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• W1978734850 abstract "•EPCR binding is retained by PfEMP1 CIDRα1 domains despite huge sequence variation•Diverse CIDRα1 domains retain structural and chemical features to bind to EPCR•CIDRα1 domains mimic features of a natural ligand of EPCR and block its binding•Patient sera contain neutralizing antibodies that prevent parasite binding to EPCR The PfEMP1 family of surface proteins is central for Plasmodium falciparum virulence and must retain the ability to bind to host receptors while also diversifying to aid immune evasion. The interaction between CIDRα1 domains of PfEMP1 and endothelial protein C receptor (EPCR) is associated with severe childhood malaria. We combine crystal structures of CIDRα1:EPCR complexes with analysis of 885 CIDRα1 sequences, showing that the EPCR-binding surfaces of CIDRα1 domains are conserved in shape and bonding potential, despite dramatic sequence diversity. Additionally, these domains mimic features of the natural EPCR ligand and can block this ligand interaction. Using peptides corresponding to the EPCR-binding region, antibodies can be purified from individuals in malaria-endemic regions that block EPCR binding of diverse CIDRα1 variants. This highlights the extent to which such a surface protein family can diversify while maintaining ligand-binding capacity and identifies features that should be mimicked in immunogens to prevent EPCR binding. The PfEMP1 family of surface proteins is central for Plasmodium falciparum virulence and must retain the ability to bind to host receptors while also diversifying to aid immune evasion. The interaction between CIDRα1 domains of PfEMP1 and endothelial protein C receptor (EPCR) is associated with severe childhood malaria. We combine crystal structures of CIDRα1:EPCR complexes with analysis of 885 CIDRα1 sequences, showing that the EPCR-binding surfaces of CIDRα1 domains are conserved in shape and bonding potential, despite dramatic sequence diversity. Additionally, these domains mimic features of the natural EPCR ligand and can block this ligand interaction. Using peptides corresponding to the EPCR-binding region, antibodies can be purified from individuals in malaria-endemic regions that block EPCR binding of diverse CIDRα1 variants. This highlights the extent to which such a surface protein family can diversify while maintaining ligand-binding capacity and identifies features that should be mimicked in immunogens to prevent EPCR binding. Parasites, such as the Plasmodium species that cause malaria, have developed strategies to aid survival in a mammalian host and to multiply in the nutrient-rich blood. They must make specific interactions with host molecules, enabling them to invade cells, acquire nutrients, and populate protected environments. At the same time, they must avoid detection by components of the innate and acquired immune systems. A common evolutionary strategy, employed by many unicellular eukaryotic parasites, is expansive development of a family of surface proteins, which lie at the interface between host and parasite. Examples include PfEMP1 (Leech et al., 1984Leech J.H. Barnwell J.W. Miller L.H. Howard R.J. Identification of a strain-specific malarial antigen exposed on the surface of Plasmodium falciparum-infected erythrocytes.J. Exp. Med. 1984; 159: 1567-1575Google Scholar), RIFIN (Kyes et al., 1999Kyes S.A. Rowe J.A. Kriek N. Newbold C.I. Rifins: a second family of clonally variant proteins expressed on the surface of red cells infected with Plasmodium falciparum.Proc. Natl. Acad. Sci. USA. 1999; 96: 9333-9338Google Scholar), and STEVOR (Cheng et al., 1998Cheng Q. Cloonan N. Fischer K. Thompson J. Waine G. Lanzer M. Saul A. stevor and rif are Plasmodium falciparum multicopy gene families which potentially encode variant antigens.Mol. Biochem. Parasitol. 