Abstract
Invasion of human red blood cells by Plasmodium falciparum involves interaction of the merozoite form through proteins on the surface coat. The erythrocyte binding-like protein family functions after initial merozoite interaction by binding via the Duffy binding-like (DBL) domain to receptors on the host red blood cell. The merozoite surface proteins DBL1 and -2 (PfMSPDBL1 and PfMSPDBL2) (PF10_0348 and PF10_0355) are extrinsically associated with the merozoite, and both have a DBL domain in each protein. We expressed and refolded recombinant DBL domains for PfMSPDBL1 and -2 and show they are functional. The red cell binding characteristics of these domains were shown to be similar to full-length forms of these proteins isolated from parasite cultures. Futhermore, metal cofactors were found to enhance the binding of both the DBL domains and the parasite-derived full-length proteins to erythrocytes, which has implications for receptor binding of other DBL-containing proteins in Plasmodium spp. We solved the structure of the erythrocyte-binding DBL domain of PfMSPDBL2 to 2.09 Å resolution and modeled that of PfMSPDBL1, revealing a canonical DBL fold consisting of a boomerang shaped α-helical core formed from three subdomains. PfMSPDBL2 is highly polymorphic, and mapping of these mutations shows they are on the surface, predominantly in the first two domains. For both PfMSPDBL proteins, polymorphic variation spares the cleft separating domains 1 and 2 from domain 3, and the groove between the two major helices of domain 3 extends beyond the cleft, indicating these regions are functionally important and are likely to be associated with the binding of a receptor on the red blood cell.
Highlights
Plasmodium falciparum invades red blood cells by the binding of specific parasite ligands to host cell receptors
PfMSPDBL2 Is a Red Cell-binding Protein on the Merozoite Surface That Contains Duffy binding-like (DBL) and SPAM Domains—The merozoite surface protein PfMSPDBL1 (PF10_0348), which binds the erythrocyte surface, consists of DBL and SPAM domains [14], and the SPAM domain is characterized by an acid-rich region followed by a leucine zipper-like region (Fig. 1)
The effect of Ca2ϩ on binding was demonstrated by titrating EGTA over a concentration range from 0 to 60 mM. This effect was specific for rDBL1 as the binding of recombinant PfRh4.9 to erythrocytes was unaffected by the level of EGTA under similar assay conditions (Fig. 4G). These results show that the recombinant DBL domains of PfMSPDBL1 and PfMSPDBL2 have the same functional properties as the full-length mature forms of the PfMSPDBL proteins derived from parasite culture supernatants (Fig. 2A), and the results suggest that the affinity for receptor binding of both PfMSPDBL1 and PfMSPDBL2 is influenced by the presence of specific metal ions
Summary
Plasmodium falciparum invades red blood cells by the binding of specific parasite ligands to host cell receptors. Two hypothetical proteins containing single DBL domains have been identified in P. falciparum (PF10_0348 and PF10_0355) Both proteins have been localized to the merozoite surface [14, 15], and functional studies have shown that the full-length form of the PF10_0348 protein has erythrocyte binding activity when expressed on the surface of COS cells [14]. Population genomic analysis has revealed a large number of polymorphisms in both genes, within the dbl region, and that both genes are subject to strong balancing selection, an indicator that they are likely to be under intense immune pressure, consistent with their presence on the surface of the merozoite [17] These DBL-containing merozoite surface proteins are of interest as potential malaria vaccine candidates, and as such, it will be useful to obtain a better understanding of their functional role during parasite invasion. We have derived the crystal structure of the erythrocytebinding DBL domain of PfMSPDBL2 to 2.09 Å resolution
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