Abstract
Plasmodium parasites are unicellular organisms that cause malaria and ensuing morbidity in afflicted regions of the world. Although the most dreadful of human malaria parasites is P. falciparum, its variant P. vivax is common in India, South East Asia and Latin America. In addition, a fifth human parasite called P. knowlesi is able to cause infections in humans in some regions. P. vivax has a wide distribution with ~40% of world’s population at risk of infection and 70-130 million annual cases. The molecular mechanisms by which P. knowlesi and P. vivax invade human red blood cells have long been studied. Malaria parasite erythrocytic stages comprise of repeated propagation of parasites via cyclical invasion of host RBCs using dedicated receptor-ligand interactions. A family of erythrocyte-binding proteins (EBPs) that include P. knowlesi and P. vivax Duffy-binding proteins (PvDBP and PkDBP respectively) attach to duffy antigen (DARC) on human erythrocytes for invasion via their duffy binding-like domains (DBLs). Here, we provide a comprehensive overview that presents new insights on the atomic resolution interactions that underpin the binding of Duffy antigen on human red blood cells with P. knowlesi and P. vivax DBL domains. Using extensive structural and biochemical data from the past decade, we provide a novel, testable and overarching model that fully rationalizes even contradictory pieces of evidence that have so far existed in the literature. We resolve the conundrum of how parasite-encoded DBL domains recognize human DARC and its two sulfated tyrosine residues. We provide evidence of two distinct DARC binding sites on P. knowlesi and P. vivax DBLs that together likely engage the extracellular domain of DARC. These analyses are important for both malaria vaccine and inhibitor development efforts that are targeted at abrogating DARC-DBL interactions as an avenue to prevent invasion of malaria parasites into human red blood cells.
Duffy antigen receptor for chemokines (DARC) exploited of ingress of malaria parasites
The duffy blood group antigen for chemokine (DARC) is a seven transmembrane protein present on surface of erythrocytes and endothelial cells (Fig 1) [1]. It is a promiscuous cytokine/chemokine receptor involved in pro-inflammatory processes of the immune system, where it acts as a scavenger, reducing excess amounts of toxic chemokines produced in some pathological conditions [2]. DARC is also used as an entry vehicle by the malaria parasites P. knowelsi and P. vivax (Pk/Pv) (Fig. 1) [3-5]. The DARC regions spanning its soluble domain from 1-60 contain two key tyrosyl residues at positions 30 and 41, where post-translational modification in the form of sulfation occurs [6]. Of tyrosines 30 and 41, it is critically the sulfation of the latter which seems essential for high affinity binding to Pk/Pv proteins [6]. The Pk/Pv erythrocyte binding proteins (PvEBPs) present on merozoite surface are responsible for binding to the DARC receptor on reticulocytes, and then mediating an irreversible junction formation that is vital for the parasite invasion process (Fig. 1) [7-9]. EBPs generally contain one or two extracellular cysteine-rich domains (region II), a second extracellular cysteine-rich domain (region VI), type I trans-membrane domain and a short cytoplasmic domain [10-12]. The key molecular players – EBPs - encoded by Pk/Pv for erythrocyte colonization contain Duffybinding-like (DBL) domains that specifically recognize DARC via intermolecular interactions [11-13]. Pk/Pv DBLs are organized into 3 subdomains, and are typified (mostly) by twelvecysteine residues that are disulfide linked [11, 13-16]. The importance of DBL-DARC pairing is underscored by human genetic data where DARC negative individuals tend to be protected from P. vivax infection [17-21]. Contrary to the established DBL–DARC invasion pathway, there is evidence for DARC-independent invasion pathway in case of P. vivax infections, calling for new caution in assessing the utility of DARC recognizing Pv DBL as a P. vivax vaccine candidate [22, 23].
Binding sites on Pk/Pv DBLs for DARC sulfated tyrosines based on analysis of crystal structures
Pk/Pv DBL subdomains contain majority of the conserved cysteine residues that are (mostly) linked into disulfides and likely contribute to the structural integrity of DBLs [11,12,16]. In addition, conservation of hydrophobic residues within DBLs allows for a parasite-specific evolutionary motif that is both constant (in structural terms) and variable (in sequence). Metaphorically, hence, DBLs are built using same principles as antibody structures, where the overall 3D core structures remain the same but sequence variation in exposed residues and loop regions allows for surface diversity that can thus engage with plethora of biomolecular receptors[11,16].
