Abstract: The life cycle of the malaria parasite (Plasmodium) is remarkably complex. Malaria parasites must engage in highly specific and varied interactions with cell types of both the mammalian host and the mosquito vector. In this issue of Cell, Tolia et al., 2005Tolia N.H. Enemark E.J. Sim B.K.L. Joshua-Tor L. Cell. 2005; 122 (this issue): 183-193Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar report detailed molecular insights into an intimate interaction between a malaria parasite protein and its host cell receptor that enables the parasite to invade erythrocytes. The life cycle of the malaria parasite (Plasmodium) is remarkably complex. Malaria parasites must engage in highly specific and varied interactions with cell types of both the mammalian host and the mosquito vector. In this issue of Cell, Tolia et al., 2005Tolia N.H. Enemark E.J. Sim B.K.L. Joshua-Tor L. Cell. 2005; 122 (this issue): 183-193Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar report detailed molecular insights into an intimate interaction between a malaria parasite protein and its host cell receptor that enables the parasite to invade erythrocytes. Recognition of target host cells by the malaria parasite is required for invasion and is a prelude to parasite growth and multiplication in the host cell. Malaria parasite invasion of host cells is clearly something that research aims to prevent, but it is not the only source of pathology. The species of malaria parasite that is responsible for the bulk of malaria-related human mortality, Plasmodium falciparum, is also a well-documented exponent of the art of sequestration. During sequestration, Plasmodium-infected red blood cells bind to capillary endothelial cells of host tissues such that the malaria parasite is effectively removed from the host circulation. Sequestration may result in severe, often fatal pathologies such as cerebral malaria. P. falciparum possesses a repertoire of genes encoding proteins that are dedicated to binding to host cell receptors during either invasion or sequestration. These proteins are capable of recognizing specific structures on the surface of the target cell. Many of the host molecules targeted by the malaria parasite are glycosylated. Indeed, this pathogen seems to have developed a taste for these host receptor-linked sugars. Plasmodium frequently exploits the presence of sugars on host cell receptors by expressing parasite-derived ligands that specifically bind to glycosyl moieties. This realization prompted an intense investigation to discover these parasite ligands and to understand their interactions at the molecular level. Such information should speed the discovery and design of inhibitors that block invasion or sequestration. The benefit of impairing either of these activities is self-evident. In their new study, Tolia et al., 2005Tolia N.H. Enemark E.J. Sim B.K.L. Joshua-Tor L. Cell. 2005; 122 (this issue): 183-193Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar report the crystal structure of the binding domains of the erythrocyte binding antigen (EBA-175) of P. falciparum. During invasion of red blood cells, this parasite protein binds to a major erythrocyte glycoprotein called glycophorin A. In particular, EBA-175 binds to sialic acid sugar residues, which are distributed on O-linked tetrasaccharides as part of the mucin domain of glycophorin A. EBA-175 is a member of the erythrocyte binding protein (EBP) family of related multidomain transmembrane proteins (Adams et al., 1992Adams J.H. Sim B.K. Dolan S.A. Fang X. Kaslow D.C. Miller L.H. Proc. Natl. Acad. Sci. USA. 1992; 89: 7085-7089Crossref PubMed Scopus (393) Google Scholar, Adams et al., 2001Adams J.H. Blair P.L. Kaneko O. Peterson D.S. Trends Parasitol. 2001; 17: 297-299Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Merozoites (the invasive blood stage form of the malaria parasite) use EBPs as part of a cascade of contact molecules involved in the multiprotein, multistep invasion process that ultimately sees the parasite gain access to the erythrocyte cytosol and establish a vacuole. Furthermore, the six EBP family members share an overall domain structure (see Figure 1 in Tolia et al., 2005Tolia N.H. Enemark E.J. Sim B.K.L. Joshua-Tor L. Cell. 2005; 122 (this issue): 183-193Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar), yet each has its own target specificity and may bind to different erythrocyte receptor molecules. For example, an EBP family member called BAEBL binds to sialic acid residues on glycophorin C (Lobo et al., 2003Lobo C.A. Rodriguez M. Hou G. Perkins M. Oskov Y. Lustigman S. Blood. 