Oct 20, 2007 - Mapping a common interaction site used by Plasmodium falciparum Duffy binding-like domains to bind diverse host receptors. Dasein P.-G.
Molecular Microbiology (2008) 67(1), 78–87 䊏
doi:10.1111/j.1365-2958.2007.06019.x First published online 30 November 2007
Mapping a common interaction site used by Plasmodium falciparum Duffy binding-like domains to bind diverse host receptors Dasein P.-G. Howell,1,2 Emily A. Levin,1 Amy L. Springer,3 Susan M. Kraemer,1† David J. Phippard,2 William R. Schief4 and Joseph D. Smith1,2* 1 Seattle Biomedical Research Institute, 307 Westlake Ave N, Ste 500, Seattle, WA 98109-5219, USA. 2 Department of Pathobiology, University of Washington, Box 357238, Seattle, WA 98195, USA. 3 Biology Department, Mount Holyoke College, 50 College St, South Hadley, MA 01075, USA. 4 Department of Biochemistry, University of Washington, Box 357350, Seattle, WA 98195, USA.
Summary The Duffy binding-like (DBL) domain is a key adhesive module in Plasmodium falciparum, present in both erythrocyte invasion ligands (EBLs) and the large and diverse P. falciparum erythrocyte membrane protein 1 (PfEMP1) family of cytoadherence receptors. DBL domains bind a variety of different host receptors, including intercellular adhesion molecule 1 (ICAM-1), a receptor interaction that may have a role in infected erythrocyte binding to cerebral blood vessels and cerebral malaria. In this study, we expressed the nearly full complement of DBLb-C2 domains from the IT4/25/5 (IT4) parasite isolate and showed that ICAM1-binding domains (DBLb-C2ICAM-1) were confined to group B and group C PfEMP1 proteins and were not present in group A, suggesting that ICAM-1 selection pressure differs between PfEMP1 groups. To further dissect the molecular determinants of binding, we modelled a DBLb-C2ICAM-1 domain on a solved DBL structure and created alanine substitution mutants in two DBLb-C2ICAM-1 domains. This analysis indicates that the DBLb-C2::ICAM-1 interaction maps to the equivalent glycan binding region of EBLs, and suggests a general model for how DBL domains evolve
Accepted 20 October, 2007. *For correspondence. E-mail joe.smith@ sbri.org; Tel. (+1) 206 256 7384; Fax (+1) 206 256 7229. †Present address: Malaria Research Institute, Department of Molecular Microbiology and Immunology, Johns Hopkins University, Bloomberg School of Public Health, 615 N Wolfe Steet, Baltimore, MD 21205, USA.
© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd
under dual selection for host receptor binding and immune evasion.
Introduction During blood stage development, Plasmodium falciparum parasites invade erythrocytes and cause them to sequester from blood circulation by binding to host endothelium (Miller et al., 2002). These two processes require the binding function of a common adhesion module called the Duffy binding-like (DBL) domain present in two different types of proteins: the erythrocyte binding ligands (EBLs), which participate in erythrocyte invasion, and the P. falciparum erythrocyte membrane protein 1 (PfEMP1) variants, which are responsible for infected erythrocyte (IE) binding (Howell et al., 2006). As both invasion and IE cytoadherence are mechanisms contributing to parasite virulence, knowledge about how DBL domains function as binding ligands may aid the development of disease interventions. PfEMP1 are large (~250–350 kDa), polymorphic proteins that vary in domain composition and binding specificity (Kraemer and Smith, 2006). They are encoded in each parasite genome by ~60 different var genes, which are expressed in a mutually exclusive fashion (Frank and Deitsch, 2006). Switches in var gene expression allow parasites to evade immunity and sequester at different sites in the body. The completion of the P. falciparum genome sequence has led to a rapid advance in our understanding of var gene organization, revealing that var genes can be classified into three major groups – A, B and C – on the basis of chromosome location, upstream gene flanking sequence (Ups type) and gene orientation (Gardner et al., 2002). A and B gene groups are subtelomeric, while C genes are found in central chromosomal locations. There is also a subset of var genes, the BC group, which has UpsB-like promoters but is present in central chromosomal regions. This general gene organization is retained across parasite isolates (Kraemer et al., 2007) and it has been postulated to influence the functional and antigenic specialization of PfEMP1 proteins through gene recombination hierarchies (Kraemer and Smith, 2003; Lavstsen et al., 2003; Robinson et al., 2003). For instance, group A PfEMP1 proteins have
Interaction site for DBLb-C2 and ICAM-1 79
diverged in sequence from other PfEMP1 proteins and do not bind the major microvasculature receptor, CD36 (Robinson et al., 2003). Whether there are additional host adherence selection pressures that differ between var groups is unknown, but has important implications for understanding the molecular basis for disease. Cerebral malaria is a severe and potentially fatal complication of P. falciparum infections associated with IE sequestration in brain microvasculature (Medana and Turner, 2006). Although the specific parasite–host interactions responsible for cerebral sequestration are still being studied, intercellular adhesion molecule 1 (ICAM-1) is a candidate for IE binding to brain endothelium (Turner et al., 1994; Chakravorty and Craig, 2005). To date, three different PfEMP1 variants from the IT4/25/4 (IT4) parasite isolate and one from an Indian field isolate (JDP8) have been shown to bind ICAM-1 (Berendt et al., 1989; Ockenhouse et al., 1991; Gardner et al., 1996; Chattopadhyay et al., 2004). Of interest, all four ICAM-1 binders use a tandem DBLb-C2 domain for binding, and the three ICAM-1 binders from the IT4 isolate all have UpsB type promoters (Smith et al., 2000a; Chattopadhyay et al., 2004; Springer et al., 2004). However, DBLb-C2 domains are found in group A, B or C PfEMP1 variants (Kraemer et al., 2007), which poses the question whether the different var groups differ in ICAM-1 binding capacity. In addition, the four DBLb-C2ICAM-1 domains share only ~45% sequence identity, making it difficult to delineate critical binding residues. More binding data are therefore needed to define an ICAM-1 binding paradigm of PfEMP1 DBLb-C2 domains. Similar to PfEMP1 proteins, DBL domains in the parasite EBLs (referred to as EBL-DBLs) bind a variety of substrates, both protein and carbohydrate (Miller et al., 2002). Two different EBL-DBL crystal structures have been solved: the tandem DBL domains (referred to as F1 and F2) of the P. falciparum EBA-175 protein (Tolia et al., 2005) and the single DBL domain (PkaDBL) of the P. knowlesi Duffy binding protein (PkaDBP) (Singh et al., 2006). The DBL fold of both structures is very similar despite limited sequence similarity and is characterized by a predominantly a-helical scaffold core anchored by multiple disulfide bonds. EBA-175 and PkaDBP bind different host receptors on the erythrocyte surface and appear to use different mechanisms of binding. Whereas EBA-175 binds glycophorin A as a dimer, the PkaDBP/ Duffy blood group antigen/receptor for chemokines (DARC) interaction is monomeric (Tolia et al., 2005; Singh et al., 2006). Moreover, the EBA-175 glycan binding site maps to the opposite face of the DBL fold from the DARC binding site. Therefore, these comparisons suggest that a single binding site is not sufficient to explain all DBL domain interactions with their varied host receptors.