1998; 97: 161-176Google Scholar) of Plasmodium falciparum, VIR of P. vivax (del Portillo et al., 2001del Portillo H.A. Fernandez-Becerra C. Bowman S. Oliver K. Preuss M. Sanchez C.P. Schneider N.K. Villalobos J.M. Rajandream M.A. Harris D. et al.A superfamily of variant genes encoded in the subtelomeric region of Plasmodium vivax.Nature. 2001; 410: 839-842Google Scholar), variant surface glycoproteins (VSGs) of Trypanosoma brucei (Schwede and Carrington, 2010Schwede A. Carrington M. Bloodstream form Trypanosome plasma membrane proteins: antigenic variation and invariant antigens.Parasitology. 2010; 137: 2029-2039Google Scholar), MASP (El-Sayed et al., 2005El-Sayed N.M. Myler P.J. Bartholomeu D.C. Nilsson D. Aggarwal G. Tran A.N. Ghedin E. Worthey E.A. Delcher A.L. Blandin G. et al.The genome sequence of Trypanosoma cruzi, etiologic agent of Chagas disease.Science. 2005; 309: 409-415Google Scholar) and SAP (Carmo et al., 2001Carmo M.S. Santos M.R. Cummings L.M. Araya J.E. Yamauchi L.M. Yoshida N. Mortara R.A. Franco da Silveira J. Isolation and characterisation of genomic and cDNA clones coding for a serine-, alanine-, and proline-rich protein of Trypanosoma cruzi.Int. J. Parasitol. 2001; 31: 259-264Google Scholar) of Trypanosoma cruzi, and SAGs of Toxoplasma gondii (Kasper et al., 1983Kasper L.H. Crabb J.H. Pfefferkorn E.R. Purification of a major membrane protein of Toxoplasma gondii by immunoabsorption with a monoclonal antibody.J. Immunol. 1983; 130: 2407-2412Google Scholar). Expression switching between family members allows parasites to display a series of antigenically distinct surfaces, posing challenges for the immune system and for rational development of vaccines. The PfEMP1 protein family of Plasmodium falciparum is one of the most closely studied surface protein families, with about 60 members encoded in each genome (Smith et al., 2013Smith J.D. Rowe J.A. Higgins M.K. Lavstsen T. Malaria’s deadly grip: cytoadhesion of Plasmodium falciparum-infected erythrocytes.Cell. Microbiol. 2013; 15: 1976-1983Google Scholar, Gardner et al., 2002Gardner M.J. Hall N. Fung E. White O. Berriman M. Hyman R.W. Carlton J.M. Pain A. Nelson K.E. Bowman S. et al.Genome sequence of the human malaria parasite Plasmodium falciparum.Nature. 2002; 419: 498-511Google Scholar). They are expressed on the surfaces of infected erythrocytes where they interact with various human endothelial receptors, tethering these erythrocytes to blood vessel or tissue surfaces. This prevents spleen-mediated clearance of the parasite and allows the infection to build. It also leads to the most severe symptoms of the disease, resulting in inflammation of the brain and the placenta during cerebral or pregnancy-associated malaria (Miller et al., 2002Miller L.H. Baruch D.I. Marsh K. Doumbo O.K. The pathogenic basis of malaria.Nature. 2002; 415: 673-679Google Scholar). PfEMP1 are therefore under dual selection pressure to retain the ability to bind to the vasculature while diversifying into a family of antigenically distinct proteins. The extracellular ectodomains of the PfEMP1 proteins contain 2–10 copies of two Plasmodium-specific domain types, the Duffy-binding-like (DBL) and cysteine-rich interdomain region (CIDR) domains (Baruch et al., 1995Baruch D.I. Pasloske B.L. Singh H.B. Bi X. Ma X.C. Feldman M. Taraschi T.F. Howard R.J. Cloning the P. falciparum gene encoding PfEMP1, a malarial variant antigen and adherence receptor on the surface of parasitized human erythrocytes.Cell. 1995; 82: 77-87Google Scholar, Smith et al., 1995Smith J.D. Chitnis C.E. Craig A.G. Roberts D.J. Hudson-Taylor D.E. Peterson D.S. Pinches R. Newbold