspecific glycan-binding proteins for the next reason : the large impact of low affinity charge-charge interactions.
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