To develop a more complete picture of the PfEMP1::ICAM-1 interaction, we utilized the recently sequenced var repertoire of the IT4 strain to test DBLb-C2 domains for binding and performed site-directed mutagenesis to identify critical binding residues. Our findings support a model that pre-existing binding sites present in EBL-DBLs were adapted in PfEMP1 variants to engage a large variety of protein and carbohydrate receptors and suggest that selection for ICAM-1 binding differs between PfEMP1 groups.
Results Identification of new ICAM-1 binding DBLb-C2 domains from the IT4 strain To determine the distribution of DBLb-C2 domains in a single parasite strain capable of binding to ICAM-1 (DBLbC2ICAM-1), we expressed 21 DBLb-C2 recombinant proteins from group A, B and C var genes of the IT4 parasite isolate (Kraemer et al., 2007). COS-7 cells expressing the recombinant proteins on their surface were incubated with magnetic beads coated with recombinant ICAM-1/Fc. Results are reported as the percentage of surfacepositive transfected cells bound to beads. In addition to three known DBLb-C2ICAM-1 domains from the IT4 isolate, four additional DBLb-C2ICAM-1 domains were identified in our screen (Table 1). Of interest, six of the seven ICAM-1 binders have UpsB type promoters and the only group C PfEMP1 variant in the IT4 genome that contained a DBLb-C2 domain was a binder (Table 1). In contrast, none of the nine DBLb-C2 domains expressed from A type PfEMP1 proteins bound ICAM-1 (Table 1). Two of the UpsB binders have been mapped to subtelomeric locations (IT4var14 and IT4var31) and two to central chromosomal locations (IT4var27 and IT4var1) (Kraemer et al., 2007). The chromosomal location of the remaining three binders is unknown. Therefore, DBLb-C2 domains capable of binding to ICAM-1 are encoded in both group B and C PfEMP1 variants, but may be absent or not under strong selection in A type PfEMP1 variants.
Phylogenetic and sequence analysis of ICAM-1 binding and non-binding DBLb-C2 domains Previously, the CIDR::CD36 interaction has been studied from a large number of PfEMP1 variants, and it has been shown that CD36 binding sequences cluster distinct from non-binding sequences using traditional phylogenetic approaches (Robinson et al., 2003). A neighbour-joining analysis of ICAM-1 binding and non-binding DBLb-C2 sequences showed that binders sometimes grouped together, although as a whole, they did not group separately from non-binders (Fig. S1). In addition, DBLb-C2
© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 67, 78–87
80 D. P.-G. Howell et al. 䊏
Table 1. ICAM-1 binding analysis of IT4var DBLb-C2 domains. var groupa
% Transfectionb
Intensityc
IT4var2, i IT4var2, ii IT4var7, i IT4var7, ii IT4var8 IT4var18, i IT4var18, iif IT4var22 IT4var35/VAR1CSA
A1 A1 A1 A1 A1 A1 A1 A1 A2
18 ⫾ 8 10 ⫾ 0 12 ⫾ 6 16 ⫾ 6 22 ⫾ 12 6⫾5 12 ⫾ 4 15 ⫾ 1 Springer et al. (2004)
2 2+ 2+ 2 2 1+ 1 2
IT4var12/ItG var1 IT4var14/A4var IT4var31/A4tres IT4var6 IT4var10/varph17 IT4var11 IT4var13 IT4var17 IT4var19 IT4var41 IT4var44p IT4var16/IT-ICAM
B1T B1T B1T B1 B1 B1 B1 B1 B1 B1 B1 B2
9⫾3 Smith et al. (2000a) Smith et al. (2000a) 24 ⫾ 0 8⫾1 23 ⫾ 2 20 ⫾ 1 24 ⫾ 6 17 ⫾ 4 25 ⫾ 1 7⫾2 Springer et al. (2004)
1+
IT4var15/FCR3 var2 IT4var20/FCR3 var3 IT4var27 IT4var1
B1C B2C B3C C1C
9⫾2 23 ⫾ 1 18 ⫾ 4 19 ⫾ 8
Gene name/alias e
2 1+ 2+ 2+ 2+ 1+ 1+ 2+ 2 2+ 2+ 2
% Bindingd 0 0 0 0 0 0 0 0 0 0 0 100 0 0 0 100 ⫾ 0 0 0 96 ⫾ 3 0 100
Previously tested domains
Non-binder Antibodies to DBLb blocked binding ICAM-1 binder
ICAM-1 binder
0 0 100 ⫾ 0 98 ⫾ 4
a. var group is defined in Kraemer et al. (2007). b. The percentage of surface positive transfected cells in 300 DAPI-labelled cells. c. Visual score of the intensity of surface fluorescence using an antibody against the recombinant protein. d. Percentage of 50 transfected cells associated with five or more ICAM-1/Fc-coated beads. e. i and ii denote the 1st or 2nd DBLb-C2 domain encoded in this gene. f. This DBL domain is g type, not b type. DBLb-C2 recombinant proteins were expressed at the surface of COS-7 cells and tested for binding to ICAM-1/Fc-coated beads. Binding was controlled with mock-treated beads that did not receive ICAM-1/Fc. Recombinant protein surface expression was confirmed by immunofluorescence. Results are expressed as the mean ⫾ SD of two independent experiments.
domains from group A PfEMP1 variants tended to group together, although they were not confined to a single branch of the tree (Fig. S1). Therefore, traditional phylogenetic criteria were not able to distinguish ICAM-1 binding from non-binding DBLb-C2 sequences. The seven IT4 DBLb-C2ICAM-1 domains share only 45% amino acid identity, making it difficult to identify characteristics responsible for binding function. We could not identify specific residues that strictly distinguished binding and non-binding sequences using multiple sequence alignments and sequence motif finders (Fig. 1). However, we did observe a concentration of polar residues, ‘T(S/ T)(Y/D)(S/T) motif’, between the C1 and C4 cysteine residues that were more prevalent in DBLb-C2ICAM-1 sequences (identified by asterisks in Fig. 1). Chimera and truncation analysis has shown that residues in both the DBLb and C2 domains are important for binding and/or proper folding, but that C2 domain sequences are not interchangeable between DBLb-C2ICAM-1 sequences (Chattopadhyay et al., 2004; Springer et al., 2004). Therefore, ICAM-1 binding may be sequence context dependent.