C.I. Miller L.H. Switches in expression of Plasmodium falciparum var genes correlate with changes in antigenic and cytoadherent phenotypes of infected erythrocytes.Cell. 1995; 82: 101-110Google Scholar, Su et al., 1995Su X.Z. Heatwole V.M. Wertheimer S.P. Guinet F. Herrfeldt J.A. Peterson D.S. Ravetch J.A. Wellems T.E. The large diverse gene family var encodes proteins involved in cytoadherence and antigenic variation of Plasmodium falciparum-infected erythrocytes.Cell. 1995; 82: 89-100Google Scholar, Gardner et al., 2002Gardner M.J. Hall N. Fung E. White O. Berriman M. Hyman R.W. Carlton J.M. Pain A. Nelson K.E. Bowman S. et al.Genome sequence of the human malaria parasite Plasmodium falciparum.Nature. 2002; 419: 498-511Google Scholar). Individual domains frequently act as discrete ligand-binding modules, with a diverse set of host endothelial surface proteins and carbohydrates identified as partners for different domains (Smith et al., 2013Smith J.D. Rowe J.A. Higgins M.K. Lavstsen T. Malaria’s deadly grip: cytoadhesion of Plasmodium falciparum-infected erythrocytes.Cell. Microbiol. 2013; 15: 1976-1983Google Scholar). DBL and CIDR domains have been divided into specific classes based on sequence similarity and the presence of constituent homology blocks (Smith et al., 2000Smith J.D. Subramanian G. Gamain B. Baruch D.I. Miller L.H. Classification of adhesive domains in the Plasmodium falciparum erythrocyte membrane protein 1 family.Mol. Biochem. Parasitol. 2000; 110: 293-310Google Scholar, Rask et al., 2010Rask T.S. Hansen D.A. Theander T.G. Gorm Pedersen A. Lavstsen T. Plasmodium falciparum erythrocyte membrane protein 1 diversity in seven genomes—divide and conquer.PLoS Comput. Biol. 2010; 6: e1000933Google Scholar). Specific domain subclasses interact with specific endothelial receptors (Smith et al., 2000Smith J.D. Subramanian G. Gamain B. Baruch D.I. Miller L.H. Classification of adhesive domains in the Plasmodium falciparum erythrocyte membrane protein 1 family.Mol. Biochem. Parasitol. 2000; 110: 293-310Google Scholar). However, even within a domain subclass, sequence diversity is high, making it challenging to identify conserved functional regions required to mediate binding to a particular receptor based on sequence analysis (Robinson et al., 2003Robinson B.A. Welch T.L. Smith J.D. Widespread functional specialization of Plasmodium falciparum erythrocyte membrane protein 1 family members to bind CD36 analysed across a parasite genome.Mol. Microbiol. 2003; 47: 1265-1278Google Scholar, Howell et al., 2008Howell D.P. Levin E.A. Springer A.L. Kraemer S.M. Phippard D.J. Schief W.R. Smith J.D. Mapping a common interaction site used by Plasmodium falciparum Duffy binding-like domains to bind diverse host receptors.Mol. Microbiol. 2008; 67: 78-87Google Scholar, Higgins and Carrington, 2014Higgins M.K. Carrington M. Sequence variation and structural conservation allows development of novel function and immune evasion in parasite surface protein families.Protein Sci. 2014; 23: 354-365Google Scholar). Despite significant PfEMP1 sequence diversity, natural immunity to severe malaria is acquired after only one or two severe infections, and immunoglobulin G (IgG) that binds PfEMP1 and prevents adhesion plays a significant role (Bull et al., 1998Bull P.C. Lowe B.S. Kortok M. Molyneux C.S. Newbold C.I. Marsh K. Parasite antigens on the infected red cell surface are targets for naturally acquired immunity to malaria.Nat. Med. 1998; 4: 358-360Google Scholar, Salanti et al., 2004Salanti A. Dahlbäck M. Turner L. Nielsen M.A. Barfod L. Magistrado P. Jensen A.T. Lavstsen T. Ofori M.F. Marsh K. et al.Evidence for the involvement of VAR2CSA in pregnancy-associated malaria.J. Exp. Med. 2004; 200: 1197-1203Google Scholar, Lusingu et al., 2006Lusingu J.P. Jensen A.T. Vestergaard L.S. Minja D.T. Dalgaard M.B. Gesase S. Mmbando B.P. Kitua A.Y. Lemnge M.M. Cavanagh D. et al.Levels of plasma immunoglobulin G with specificity against the cysteine-rich interdomain regions of a semiconserved Plasmodium falciparum erythrocyte membrane protein 1, VAR4, predict protection against malarial anemia and febrile episodes.Infect. Immun. 