2003; 101: 4628-4631Crossref PubMed Scopus (126) Google Scholar). The six EBP family members may bind to different elements of receptors as shown using protease- or glycosylase-treated erythrocytes (Mayer et al., 2004Mayer D.C. Mu J.B. Kaneko O. Duan J. Su X.Z. Miller L.H. Proc. Natl. Acad. Sci. USA. 2004; 101: 2518-2523Crossref PubMed Scopus (87) Google Scholar, Gaur et al., 2004Gaur D. Mayer D.C. Miller L.H. Int. J. Parasitol. 2004; 34: 1413-1429Crossref PubMed Scopus (174) Google Scholar). Thus, the EBP family offers the parasite alternative pathways for erythrocyte invasion, increasing the likelihood of success. EBA-175 operates in one parasite invasion pathway that is sialic acid dependent. This parasite protein is a potential vaccine and drug target candidate. Experiments with EBA-175-specific antibodies, recombinant glycophorin A, and certain EBA-175 regions (when presented as peptides) all prevent either erythrocyte invasion or binding of recombinant EBA-175 to red blood cells (Jakobsen et al., 1998Jakobsen P.H. Heegaard P.M. Koch C. Wasniowska K. Lemnge M.M. Jensen J.B. Sim B.K. Infect. Immun. 1998; 66: 4203-4207PubMed Google Scholar, Narum et al., 2000Narum D.L. Haynes J.D. Fuhrmann S. Moch K. Liang H. Hoffman S.L. Sim B.K. Infect. Immun. 2000; 68: 1964-1966Crossref PubMed Scopus (73) Google Scholar). The glycan binding domain of EBA-175 is known as Region II (RII) and contains two Duffy binding-like (DBL) domains called F1 and F2. Duffy binding domains—first described in proteins of a malaria parasite from a different phylogenetic clade that includes Plasmodium vivax—were shown to bind to another human erythrocyte surface molecule, the Duffy antigen. DBL domains are found not only in proteins of different species but also in multiple proteins within a single species (Adams et al., 1992Adams J.H. Sim B.K. Dolan S.A. Fang X. Kaslow D.C. Miller L.H. Proc. Natl. Acad. Sci. USA. 1992; 89: 7085-7089Crossref PubMed Scopus (393) Google Scholar, Adams et al., 2001Adams J.H. Blair P.L. Kaneko O. Peterson D.S. Trends Parasitol. 2001; 17: 297-299Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). The presence of a DBL domain defines the ebl superfamily that includes not only the EBP proteins but also the var family. PfEMP-1, a P. falciparum protein encoded by var, is expressed on the surface of infected red blood cells and is responsible for the sequestration-associated pathology of this parasite. Only one of a rapidly recombining and mutating pool of 60 var genes is expressed by each individual parasite. Switching var gene expression results in clonal antigenic variation of PfEMP-1, which helps the parasite to evade the host immune response. Thus, the structural resolution of the EBA-175 protein containing DBL domains and elucidation of its interaction with glycosyl targets by Tolia et al., 2005Tolia N.H. Enemark E.J. Sim B.K.L. Joshua-Tor L. Cell. 2005; 122 (this issue): 183-193Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar is of broad importance. The EBA-175 structure has implications for understanding not only the basic biology of parasite invasion of host cells but also disease-associated pathology. The new work may also contribute to prevention of invasion and sequestration through immunization or through the design of small molecule inhibitors that block DBL-mediated parasite interactions with host cells. The new study (Tolia et al., 2005Tolia N.H. Enemark E.J. Sim B.K.L. Joshua-Tor L. Cell. 2005; 122 (this issue): 183-193Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar) reveals the crystal structure of the extracellular domain of EBA-175 in its pure monomeric and dimeric forms, as well as the dimer structure in a complex with a sialic acid derivative (α-2,3-sialyl lactose). In this way, the investigators elucidate the amino acid residues of EBA-175 that are important for the DBL monomeric structure, dimerization, and glycan binding. The monomeric form reveals the intramolecular disulphide bridges that are largely conserved in all DBL domains of EBPs (see Figure 1 in Tolia et al., 2005Tolia N.H. Enemark E.J. Sim B.K.L. Joshua-Tor L. Cell. 2005; 122 (this issue): 183-193Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). These bridges give specific structure to the F1 and F2 DBL domains, with each domain containing the same novel protein fold. The F1 and F2 DBL domains each have two subdomains and form independent, largely helical structures within the RII region of EBA-175. These structures are brought together in the dimer to produce two prominent central channels that contain four of the six glycan binding sites. Both F1 and F2 in the RII monomer form what the authors term β