Homology modelling and mutagenesis of IT4 var DBLb-C2 domains reveal important residues for binding to ICAM-1 Duffy binding-like domains are key players in two pathogenic processes, erythrocyte invasion and IE cytoadhesion. While all Plasmodium species invade erythrocytes, PfEMP1 proteins are confined to P. falciparum and P. reichenowi, suggesting that PfEMP1-DBL domains evolved from EBL-DBL domains. Therefore, we hypothesized that pre-existing binding sites in EBL proteins were adapted for PfEMP1 binding (Howell et al., 2006). To test this hypothesis and determine which residues were functionally important for DBLb-C2ICAM-1 domain binding to ICAM-1, we performed site-directed alanine substitution mutagenesis of the IT4var16/IT-ICAMvar (var16) DBLbC2ICAM-1 domain. We created a homology model of the var16 DBLb-C2 domain based upon the P. falciparum EBA-175 F2 DBL domain crystal structure (Fig. 2) (Tolia et al., 2005). This model was not intended as a highresolution prediction for the var16 DBLb-C2 domain structure. Rather, the model helped us identify positions
© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 67, 78–87
Interaction site for DBLb-C2 and ICAM-1 81 C 1 C 2 C3 C 4
C5
C6
C6a
C7-10
C11
C12-13
EBA-175 F2 DBL Gly5 C1
Gly1
D1 C4
Gly3
D2
C5
C5a
C6
C6a
C7,7a,8 C8a-10
WDCKKKND--RSNYVCIPDRRIQLCIVN WECKK-PYKLSTKDVCVPPRRQELCLGN
MGNDMDF-GGYSTKAENKIQEVFKGAHGEI-------------------SEHKIKNFR GGTDYWNDLS-NRKLVGKINTNS-------------------NYVH--RNKQNDKLFR
LSKEYETQKVPKEN------AE--NYLIKISENKNDAKVS-LLL QAKQYQEYQKGNNY--KMYSEFKSIKPEVYLKKYSEKCSNLNFE
17
160 * ** * * * * * * RGRDLWDLDEGSKKMEGHLKKIFKQIKEKHP----G-V--QEKYNSDNDY-NKYINLR RGRDLWEH-GDQTKLQGHLQIIFGKIKEEIKKKHPG-INGNDKYKGDEKNNPPYKQLR KGTDLWDKDSGEQKTQRNLVTIFGKIKVQRK----G-I-DTSKYTNT---DGKHNQLR RGTDMWDKDEGSKKMDVILKKIFGKIKQELPK---E-IQ--KKYKNP---DGKHTQLR RGRDLWDLDEGSKKMEGHLKKIFKQIKEKHP----G-V--QEKYNSDNDY-NKYINLR RGRDMWDEDKSSTDMETRLITVFKNIKE----KHDG-IKDNPKYTGDESKKPAYKKLR RGRDMWDLNDGSQKIEKNLKDIFDKIKDNLPD---G-I--KDNRQYNGD--PNHIKLR KGTDLWDQNKGETDTQSNLVTIFGKIKGTLN----D----TSKYN-----DGKHLELR
283
var16 var13 var14 var31 var41 var27 var1 JDP8
43 ** ** WKGG-EQVSTSYSDVFLPPRRQHMCTSN WKIG-EKVETTDTDAYIPPRRQHMCTSN WTNIVKKKTTSYKDVFLPPRREHMCTSN WSKVG-EDKTTYSDVYLPPRRQHMCTSN WKGG-EQVSTSYSDVFLPPRRQHMCTSN WSNIEGKKQTSYKNVFLPPRREHMCTSN WSKV-GQNKTSYSDVFLPPRREHMCTSN WKNGGE-VKMSDTHSYMPPRREHFCTSN
111*
Binders
minC2
Non-Binders
EBLs
DBLβ-C2
var2i var2ii var7i var7ii var8 var18i var11 var12 var17 var19 var44 var15 var20 var6 var10
WKDGDF-VSTTHKEVFMPPRREHICTSN WSYGEKEKEKTHPKVYMPPRREHMCTSN WSHVN-EKKTTYTDVYLPQRREHMCTSY WKDKDF-VNPKYPGIYMPPRRQHMCTSN WKTGKD-VKMTENEAYMPPRRQHMCTSN WTYANGKNTTTYSDVYLPQRRQHICTSN WND-RT-SQSSTPNVYVRPRREHMCTSN WKPAG-FISKTYKDIYMPPRRQHFCTSN WETGGT-VQMTETEAYMPPRRQHMCTSN WSYGEKKKEMTHPKVYMPPRREHMCTSN WEDE-N-SKSNTLGMHIRPRRKHMCTSN WKNGGE-VKMSHTDLYLPPRRQHFCTSN WKPGRQI-QMSAEDIYMPPRRQHMCTSN WTHLQNA-KTTYSDVFLPPRRQHMCTSN WEGDNF-VSATHKNLYIPPRRQHMCTSN
RGKDLWDH-KDFKKLEKHLQKIFGKIKEELKSK----I--NDKYED--NSEGKHTKFR RGKDMWEH-KDQNKLQGYLQTIFDKIKDELKSK----L--GDKYAS--E-DKPYTQLR RGRDMWDLDDGSKKMEDIFKKIFGTLHKSLD----G-IK--DKYKE----GEPYTKLR RGTDLWDINGDVTGVQSNLQTVFGKIKKQF----------NGKYT----NDSKHTQLR RGKDLWDH-RDFKNLERDLVTIFGKIKEGITDET-----IKEKYDS--YKDNKHIQLR RGRDMWVQNYGENTTQGNLKDIFGTIKNKNP----E-IQ--GKYKHI--DDEKHTQLR RGRDLWDRDSGSTDMETRLKNIFKKIKEQIP----E-IH--DKYKDDENKTPPYKQLR KGTDMWDNDSGESKTRDKLREIFDTIKKKHP----G-IK--EIYKE----DTPYTKLR RGRDLWDN-KDQVTLQDHLKTIFGKIKGELKG--------EDKYNRDDKKSPPYKQLR RGRDMWDKDSGSKKMDVILKNVFGTLHKSLEG-----IRNHPKYAYDKNKTSPYKQLR RGRDLWEN-GEAKSLQGNLVTIFGHIHSSLNG--------KGKYASDEKNNPPYKQLR RGTDLWDINGDATGVQNNLKDIFSKITEELKKQHPDKFNDNDKYT----NDSKHTKLR RGRDLWDKNSDAKRLQTNLKEIFTKIKEELPE---D-IK--KKYDKD---GTDHKLLR RGRDMWDKDDGAQKMDVILKNVFGTLHKSLEG-----IRNHPKYAYDKNKTSPYKQLR RGRDMWDKDDGAQKMEDIFKKIFGNLYESLP----G-IK--GKYDGDDQRTPQYKQLR
IKQKYDDLYQKAKQNG-V-----------TS-SDKDADVV-KFL IKTKYEQLYKKVQDDV-TN-------TSSDG-SKDETDVV-SFL IRAKYKKIYEHARVDIAAN-GG-LNTSTAIND-NEDKPVI-EFL IKEKYQKLYEQAKN----N-TG-----ATTS-DSNDQQVI-EFL MSKKYKQLYEQAKK----D-TN-----CSTL-TKQDQDVV-AFL ISKKYEKLYKQAHIT-AIN-GG-PDASNGLVD-DEDKPVI-EFL MQLQYLILYHLANTTG-PH-G--INSYGGAVG-EKDKPVV-QFL MEIKYKLLYLQAQTT-AAN-GG-PDTYSGLVD-ENEKPVV-NFL MEIKYIPLYLQAKNAY-YG-----IS---FP-GADYQLMV-DFL IKGKYEELYKKALDS-VNG-NG-KGEKSTTSGTKDEKDVV-DFL MDMKYTPLYLQAKNPY-PG-----IV---FP-GADYQLMV-DFL ISNKYQILYWQAKIAA-IN-GG-TEKSTTTK-DDKDKNVI-DFL IKNKYAQLYKKALDS-VNG-KE-ESKKKTASD-AKDQQVV-HFL IKDKYKTLYEQATKN---------GETSGTPN-EKDKDVV-DFL MLLQYTLLYWQAETTA-RY-GG-TRAYSGDVG-DKDKPVV-QFL
F1 F2
322 ** * * MEIKYLTLYAYAQMAS-NN-KGDMSIFGNAVG-PKDKPVV-QIL IKDKYEELYLQAKIAF-AG----T--SFGGGD-RDYQQMV-HFF LELEYALSYLHAKNDS-R----RM--AFGGTD-PDYQQVV-HFF ISDKYQFLYLQAKTA-AAN-GG-PHASSGDVG-EKDKPVV-NFL MEIKYLTLYAYAQMAS-NN-KGDMSIFGNAVG-PKDKPVV-QIL ISDKYNLLYLQAKTTS-TN-PGRT--VLGDDD-PDYQQMV-DFL MQLKYLYFYHEAKTTS-RH-G--IDAYSGAVE-PKDKPVV-KFL MELKYILLYANAKTTS-TN-AGRT--VLGDAS-PDYQQML-DFF