2006; 74: 2867-2875Google Scholar, Cham et al., 2009Cham G.K. Turner L. Lusingu J. Vestergaard L. Mmbando B.P. Kurtis J.D. Jensen A.T. Salanti A. Lavstsen T. Theander T.G. Sequential, ordered acquisition of antibodies to Plasmodium falciparum erythrocyte membrane protein 1 domains.J. Immunol. 2009; 183: 3356-3363Google Scholar, Gupta et al., 1999Gupta S. Snow R.W. Donnelly C.A. Marsh K. Newbold C. Immunity to non-cerebral severe malaria is acquired after one or two infections.Nat. Med. 1999; 5: 340-343Google Scholar, Nielsen et al., 2002Nielsen M.A. Staalsoe T. Kurtzhals J.A. Goka B.Q. Dodoo D. Alifrangis M. Theander T.G. Akanmori B.D. Hviid L. Plasmodium falciparum variant surface antigen expression varies between isolates causing severe and nonsevere malaria and is modified by acquired immunity.J. Immunol. 2002; 168: 3444-3450Google Scholar, Gonçalves et al., 2014Gonçalves B.P. Huang C.Y. Morrison R. Holte S. Kabyemela E. Prevots D.R. Fried M. Duffy P.E. Parasite burden and severity of malaria in Tanzanian children.N. Engl. J. Med. 2014; 370: 1799-1808Google Scholar). This raises hope that it will be possible to develop a vaccine to mimic this natural immunity and to prevent severe disease. However, such a vaccine must raise antibodies that recognize a diverse set of PfEMP1 proteins, and rational design of constituent immunogens requires an understanding of this diversity and detailed knowledge of the structures of conserved features that should be targeted by inhibitory antibodies. The lack of a structure of a PfEMP1 protein domain in complex with a protein ligand has made such an analysis impossible. In this study, we have combined sequence analysis with structural and biochemical studies to determine the extent to which PfEMP1 domains that interact with a particular receptor can diversify and to identify features that remain conserved. We have focused on the interaction between CIDRα1 domains and endothelial protein C receptor (EPCR), as the expression of CIDRα1-containing PfEMP1s and the EPCR-binding phenotype are both associated with severe childhood malaria (Lavstsen et al., 2012Lavstsen T. Turner L. Saguti F. Magistrado P. Rask T.S. Jespersen J.S. Wang C.W. Berger S.S. Baraka V. Marquard A.M. et al.Plasmodium falciparum erythrocyte membrane protein 1 domain cassettes 8 and 13 are associated with severe malaria in children.Proc. Natl. Acad. Sci. USA. 2012; 109: E1791-E1800Google Scholar, Turner et al., 2013Turner L. Lavstsen T. Berger S.S. Wang C.W. Petersen J.E. Avril M. Brazier A.J. Freeth J. Jespersen J.S. Nielsen M.A. et al.Severe malaria is associated with parasite binding to endothelial protein C receptor.Nature. 2013; 498: 502-505Google Scholar). Indeed, the key role of this interaction in malaria pathogenesis is substantiated by the discovery of altered brain endothelial EPCR expression in cerebral malaria patients (Moxon et al., 2013Moxon C.A. Wassmer S.C. Milner Jr., D.A. Chisala N.V. Taylor T.E. Seydel K.B. Molyneux M.E. Faragher B. Esmon C.T. Downey C. et al.Loss of endothelial protein C receptors links coagulation and inflammation to parasite sequestration in cerebral malaria in African children.Blood. 