Crystal structure of PkDBL had suggested a region on its subdomain 2 that could accommodate the DARC’s sulfated tyrosine 41 [11], here referred to as Site 1 (Fig 2a). The PkDBL subdomain 2 presents a remarkably surface exposed region of highly conserved residues that arrange into distinct regions lying adjacent to each other – positively charged residues (Lys 96, Lys 100 and Arg 103 and Lys 133) and non-polars (Tyr 94, Leu 168 and Ile 175) [11]. These two dual character surfaces on Pk and Pv DBLs were proposed to bind sulfated tyrosyl 41 based on structural considerations emanating from collation of mutagenesis data from two distinct groups at the time [15, 24]. Further, the proposed residues on Pk and Pv DBLs remain fully conserved in these DARC binding DBLs, hence lending support to the proposal of their essential role in binding to DARC’s sulfated tyrosine 41 [11,15,16]. These data, coupled with the observation that sulfation of DARC’s Tyr41 significantly enhanced binding to Pk/Pv DBLs led to a model of DARC’s docking onto Pk/Pv DBLs via the identified site on PkDBL (now labeled as Site 1, Fig 2a-c).
The crystal structure of Pv DBL later identified another site on DARC-binding DBL (Fig 2b, d), hereafter labeled Site 2, where based on the binding of crystallization liquor phosphate/selenate (ostensibly mimicking the sulfate of DARC’s tyr 30/41), it was proposed as the key site for DARC recognition [25]. The DARC-DBL engagement was further proposed to lead to dimerization of PkDBL [25]. Indeed, the existence of this putative sulphotyrosine binding site (Site 2) was proposed based on the evidence for the bound phosphate/selenate groups at the (proposed) dimer interface of PvDBP [25]. In a latter study of PvDBL-DARC peptide complex, DARC resides were indeed found in proximity to Site 2 (Fig 2b, d) but of the DARC region that contains Tyr30 (unsulfated) and not Tyr41 [26]. Hence, the conundrum of where the binding site for the DARC peptide that contains the key Tyr41 resides on Pk/Pv DBLs had remained unsolved. It is further noteworthy that although it is feasible that the DBL engagement with DARC in vivo drives oligomerization of the DBLs (and speculatively of the Pk/Pv EBPs), both biochemical evidence from our laboratory and PISA calculations do not support dimerization of Pk/PvDBLs regardless of the presence of either sulfates, phosphates or selenates [11, 25, 26]. Indeed, the proposed trimeric/tetrameric oligomer states for PvDBL-DARC based on crystal structure of DARC peptide with PvDBL are also unsupported by PISA [27], a gold-standard software in macromolecular crystallography that takes into account protomer packing, stability and monomer buried surface area values into consideration for assessing oligomeric states [25,26].
A new overarching model for coupling of Pk/Pv DBLs with DARC via its sulfated tyrosines
Driven by the observation of bound phosphates/selenates in Pv DBL, we investigated the Pk DBL crystal structure and observed a bound sulfate at Site 1 (Fig 2b, c) which overlaps with earlier our proposed model [11], where the highly conserved positively charged residues were earlier mapped [11], and which were identified in two different mutagnesis screens [11, 15, 23]. The presence of bound sulfate at Site 1 in Pk DBL is a striking observation and it immediately opens the possibility of reinterpreting the engagement rules for DARC recognition by Pk/Pv DBLs. Hence, based on (a) Pk DBL-sulfate complex (Site 1, Fig. 2b, c, e), (b) PvDBL structure with bound phosphate (in Site 2, Fig. 2b, d), (c) PvDBL structure with bound DARC and tyr 30 (Site 2, Fig. 2b, d) and available mutagenesis data on DARC/DBL interactions, we propose a simple, novel, testable and fully rationalized resolution to the conundrum of DARC-DBL coupling (Fig. 2e, f). We envisage that DARC peptide 1-60 may dock on to Pk/Pv DBLs via its sulphated tyro 41 on Site 1 (Fig. 2b, e, f) and on to Site 2 via its tyr 30 (Fig 2b, e, f). Based on structural, geometrical and molecular size considerations, it is both reasonable and feasible that the DARC peptide stretches from Site 1 to Site 2 on Pk/Pv DBLs and hooks with both via its sulfated tyr41 and tyr 30 (Fig 2f.) Indeed, the side chains in Pk/Pv DBLs that constitute Site 1 are identical (4/4) between Pk and Pv DBLs, and mostly (2/3) conserved for Site 2 (Fig 2). Invariance in Site 1 residues from Pk and Pv DBL - Lys96, Lys100, Arg103 and Lys 177 (Pk DBL numbering)[11], that directly contact the bound sulphate moiety (Fig 2e), and their implication via binding data based on earlier mutagenesis experiments provide very strong support for the primacy of Site 1 in DARC-DBL binding.