Fig. 1. Comparison of ICAM-1 binding and non-binding DBLb-C2 sequences to EBA-175 DBL domains. The cartoon compares EBA-175 F2 DBL domain (residues C318-V601) with a generic DBLb-C2 domain. The approximate locations of the Gly5, Gly1 and Gly3 pockets in EBA-175 (Tolia et al., 2005) and the DARC1 (D1) and DARC2 (D2) pockets in the P. knowlesi Duffy binding protein (PkaDBP) (Singh et al., 2006) are labelled under the F2 DBL domain. Conserved cysteines are numbered as previously reported (Smith et al., 2000b). Semi-conserved homology blocks in DBL domains are delineated by colour blocks as follows: A, pink; B, red; C, orange; D, yellow; E, light green; F, green; G, light blue; H, blue; I, cyan; J, purple. Only the minimum region of the C2 domain (first ~1/3 of the full-length C2 sequence, minC2) necessary for binding to ICAM-1 (Springer et al., 2004) is depicted in magenta. The minC2 is predicted in homology models to fold as part of the DBL domain (see Fig. 2). The locations compared in the sequence alignments are indicated by brackets underneath the DBLb-C2 cartoon. MCOFFEE was used to create this alignment (Notredame et al., 2000; Wallace et al., 2006). In the alignment, dots over residues in F1 and F2 were shown to have a role during binding (black) or dimerization (blue) of EBA-175 (Tolia et al., 2005). Residues mutated in IT4var16/IT-ICAMvar DBLb-C2 in this study that had an impact on binding are indicated with an asterisk. Homology block boundaries of the var16 sequence are delineated above its sequence with appropriately coloured lines. Numbering of the var16 sequence starts with 1 at the first conserved cysteine. The C2 domain begins at L288. Colours of the amino acids are as follows: red, positive charge; blue, negative charge; green, hydrophobic; grey blue, polar; gold, cysteine; brown, small.
in the sequence that corresponded to the equivalent glycan and DARC binding sites of the EBA-175 and PkaDBP DBL domains respectively (Figs 1 and 2). EBA175 binds to sialic acid presented on glycophorin A. The glycan binding site is formed at the dimer interface between F1 and F2 DBL domains. A combination of residues from three discontinuous regions of the EBA175 F1 and F2 DBL domain sequences comes together to form the glycan binding site, consisting of three distinct binding pockets (Tolia et al., 2005), referred to as Gly5, Gly1 and Gly3 (Fig. 1). However, as the F2 domain provides the majority of glycan contact residues in each pocket (15 out of 21 residues), we concentrated our mutagenesis of the var16 DBLb-C2 domain on the equivalent F2 glycan binding pockets. Similarly, the PkaDBP DBL domain uses residues from two discontinuous regions of the sequence to bind DARC, referred to as
DARC1 (D1) and DARC2 (D2). In both glycan and DARC binding regions, binding residues are located on flexible loops (Fig. 2A) (Tolia et al., 2005; Singh et al., 2006). We introduced a nested series of single and multiple alanine substitutions of residues in the equivalent Gly5,1,3 and DARC1,2 regions of the wild-type var16 sequence and assayed the mutants for binding to recombinant ICAM-1 (Fig. 3). Overall, the majority of single residue substitutions did not reduce binding to a significant degree. The only exceptions were an aspartate 150 (D150) substitution in the Gly1 pocket, a Y153 substitution in the Gly 1 pocket, and a D303 substitution in the Gly3 pocket that reduced ICAM-1 binding approximately 50% compared with the wild-type protein. The first two residues are from the DBLb region, while the D303 residue is from the first part of the C2 region (Fig. 1). By comparison, several mutants with
© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 67, 78–87
82 D. P.-G. Howell et al. 䊏 A
B
DARC binding residues
Equivalent glycan binding site
Glycan binding residues
C
150°
multiple substitutions had additive and significant reductions in binding (50% or below, Fig. 3). In addition, three mutants with combined substitutions in two or more pockets, Gly5 + Gly1 (T25, S26, S28, D29, D119, E120, S122), Gly1 + Gly3 (W116, K130, D303) and Gly5 + Gly1 + Gly3 (S28, W116, Q295), abrogated binding (5% or less, Fig. 3). One of the Gly5 + Gly1 mutants included substitutions in the ‘T(S/T)(Y/D)(S/T) motif’, which on its own was responsible for a drop to ~57% binding (T25, S26, S28, D29 mutant). In contrast, a mutant engineered with three substitutions (K123, K137, D150) in the Gly1 region was not significantly impaired in its ability to bind ICAM-1, indicating that these residues may not be involved in binding. The Gly3 region in all IT4 DBLb-C2ICAM-1 domains encodes an eight amino acid stretch of primarily aliphatic residues. We hypothesized that substitution of these residues with charged residues would impact ICAM-1 binding. Indeed, a mutant in which leucine and threonine in this region were mutated to lysine (L288K, T289K) had greatly reduced binding compared with the wild-type protein. Overall, multiple substitutions in all three equivalent glycan binding pockets reduced binding to ICAM-1, including residues contributed by both