2013; 122: 842-851Google Scholar). Additionally, a polymorphism in the transmembrane domain of EPCR that leads to increased plasma levels of soluble receptor also associates with protection from severe malaria in a Thai population (Naka et al., 2014Naka I. Patarapotikul J. Hananantachai H. Imai H. Ohashi J. Association of the endothelial protein C receptor (PROCR) rs867186-G allele with protection from severe malaria.Malar. J. 2014; 13: 105Google Scholar). Here, we show that EPCR-binding CIDRα1 domains are extremely diverse, even in the residues that directly contact EPCR. However, we find that conserved structural features with conserved bonding potential are retained to maintain this binding phenotype. This shows the extent to which such a parasite protein family can diversify while retaining high-affinity ligand binding and characterizes the features that should be targeted in development of therapeutics to block EPCR binding in severe malaria. Endothelial protein C receptor binding was identified as a property of CIDRα1 domain variants found in PfEMP1 proteins containing two particular combinations of domains: domain cassette 8 (DBLα2-CIDRα1.1-DBLβ12-DBLγ4/γ6) and domain cassette 13 (DBLα1.7-CIDRα1.4) (Turner et al., 2013Turner L. Lavstsen T. Berger S.S. Wang C.W. Petersen J.E. Avril M. Brazier A.J. Freeth J. Jespersen J.S. Nielsen M.A. et al.Severe malaria is associated with parasite binding to endothelial protein C receptor.Nature. 2013; 498: 502-505Google Scholar). These cassettes are present in PfEMP1s expressed in a large proportion of tested children suffering from severe malaria (Lavstsen et al., 2012Lavstsen T. Turner L. Saguti F. Magistrado P. Rask T.S. Jespersen J.S. Wang C.W. Berger S.S. Baraka V. Marquard A.M. et al.Plasmodium falciparum erythrocyte membrane protein 1 domain cassettes 8 and 13 are associated with severe malaria in children.Proc. Natl. Acad. Sci. USA. 2012; 109: E1791-E1800Google Scholar, Bertin et al., 2013Bertin G.I. Lavstsen T. Guillonneau F. Doritchamou J. Wang C.W. Jespersen J.S. Ezimegnon S. Fievet N. Alao M.J. Lalya F. et al.Expression of the domain cassette 8 Plasmodium falciparum erythrocyte membrane protein 1 is associated with cerebral malaria in Benin.PLoS ONE. 2013; 8: e68368Google Scholar) and also in parasites selected for adhesion to brain endothelial cells (Avril et al., 2012Avril M. Tripathi A.K. Brazier A.J. Andisi C. Janes J.H. Soma V.L. Sullivan Jr., D.J. Bull P.C. Stins M.F. Smith J.D. Smith J.D. A restricted subset of var genes mediates adherence of Plasmodium falciparum-infected erythrocytes to brain endothelial cells.Proc. Natl. Acad. Sci. USA. 2012; 109: E1782-E1790Google Scholar, Claessens et al., 2012Claessens A. Adams Y. Ghumra A. Lindergard G. Buchan C.C. Andisi C. Bull P.C. Mok S. Gupta A.P. Wang C.W. et al.A subset of group A-like var genes encodes the malaria parasite ligands for binding to human brain endothelial cells.Proc. Natl. Acad. Sci. USA. 2012; 109: E1772-E1781Google Scholar), suggesting a pivotal role in severe outcomes of P. falciparum infections. EPCR binding by PfEMP1s was mapped exclusively to their CIDRα1.1 and CIDRα1.4 domains. Indeed, the CIDRα1.1 domain of the IT4var20 PfEMP1 protein bound to EPCR with an affinity comparable to that of the whole ectodomain (Turner et al., 2013Turner L. Lavstsen T. Berger S.S. Wang C.W. Petersen J.E. Avril M. Brazier A.J. Freeth J. Jespersen J.S. Nielsen M.A. et al.Severe malaria is associated with parasite binding to endothelial protein C receptor.Nature. 2013; 498: 502-505Google Scholar). Other CIDR domain classes, not present in DC8 and DC13 domain cassettes, such as the CIDRα2 and CIDRα3 domains, did not interact with EPCR but bound to CD36 (Turner et al., 2013Turner L. Lavstsen T. Berger S.S. Wang C.W. Petersen J.E. Avril M. Brazier A.J. Freeth J. Jespersen J.S. Nielsen M.A. et al.Severe malaria is associated with parasite binding to endothelial protein C receptor.Nature. 