Our presented model resolves the puzzle of how parasite-encoded DBL domains recognize human DARC and its two sulfated tyrosine residues. Our analysis provides new evidence for two distinct DARC binding sites on P. knowlesi and P. vivax DBLs that together likely engage the extracellular domain of DARC via its tyrosine 30 and sulfated tyrosine 41. Our analyses shall be important for both malaria vaccine and inhibitor development efforts that are targeted at abrogating DARC-DBL interactions as an avenue to prevent invasion of malaria parasites into human red blood cells.
Conclusions
Structural investigations of receptor-ligand interactions that allow ingress of malaria parasites into human erythrocytes have yielded substantial information on the residue-level engagements that drive invasion of P. knowlesi and P. vivax. Over the past decade, it has become clear that the intricacies of DARC-DBL are yet to be fully revealed, despite substantial advances. We consolidated structural and biochemical data on Pk/Pv DBL interactions with DARC via its extracellular peptide spanning residues 1-60. We specifically focused on rationalizing the potentially disparate sets of binding sites for tyrosines 30 and 41 of DARC on Pk/PvDBLs. We have resolved the possible muddle in the literature on mechanics of how parasite-encoded DBL domains recognize human DARC and its two sulfated tyrosine residues. Our analysis collates structural data emanating from PK/PV DBL Sites 1 and 2, from bound sulfate in Site 1 and from bound DARC peptide in Site 2 to provide an architectural framework that suggests twin binding for DARC’s tyrosines on Pk/Pv DBLs. These analyses will allow a yet deeper dissection of DARC-DBL interactions that are vital for invasion of many strains of P. vivax malaria parasites into human red blood cells.
Key learning points
P. knowlesi and P. vivax cause malaria in humans and these parasites exploit their encoded DBL domains to recognize and bind human DARC to invade red blood cells.
In most human infections of P. knowlesi and P. vivax, parasite ingress may be prevented if the DBL engagement with DARC can be intercepted.
Post-translational modification of human DARC at tyrosines 30 and 41 occurs, and of these it the DARC’s sulfated Tyr 41 that confers strength to binding between DARC and Pv/Pk DBLs.
A resolution to the existing conundrum of how both DARC tyrosines 30 and 41 hook with DBLs is presented here. We show that Pk/Pv DBLs possess dual binding sites where Site 1 and Site 2 link with DARC’s tyrosines 41 and 30.
Our new model rationalizes available structural and biochemical data on the intricacies of DBL-DARC interactions, and presents a novel, testable and overarching framework for understanding DBL-DARC engagement.
Key papers in the field
Hans D, Pattnaik P, Bhattacharyya A, et al. Mapping binding residues in the Plasmodium vivax domain that binds Duffy antigen during red cell invasion. Mol Microbiol. 2005;55(5):1423-1434. doi:10.1111/j.1365-2958.2005.04484.x.
Singh SK, Hora R, Belrhali H, Chitnis CE, Sharma A. Structural basis for Duffy recognition by the malaria parasite Duffy-binding-like domain. Nature. 2006;439(7077):741-744. doi:10.1038/nature04443.
Chitnis CE, Sharma A. Targeting the Plasmodium vivax Duffy-binding protein. Trends Parasitol. 2008;24(1):29-34. doi:10.1016/j.pt.2007.10.004.
Batchelor JD, Zahm JA, Tolia NH. Dimerization of Plasmodium vivax DBP is induced upon receptor binding and drives recognition of DARC. Nat Struct Mol Biol. 2011;18(8):908-914. doi:10.1038/nsmb.2088.
Batchelor JD, Malpede BM, Omattage NS, DeKoster GT, Henzler-Wildman KA, Tolia NH. Red Blood Cell Invasion by Plasmodium vivax: Structural Basis for DBP Engagement of DARC. PLoS Pathog. 2014;10(1). doi:10.1371/journal.ppat.1003869.
Ntumngia FB, Thomson-Luque R, Pires C V., Adams JH. The role of the human duffy antigen receptor for chemokines in malaria susceptibility: Current opinions and future treatment prospects. J Receptor Ligand Channel Res. 2016;9: 1–10. doi:10.2147/JRLCR.S99725