Fig. 2. Three dimensional model of ICAM-1 binding residues in the DBLb-C2 domain. A homology model of the IT4var16/IT-ICAMvar DBLb-C2 domain was based upon the EBA-175 F2 DBL domain structural template. A. The EBA-175 F2 domain (C318-V601) is illustrated as a ribbon coloured according to semi-conserved homology blocks, as follows: A, pink; B, red; C, orange; D, yellow; E, light green; F, green; G, light blue; H, blue; I, cyan; J, purple. These are the same blocks illustrated in Fig. 1. The residues that impact binding to glycophorin A-sialic acid and DARC are depicted as ball-and-stick (side-chain atoms only). Atom colours are: C, gold; N, cyan; S, yellow and slightly larger diameter; O, red. B. The var16 DBLb-C2 domain homology model (C1-A355) is delineated as in (A). The minC2 domain illustrated in Fig. 1 is coloured magenta in the ribbon. Mutated residues that had an effect on binding are depicted as ball-and-stick. These residues are indicated with an asterisk in Fig. 1. C. The var16 DBLb-C2 domain homology model is illustrated as a space-filled model. Red residues were shown to reduce binding to ICAM-1 (the same residues depicted in (B) as ball-and-stick), green residues are located in the equivalent DARC binding region and did not significantly impact binding, and blue residues are predicted to be surface-exposed and also did not significantly impact binding to ICAM-1. Space-filled models in (C) are rotated 150° from one another and provide a slightly different perspective from the ribbon model in (B).
DBLb and the first part of the C2 region (asterisked residues in Fig. 1). Of the 18 asterisked residues in Fig. 1 which were found to diminish binding, only two residues, W116 and Y147, are conserved in all DBLb type domains, and therefore may be important for structure. Significantly, homology modelling suggests that the first part of the C2 region is actually part of the DBL domain and comprises a loop and the third helix in a triple helical bundle in DBL subdomain 3 (blue, purple and magenta helices in Fig. 2B). The third helix is not highly conserved in sequence and assumes more or less degenerate forms in the three solved DBL domain structures, being a split helix in the EBA-175 F2 domain (Fig. 2A) (Tolia et al., 2005; Singh et al., 2006). In contrast to the Gly region substitutions, none of the substitutions in the equivalent DARC binding sites influenced binding, even when multiple substitutions were introduced into both DARC1 and DARC2 regions (Fig. 3B). We also created control mutants with substitutions of several residues that were predicted to be surface exposed and not present in either of the equivalent Gly or DARC regions. These control mutants were also not significantly impaired in their ability to bind
© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 67, 78–87
Interaction site for DBLb-C2 and ICAM-1 83 A
B
Gly
Int
%Tsf
2+
14,10
Pos
Int
%Tsf
2+ 22,13 2
14,8
2
11,6
5
2
9,4
D1,2
2+
7,4
1
2+
8,1
D2
2
12,4
1
2+
10,1
D2
2
10,3
5,1
2
5,0
D1,2
2
14,4
1
2
6,1
D2
2+
16,3
5,1
2+
7,1
D1,2
2+
8,4
3
2
8,3
D1,2
2+
11,6
3
2
21,9
S
2
25,14
5,1
2+
11,6
S
2+
21,3
5,1,3
2
11,3
S
2
15,6
1,3
2
16,7
S
2
12,3
5
2+
17,5
5,1
2+
12,7
1
2
16,4
1
2
13,6
5,1
2
7,4
Int
%Tsf
5,1
2
8,4
1
2
17,6
3
2+
21,19
1
1+
17,4
1
2+
16,5
1
2+
19,7
1
2+
22,6
5,3
2
14,5
C Gly
L288K T289K
2+
11,5
2+
14,9
1
2
12,4
5
2+
18,3
5
2
11,10
5,1
2
9,11
Fig. 3. ICAM-1 binding analysis of mutant IT4var16/IT-ICAMvar and IT4var31/A4tres DBLb-C2 domains. A. Alanine-substitution mutations made in the equivalent glycan binding site of the var16 DBLb-C2 domain were tested for binding to ICAM-1/Fc coated beads. Binding was controlled using mock-treated beads that did not receive ICAM-1/Fc. The C2 domain begins with L288. Gly indicates the equivalent glycan binding pocket (5, 1, 3 as in Fig. 1); Int is the relative intensity of the immunofluorescent cell surface stain; and %Tsf is the mean and SD of transfection efficiency of 2 or 3 independent experiments [except for the FCR3 (FCR3 VAR1CSA, negative control) and wild-type values, which are the means of all combined experiments]. TSSD denotes T25, S26, S28, D29. B. Substitutions in the var16 DBLb-C2 domain were made in the equivalent DARC binding region or in positions predicted to be surface-exposed. Pos indicates the position of the substitution, where D1, 2 are the two DARC regions and S are surface controls. Int and %Tsf are as in (A). C. Mutants of the var31 DBLb-C2 domain were created with substitutions in similar Gly regions as the var16 substitutions. Gly, Int and %Tsf are as in (A). TTSD denotes T25, T26, S28, D29.
ICAM-1 (Fig. 3B). When mapped on the DBLb-C2 model, the residues that affect binding come together on one side of the DBL fold, involving four loops and parts of the a-helical scaffold (Fig. 2). Overall, these results support the hypothesis that residues located in the equivalent glycan binding site of the EBA-175 F2 DBL domain were adapted in the context of the PfEMP1 family to bind new substrates. To determine if residues important for ICAM-1 binding were also present in the equivalent glycan binding region of another IT4 DBLb-C2ICAM-1 domain, we created mutants of the A4tres DBLb-C2ICAM-1 domain with alanine substitutions in similar residues of the Gly5 and Gly1 regions (Fig. 1). Double mutations in either the Gly5 or Gly1 pockets alone had no effect on binding (Fig. 3C). However, a combination of four amino acid substitutions in the Gly 5 pocket (including ‘T(S/T)(Y/D)(S/T) motif’) reduced binding by 80% (Fig. 3C). Binding was further impaired when these substitutions were combined with a double mutation in the Gly1 region (Gly5 + Gly1 mutant), indicating that a similar ICAM-1 binding mechanism may be shared between all DBLb-C2ICAM-1 domains.