2013; 498: 502-505Google Scholar). To test the depth of diversity of EPCR-binding domains, we expanded our collection of CIDRα1 domain sequences from the previously described 66 sequences, originating mainly from seven parasite genomes (Kraemer and Smith, 2006Kraemer S.M. Smith J.D. A family affair: var genes, PfEMP1 binding, and malaria disease.Curr. Opin. Microbiol. 2006; 9: 374-380Google Scholar, Rask et al., 2010Rask T.S. Hansen D.A. Theander T.G. Gorm Pedersen A. Lavstsen T. Plasmodium falciparum erythrocyte membrane protein 1 diversity in seven genomes—divide and conquer.PLoS Comput. Biol. 2010; 6: e1000933Google Scholar), by addition of domain sequences extracted from assemblies of whole-genome sequencing data from 226 parasite isolates collected in both Africa and Asia (Manske et al., 2012Manske M. Miotto O. Campino S. Auburn S. Almagro-Garcia J. Maslen G. O’Brien J. Djimde A. Doumbo O. Zongo I. et al.Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing.Nature. 2012; 487: 375-379Google Scholar), resulting in a total data set of 885 sequences. These domains were grouped, based on phylogenetic analysis, into eight previously defined subclasses (CIDRα1.1–1.8) with an additional splitting of CIDRα1.5, CIDRα1.6, and CIDRα1.8 variants into two, generating CIDRα1.5a/b, CIDRα1.6a/b, and CIDRα1.8a/b (Figure 1A; Table S1, available online). To determine which subclasses contain features required to bind EPCR, members of each subclass, chosen to represent the diversity across CIDRα1 domains, were produced in an insect cell system and tested for binding to EPCR and CD36 by ELISA. All proteins bound to EPCR, with the exception of CIDRα1.2 and CIDRα1.3 domains, which are both found in var1 genes considered to be pseudogenes (Figures 1A and S1A). Binding was further characterized by surface plasmon resonance (SPR), allowing determination of binding affinities and kinetic constants. We developed an SPR assay in which EPCR was produced with an N-terminal biotin, allowing coupling to a chip with an orientation matching that found on the cell surface and allowing complete regeneration between measurements. This was used to show that all members of subclasses CIDRα1.1 and CIDRα1.4–1.8 bound to EPCR. The majority of domains bound with high affinities in the range of 0.3–60 nM, but with a few weaker binders (Figures 1A and S1B, Table S2). Despite differences in affinity, it was noticeable that all domains bound with a slow off rate. Indeed, kinetic analysis showed less variation in rate constants for dissociation than in those for association, with a propensity toward slow off rates (Figure S1; Table S2), suggesting that these domains are under selection pressure to form a stable complex with EPCR. To better understand the degree of diversity of EPCR-binding domains, we analyzed 737 different CIDRα1 sequences from members of the six EPCR-binding subclasses (CIDRα1.1 and CIDRα1.4–1.8). These showed little identity between variants, with just 14 residues (6.5%) absolutely conserved and a further 22 residues conserved in more than 90% of the domains (Figure 1B; Table S3). Most conserved residues are cysteines or aromatics. This is reminiscent of the PfEMP1 DBL domains in which the small percentage of conserved cysteine and aromatic residues are found in the domain core where they play a structural role (Batchelor et al., 2011Batchelor J.D. Zahm J.A. Tolia N.H. Dimerization of Plasmodium vivax DBP is induced upon receptor binding and drives recognition of DARC.Nat. Struct. Mol. Biol. 2011; 18: 908-914Google Scholar, Batchelor et al., 2014Batchelor J.D. Malpede B.M. Omattage N.S. DeKoster G.T. Henzler-Wildman K.A. Tolia N.H. Red blood cell invasion by Plasmodium vivax: structural basis for DBP engagement of DARC.PLoS Pathog. 