fingers, which are β hairpins that interact with a cavity in the second monomer (F2 with F1, and F1 with F2) to stabilize the dimer. Dimerization also creates a more hidden pocket in the top of the structure, within which lie the two remaining glycan binding sites. It is not possible for glycans to directly access this deep pocket without significant perturbation of the protein structure. Thus, the authors propose that RII dimerization occurs around glycophorin A (which is itself a dimer) during merozoite contact with the erythrocyte. Modeling studies indicate that the full O-glycan of glycophorin A could be accommodated by the 6-glycan binding sites of the RII dimer. During merozoite invasion of an erythrocyte, the requirement for EBA-175 dimerization should increase the specificity of the interaction between the parasite and the host cell. This may provide a fail-safe mechanism to avoid potentially futile signaling through the EBA-175 cytoplasmic domains, which are essential for merozoite invasion, though not for erythrocyte engagement (Gilberger et al., 2003Gilberger T.W. Thompson J.K. Reed M.B. Good R.T. Cowman A.F. J. Cell Biol. 2003; 162: 317-327Crossref PubMed Scopus (70) Google Scholar). Signal transduction via EBA-175 is expected to trigger the later phases of invasion involving merozoite orientation and junction formation between the apical end of the merozoite and the erythrocyte. During these processes, apical organelles of the merozoite associated with invasion (rhoptries, micronemes, and dense granules) are activated. The authors next validated their observations of the resolved RII structure and its interaction with glycans. They expressed mutant RII on the surface of cultured cells in vitro and observed the ability of the mutant protein to bind to erythrocytes in a well-established assay. Mutation of the residues involved in dimer-associated electrostatic contacts between monomers of EBA-175 (that influenced the β finger/cavity interactions) or mutation of various glycan binding sites resulted in a significant reduction in the erythrocyte binding capacity of the mutant recombinant EBA-175. Thus, both dimerization and the implicated glycan binding sites of EBA-175 are important for physical engagement of the erythrocyte. The structure of the RII region of EBA-175 containing two DBL domains, and resolution of the interactions of these domains with glycans, elicits a measure of optimism about the design of small molecule inhibitors. Such inhibitors could be used to competitively block dimerization of RII or to block binding of RII to glycans of the host cell receptor. Such small molecule inhibitors would ideally prevent the merozoite from initial recognition of its erythrocyte target and the orientation steps that enable the parasite to invade red blood cells leading to disease progression. Given that merozoites have a short half-life in the bloodstream, even small degrees of interference may be sufficient to prevent invasion of erythrocytes. An increased failure rate for successful invasion by merozoites not only may prevent disease progression but also may increase the supply of substrate to the immune system, possibly enhancing antimerozoite immunity of the host. Antibodies that prevent merozoite invasion are crucial players in the general immunity established in Africans experiencing long-term and repeated exposure to malaria infection (Cohen and Butcher, 1970Cohen S. Butcher G.A. Nature. 1970; 225: 732-734Crossref PubMed Scopus (21) Google Scholar). The protein structures reported by Tolia et al., 2005Tolia N.H. Enemark E.J. Sim B.K.L. Joshua-Tor L. Cell. 2005; 122 (this issue): 183-193Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar allow the structures of DBLs, their potential dimers, and their complexes with host receptors to be modeled more accurately. Hopefully, a single molecule inhibitor might be derived that blocks the interactions of the DBL domains of all EBP family members, perhaps blocking their dimerization. Considering the physical and phylogenetic distribution of DBL domains in Plasmodium, Tolia and coworkers have provided a crucial template for the development of tools to reduce disease burden resulting from human infection with this parasite. However, this promise may be compromised by the sheer diversity of DBL domains and the structures to which they bind. In addition, the potential of the parasite to rapidly evolve such structures may mean that the design of simple molecules to inhibit specific DBL binding activities will remain a daunting problem. The author would like to thank Chris Janse and Shahid Khan for critical reading of the text.