Discussion The DBL domain is a key adhesive domain of P. falciparum parasites. In the context of the PfEMP1 variant antigen family, it is the basis of IE binding to several types of host receptors, including ICAM-1 (Kyes et al., 2007). In this study, we sought to determine the prevalence of DBLb-C2ICAM-1 domains in the entire var gene repertoire of a single parasite strain, IT4. Prior to this study, most binding studies of PfEMP1 domains have been gene-by-gene approaches, and few binding traits had been mapped to an extensive number of domains. One exception to this was a study of CIDR domain binding to CD36 (Robinson et al., 2003), which was made possible by the sequencing of the 3D7 genome (Gardner et al., 2002). Similarly, the recently published IT4 var repertoire (Kraemer et al., 2007) has allowed us to use a higher-throughput approach to study ICAM-1 binding than previously possible. Our screen of 21 newly identified domains indicates that DBLb-C2ICAM-1 domains are not largely common within one particular laboratory strain (7/25 or 28% of the domains could bind), and that
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84 D. P.-G. Howell et al. 䊏
at least seven of the 59 estimated PfEMP1 proteins in the IT4 genome encode ICAM-1 binding domains. Of interest, all of the DBLb-C2ICAM-1 domains are found in group B and C var genes, and none of the nine tested DBLb-C2 domains from group A genes binds ICAM-1. Although we cannot exclude the possibility of falsenegative binding results, this analysis suggests that the DBLb-C2::ICAM-1 interaction, like CD36 binding (Robinson et al., 2003), may be rare or absent in type A proteins and undergoing differential selection between the major var groups. While more work needs to be done to understand whether specific groups of PfEMP1 variants are associated with particular P. falciparum clinical complications, it is interesting to note that partially immune children possessed antibodies to Group A variants (Jensen et al., 2004) and that Group A genes appeared to be preferentially transcribed in individuals experiencing severe malaria or cerebral malaria complications (Kirchgatter and Portillo Hdel, 2002; Kyriacou et al., 2006). Therefore, it will be important to clarify the role of non-A type PfEMP1 variants in cerebral complications and to better understand the adhesion properties of the Group A proteins. Intercellular adhesion molecule 1 is a member of the immunoglobulin superfamily and contains five Ig-like domains. The PfEMP1::ICAM-1 interaction site has been mapped to the N-terminal Ig-like domain in ICAM-1 on the opposite face from the binding site for lymphocyte function associated antigen 1 (LFA1), but partially overlapping the fibrinogen binding site (Chakravorty and Craig, 2005). Binding and mutagenesis studies suggest PfEMP1 proteins have a relatively large contact ‘footprint’ on ICAM-1 primarily across three b strands, termed B, E and D (the BED side) (Berendt et al., 1992; Ockenhouse et al., 1992; Tse et al., 2004). This binding footprint includes residues that are predicted to be buried deep in the interface between two ICAM-1 proteins, which are thought to exist as dimers on the cell surface (Reilly et al., 1995; Miller et al., 1995). Although different PfEMP1 variant footprints overlap considerably, they differ in specific ICAM-1 contact residues, and this was shown to affect ICAM-1 binding affinity under flow conditions (Cooke et al., 1994; Gray et al., 2003; Tse et al., 2004), presumably owing to DBLb-C2 sequence variation. Our data suggest that DBLb-C2 domains use a common interaction site for ICAM-1 binding that maps to the equivalent glycan binding region in the EBA-175 F2 DBL domain. Notably, homology modelling suggests that the equivalent Gly1 pocket in DBLb-C2 domains may be considerably larger than the equivalent region in EBL proteins (compare Fig. 2A and B, loop at bottom of domains), and two important residues for ICAM-1 binding, D150 and Y153, were associated with this longer
loop extension. It is interesting to speculate that this loop may be able to reach deep within the ICAM-1 dimer interface, although we do not have evidence for specific DBLb-C2::ICAM-1 interaction residues. We note that a recent in silico modelling study of the DBLb-C2::ICAM-1 interaction found a low energy docked conformation consistent with a central role for the regions that we mutated at the DBLb-C2::ICAM-1 interface (Bertonati and Tramontano, 2007). However, the high-resolution details of that predicted model may be unreliable owing to unaccounted backbone flexibility in the loops on DBLb-C2 or ICAM-1, and therefore we cannot compare our mutational data with that model in great detail. Our data provide a molecular explanation for how DBLb-C2 domains engage ICAM-1. It will be informative to test if other PfEMP1–host interactions utilize this same interaction site (Mayor et al., 2005). Unlike erythrocyte invasion which occurs in seconds, PfEMP1 proteins are exposed to antibody pressure for many hours at the IE surface. Consequently, PfEMP1DBL domains are more polymorphic than EBL-DBLs, a quality that helps subvert the host antibody response. Homology modelling suggests that PfEMP1-DBL domains retain the core DBL fold, but have extensively diversified at flexible loops that connect the a-helical scaffolding elements (Howell et al., 2006). All of the var16 DBLb-C2 residues in the equivalent Gly1, 3 and 5 sites reside within these polymorphic loops, as do the EBL-DBL binding residues for both glycophorin A and DARC (Fig. 2, compare A and B). The structure of the DBL domain may therefore have been well suited to evolve new binding traits by allowing the creation of novel binding interactions at flexible loops while retaining structural stability with the conserved scaffold. It is interesting that multiple mutations were required for loss of binding. It is possible that this is an adaptation that allows for loss of antigenic epitopes without loss of function, either by gene mutation or recombination. Sequence comparisons suggest that var gene recombination/gene conversion mechanisms promote sequence exchange and combinatorial diversity at DBL loops (Bockhorst et al., 2007). Reshuffling polymorphic loops at binding sites coupled with new mutation is an ingenious method to change binding affinity or specificity as needed, as well as for immunoevasion. This dual advantage might thus predict that it will be difficult to get an antibody response against the DBL domain that blocks all ICAM-1 binding, but this needs to be addressed. In summary, these findings provide a clearer understanding of how PfEMP1-DBL domains are diversifying under dual selection for host receptor binding and immune evasion and may assist the rational design of vaccine or drug interventions against malaria disease.
© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 67, 78–87
Interaction site for DBLb-C2 and ICAM-1 85
Experimental procedures IT4 var DBLb-C2 domain constructs DBLb-C2 domain sequences were PCR amplified from IT4 genomic DNA using specific primers (Table S1), usually beginning seven amino acids upstream of the first conserved cysteine of the DBL domain and ending exactly at the end of the C2 domain. DNA polymerases used included Expand High-Fidelity (Roche Applied Science, Indianapolis, IN), BIO-X-ACT Long and Short Mixes (Bioline, Randolph, MA) and AccuSure (Bioline). PCR products were ligated into the T8a+(12CA5) vector (Affymax, Palo Alto, CA) for glycoslyphosphoinositol-anchored cell surface expression of the protein. Epitope tags (179 and HA) were expressed at the N- and C-termini of the cloned domain to verify surface expression. A mouse monoclonal a-179 tag antibody (courtesy of Erik Whitehorn, Affymax) and secondary antibody, goat a-mouse IgG conjugated to AlexaFluor-488 (Invitrogen, Carlsbad, CA), were used for immunofluorescence as described below. All constructs were sequence verified and compared with sequences at The Sanger Institute website IT4 genome database (http://www.sanger.ac.uk/Projects/ P_falciparum). The majority of DBLb-C2 inserts had no changes, five inserts contained a single amino acid difference and two inserts had two differences. Inserts with sequence changes were not biased to any group of var genes and amino acid substitutions typically occurred in regions of the DBL fold that tolerate amino acid variation (data not shown).
ICAM-1 binding assay To prepare beads, magnetic Dynabeads conjugated to sheep a-mouse antibodies (Invitrogen) were incubated with mouse a-human Fc-fragment-specific antibodies (Jackson ImmunoResearch, West Grove, PA) at a ratio of 1 mg antibody per 1 ¥ 106 beads. Beads were mixed by rotation for 3 h at room temperature, washed with wash buffer (0.1% bovine serum albumin in PBS) and incubated overnight with recombinant ICAM-1/Fc protein (R&D Systems, Minneapolis, MN) at a ratio of 1 mg ICAM-1 per 1 ¥ 106 cells, or mock treated for controls. Beads were washed, resuspended in binding medium (0.5% bovine serum albumin in RPMI-1640, pH 7.2) and warmed to 37°C prior to adding to cells. COS-7 cells were plated into six-well plates (Corning, Lowell, MA) 16–24 h prior to transient transfection with DBLb-C2 constructs using GeneJuice transfection reagent (Novagen, San Diego, CA), as recommended by the manufacturer. Cells were grown on 12 mm round untreated glass coverslips (Fisher Scientific Research, Pittsburg, PA) present in each well. Two days post-transfection, coverslips were transferred to an empty well and incubated with 1 ¥ 106 ICAM-1-coated or mock-coated beads in a humidified container for 1 h at 37°C. Binding medium was added to the well prior to inverting the coverslips onto stands present in each well. Coverslips remained inverted on the stands for 8 min prior to being re-inverted. To assess the percentage of transfected cells bound to beads, an immunofluorescence assay was performed on the bead-treated coverslips after fixing the cells for at least 20 min in 4% paraformaldehyde in PBS. Coverslips were
washed with wash buffer and incubated with mouse monoclonal a-179 antibody (5 mg ml-1, 45 min at room temperature) and a secondary antibody, goat a-mouse conjugated to AlexaFluor-488 (4 mg ml-1, 45 min at room temperature) in a humidified container. Coverslips were washed, mounted onto glass slides using ProLong Antifade Kit (Invitrogen), and sealed with nail polish prior to inspection by fluorescence microscopy. Binding was quantitated as the percentage of 50 green (transfected) cells associated with five or more ICAM1/Fc coated beads. Binding to control beads (mock-treated with ICAM-1) was not seen. To assess transfection efficiency, 300 DAPI-fluorescent nuclei from coverslips treated with mock-coated beads were counted and scored positive for transfection if green surface fluorescence was codetected. The relative level of green surface fluorescence was scored by eye on an arbitrary scale of 1, 1+, 2, 2+ or 3.
Phylogenetic analysis of IT4 var DBLb-C2 sequences A multiple alignment of DBLb-C2 sequences was made using ClustalX (gap opening penalty 5.0, gap extension penalty 0.05), and analysed using PAUP*4.0b10 [Phylogenetic Analysis Using Parsimony (and Other Methods)] by neighbourjoining method to create an unrooted phylogram. Bootstrap values (1000 replicates) ⱖ 80% are indicated (Fig. S1).
Creation of the IT4var16 DBLb-C2 homology model The IT4var16/IT-ICAM DBLb-C2 sequence was aligned to the EBA-175 F2 domain based on semi-conserved homology blocks present in all (EBL and PfEMP1) DBL domains (Smith et al., 2000b). The alignment was submitted to the SWISS-MODEL server (http://swissmodel.expasy.org) using the Swiss-PdbViewer 3.7 (Peitsch, 1995; Guex and Peitsch, 1997; Schwede et al., 2003). The resultant homology model was used to guide selection of residues for alanine substitution mutagenesis. The alignment in Fig. 1 was created using M-COFFEE (Notredame et al., 2000; Wallace et al., 2006). The models shown in Fig. 2 were created using RasMol 2.7 (Sayle and Milner-White, 1995; Bernstein, 2000), Molscript 2.1 (Kraulis, 1991) and Raster3D 2.7 (Merritt and Bacon, 1997).
Mutagenesis of IT4 var DBLb-C2 domains Mutagenesis reactions were performed using the QuikChange Multi Site-directed Mutagenesis kit (Stratagene, La Jolla, CA) as recommended by the manufacturer. Substitutions were confirmed by sequencing. In some cases, two rounds of mutagenesis reactions were performed to introduce multiple mutations.
Acknowledgements We would like to thank Sarel J. Fleishman for assistance with modelling and helpful discussions, and Erik Whitehorn for providing the pT8a+(12CA5) vector and mAb 179. D.P.G.H. is supported by a National Institutes of Health Public Health Diseases Training Grant awarded to the Department of Pathobiology, University of Washington (T32 AI007509). This project was supported by NIH Grant RO1 AI047953-07A1 (J.D.S.) and the MJ Murdock Charitable Trust (SBRI).