2014; 10: e1003869Google Scholar, Higgins, 2008Higgins M.K. The structure of a chondroitin sulfate-binding domain important in placental malaria.J. Biol. Chem. 2008; 283: 21842-21846Google Scholar, Higgins and Carrington, 2014Higgins M.K. Carrington M. Sequence variation and structural conservation allows development of novel function and immune evasion in parasite surface protein families.Protein Sci. 2014; 23: 354-365Google Scholar, Hodder et al., 2012Hodder A.N. Czabotar P.E. Uboldi A.D. Clarke O.B. Lin C.S. Healer J. Smith B.J. Cowman A.F. Insights into Duffy binding-like domains through the crystal structure and function of the merozoite surface protein MSPDBL2 from Plasmodium falciparum.J. Biol. Chem. 2012; 287: 32922-32939Google Scholar, Khunrae et al., 2010Khunrae P. Dahlbäck M. Nielsen M.A. Andersen G. Ditlev S.B. Resende M. Pinto V.V. Theander T.G. Higgins M.K. Salanti A. Full-length recombinant Plasmodium falciparum VAR2CSA binds specifically to CSPG and induces potent parasite adhesion-blocking antibodies.J. Mol. Biol. 2010; 397: 826-834Google Scholar, Lin et al., 2012Lin D.H. Malpede B.M. Batchelor J.D. Tolia N.H. Crystal and solution structures of Plasmodium falciparum erythrocyte-binding antigen 140 reveal determinants of receptor specificity during erythrocyte invasion.J. Biol. Chem. 2012; 287: 36830-36836Google Scholar, Malpede et al., 2013Malpede B.M. Lin D.H. Tolia N.H. Molecular basis for sialic acid-dependent receptor recognition by the Plasmodium falciparum invasion protein erythrocyte-binding antigen-140/BAEBL.J. Biol. Chem. 2013; 288: 12406-12415Google Scholar, Singh et al., 2006Singh S.K. Hora R. Belrhali H. Chitnis C.E. Sharma A. Structural basis for Duffy recognition by the malaria parasite Duffy-binding-like domain.Nature. 2006; 439: 741-744Google Scholar, Tolia et al., 2005Tolia N.H. Enemark E.J. Sim B.K. Joshua-Tor L. Structural basis for the EBA-175 erythrocyte invasion pathway of the malaria parasite Plasmodium falciparum.Cell. 2005; 122: 183-193Google Scholar, Vigan-Womas et al., 2012Vigan-Womas I. Guillotte M. Juillerat A. Hessel A. Raynal B. England P. Cohen J.H. Bertrand O. Peyrard T. Bentley G.A. et al.Structural basis for the ABO blood-group dependence of Plasmodium falciparum rosetting.PLoS Pathog. 2012; 8: e1002781Google Scholar). All residues conserved in the EPCR-binding domains are also totally conserved in CIDRα1.2 and CIDRα1.3 subclasses that do not bind EPCR, showing that CIDRα1 domains are an extremely diverse subfamily that lacks conserved residues that correlate with EPCR binding. We therefore determined cocrystal structures to allow us to understand the molecular basis for EPCR binding and to rationalize how sequence diversity is compatible with the retention of this binding phenotype. We have previously shown that a single CIDRα1 domain binds to EPCR with the same affinity as the full-length PfEMP1 protein, demonstrating that EPCR binding capability is contained entirely within CIDRα1 (Turner et al., 2013Turner L. Lavstsen T. Berger S.S. Wang C.W. Petersen J.E. Avril M. Brazier A.J. Freeth J. Jespersen J.S. Nielsen M.A. et al.Severe malaria is associated with parasite binding to endothelial protein C receptor.Nature. 2013; 498: 502-505Google Scholar). Our strategy here was to select a diverse set of domains, increasing the likelihood of identifying a complex that would crystallize, and then combine the structures we obtained with sequence analysis and biophysical studies to rationalize EPCR binding by the protein family. We therefore generated a panel of CIDRα1 domains with domain boundaries appropriate for crystallization and used SPR and isothermal titration calorimetry (ITC) to confirm binding to EPCR with nanomolar affinities and slow dissociation rates (Figures S1 and S2). These domains were reconstituted into complexes with the extracellular domain of EPCR and examined using small-angle X-ray scattering, analytical ultracentrifugation, and multi-angle laser light scattering. In each case, a 1:1 complex formed with no higher-order assemblies observed (Figure S2). Small-angle X-ray scattering of a protein containing the three membrane distal domains of DD2var32 (DBLα1.7-CIDRα1.4-DBLβ1), alone and in complex with EPCR, also revealed the formation of a 1:1 PfEMP1:EPCR complex (Table S4). In addition, molecular envelopes showed a predominantly elongated architecture for these three domains (Figure 2A). This architecture did not alter in the presence of EPCR, but instead a single additional protein density was evident, attached to the central CIDRα1 domain (Figure 2A), supporting the notion of a modular arrangement for the PfEMP1 protein with a single EPCR binding site on the CIDRα1 domain. The CIDRα1:EPCR complexes were next subjected to crystallization trials. Crystals of the HB3var03 CIDRα1.4:EPCR complex formed and diffracted to 2.65Å resolution. The structure was determined by molecular replacement using the structure of EPCR (PDB 1L8J) as a search model, followed by iterative model building and refinement (Figures 2B and S3; Table S5). The structure was consistent with an envelope obtained from solution small-angle X-ray scattering (Figures S2D–S2F), and the two copies of HB3var03 CIDRα1 in the asymmetric unit aligned with a root-mean-square deviation (rmsd) of just 0.09Å, showing them to be extremely similar. A second complex, containing IT4var07 CIDRα1.4:EPCR complex also crystallized, and crystals diffracted to 2.9Å resolution. Despite a sequence identity of 78.5% compared to the HB3var03 CIDRα1 domain, this complex crystallized in a different space group and with different crystal packing. The structure was determined using HB3var03 CIDRα1 and EPCR as separate search models in molecular replacement. HB3var03 and IT4var07 CIDRα1 are extremely similar (rmsd = ∼0.3Å), but despite differences in space groups and crystal packing, both CIDRα1 domains bind to EPCR using the equivalent surface (Figure 2C). The two CIDRα1 domains are built around a long three-helical core bundle. On one side of this bundle lies a four-stranded β sheet. On the opposite side, between the second and third core helices, an insertion folds into a kinked α helix and a long α helix that lie approximately perpendicular to the core bundle, stabilized by residues F651, V658, and W669 and forming the majority of the EPCR-binding surface (Figure 3A). The α-helical core of the CIDRα1 domain, and the EPCR-binding surface, are well ordered and well defined in the crystal structure, with B factors of 20–40 (Figures S2I and S2J). However, away from the binding surface, the domain is decorated with a variety of loops, some of which are not observed in the electron density, while others are characterized by high B factors, suggesting flexibility. Comparison with the two existing CIDR domain structures shows the CIDRα1 domains to be more similar to CIDRγ from var0 (Vigan-Womas et al., 2012Vigan-Womas I. Guillotte M. Juillerat A. Hessel A. Raynal B. England P. Cohen J.H. Bertrand O. Peyrard T. Bentley G.A. et al.Structural basis for the ABO blood-group dependence of Plasmodium falciparum rosetting.PLoS Pathog. 2012; 8: e1002781Google Scholar) (Figure 2D) than to CIDRα2 from CD36-binding MC179 (Klein et al., 2008Klein M.M. Gittis A.G. Su H.P. Makobongo M.O. Moore J.M. Singh S. Miller L.H. Garboczi D.N. The cysteine-rich interdomain region from the highly" @default.
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