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References Berendt, A.R., Simmons, D.L., Tansey, J., Newbold, C.I., and Marsh, K. (1989) Intercellular adhesion molecule-1 is an endothelial cell adhesion receptor for Plasmodium falciparum. Nature 341: 57–59. Berendt, A.R., McDowall, A., Craig, A.G., Bates, P.A., Sternberg, M.J., Marsh, K., et al. (1992) The binding site on ICAM-1 for Plasmodium falciparum-infected erythrocytes overlaps, but is distinct from, the LFA-1-binding site. Cell 68: 71–81. Bernstein, H.J. (2000) Recent changes to RasMol, recombining the variants. Trends Biochem Sci 25: 453–455. Bertonati, C., and Tramontano, A. (2007) A model of the complex between the PfEMP1 malaria protein and the human ICAM-1 receptor. Proteins 69: 215–222. Bockhorst, J., Lu, F., Janes, J.H., Keebler, J., Gamain, B., Awadalla, P., et al. (2007) Structural polymorphism and diversifying selection on the pregnancy malaria vaccine candidate VAR2CSA. Mol Biochem Parasitol 155: 103–112. Chakravorty, S.J., and Craig, A. (2005) The role of ICAM-1 in Plasmodium falciparum cytoadherence. Eur J Cell Biol 84: 15–27. Chattopadhyay, R., Taneja, T., Chakrabarti, K., Pillai, C.R., and Chitnis, C.E. (2004) Molecular analysis of the cytoadherence phenotype of a Plasmodium falciparum field isolate that binds intercellular adhesion molecule-1. Mol Biochem Parasitol 133: 255–265. Cooke, B.M., Berendt, A.R., Craig, A.G., MacGregor, J., Newbold, C.I., and Nash, G.B. (1994) Rolling and stationary cytoadhesion of red blood cells parasitized by Plasmodium falciparum: separate roles for ICAM-1, CD36 and thrombospondin. Br J Haematol 87: 162–170. Frank, M., and Deitsch, K. (2006) Activation, silencing and mutually exclusive expression within the var gene family of Plasmodium falciparum. Int J Parasitol 36: 975–985. Gardner, J.P., Pinches, R.A., Roberts, D.J., and Newbold, C.I. (1996) Variant antigens and endothelial receptor adhesion in Plasmodium falciparum. Proc Natl Acad Sci USA 93: 3503–3508. Gardner, M.J., Hall, N., Fung, E., White, O., Berriman, M., Hyman, R.W., et al. (2002) Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419: 498–511. Gray, C., McCormick, C., Turner, G., and Craig, A. (2003) ICAM-1 can play a major role in mediating P. falciparum adhesion to endothelium under flow. Mol Biochem Parasitol 128: 187–193. Guex, N., and Peitsch, M.C. (1997) SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18: 2714–2723. Howell, D.P., Samudrala, R., and Smith, J.D. (2006) Disguising itself–insights into Plasmodium falciparum binding and immune evasion from the DBL crystal structure. Mol Biochem Parasitol 148: 1–9. Jensen, A.T., Magistrado, P., Sharp, S., Joergensen, L., Lavstsen, T., Chiucchiuini, A., et al. (2004) Plasmodium falciparum associated with severe childhood malaria preferentially expresses PfEMP1 encoded by group A var genes. J Exp Med 199: 1179–1190. Kirchgatter, K., and Portillo Hdel, A. (2002) Association of
severe noncerebral Plasmodium falciparum malaria in Brazil with expressed PfEMP1 DBL1 alpha sequences lacking cysteine residues. Mol Med 8: 16–23. Kraemer, S.M., and Smith, J.D. (2003) Evidence for the importance of genetic structuring to the structural and functional specialization of the Plasmodium falciparum var gene family. Mol Microbiol 50: 1527–1538. Kraemer, S.M., and Smith, J.D. (2006) A family affair: var genes, PfEMP1 binding, and malaria disease. Curr Opin Microbiol 9: 374–380. Kraemer, S.M., Kyes, S.A., Aggarwal, G., Springer, A.L., Nelson, S.O., Christodoulou, Z., et al. (2007) Patterns of gene recombination shape var gene repertoires in Plasmodium falciparum: comparisons of geographically diverse isolates. BMC Genomics 8: 45. Kraulis, P.J. (1991) MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J Appl Cryst 24: 946–950. Kyes, S.A., Kraemer, S.M., and Smith, J.D. (2007) Antigenic variation in Plasmodium falciparum: gene organization and regulation of the var multigene family. Eukaryot Cell 6: 1511–1520. Kyriacou, H.M., Stone, G.N., Challis, R.J., Raza, A., Lyke, K.E., Thera, M.A., et al. (2006) Differential var gene transcription in Plasmodium falciparum isolates from patients with cerebral malaria compared to hyperparasitaemia. Mol Biochem Parasitol 150: 211–218. Lavstsen, T., Salanti, A., Jensen, A.T., Arnot, D.E., and Theander, T.G. (2003) Sub-grouping of Plasmodium falciparum 3D7 var genes based on sequence analysis of coding and non-coding regions. Malar J 2: 27. Mayor, A., Bir, N., Sawhney, R., Singh, S., Pattnaik, P., Singh, S.K., et al. (2005) Receptor-binding residues lie in central regions of Duffy-binding-like domains involved in red cell invasion and cytoadherence by malaria parasites. Blood 105: 2557–2563. Medana, I.M., and Turner, G.D. (2006) Human cerebral malaria and the blood–brain barrier. Int J Parasitol 36: 555–568. Merritt, E.A., and Bacon, D.J. (1997) Raster3D: photorealistic molecular graphics. Methods Enzymol 277: 505–524. Miller, J., Knorr, R., Ferrone, M., Houdei, R., Carron, C.P., and Dustin, M.L. (1995) Intercellular adhesion molecule-1 dimerization and its consequences for adhesion mediated by lymphocyte function associated-1. J Exp Med 182: 1231–1241. Miller, L.H., Baruch, D.I., Marsh, K., and Doumbo, O.K. (2002) The pathogenic basis of malaria. Nature 415: 673– 679. Notredame, C., Higgins, D.G., and Heringa, J. (2000) T-Coffee: a novel method for fast and accurate multiple sequence alignment. J Mol Biol 302: 205–217. Ockenhouse, C.F., Ho, M., Tandon, N.N., Van Seventer, G.A., Shaw, S., White, N.J., et al. (1991) Molecular basis of sequestration in severe and uncomplicated Plasmodium falciparum malaria: differential adhesion of infected erythrocytes to CD36 and ICAM-1. J Infect Dis 164: 163–169. Ockenhouse, C.F., Betageri, R., Springer, T.A., and Staunton, D.E. (1992) Plasmodium falciparum-infected erythrocytes bind ICAM-1 at a site distinct from LFA-1, Mac-1, and human rhinovirus. Cell 68: 63–69.
© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 67, 78–87
Interaction site for DBLb-C2 and ICAM-1 87
Peitsch, M.C. (1995) Protein modeling by e-mail. Bio/Tech 13: 658–660. Reilly, P.L., Woska, J.R., Jr, Jeanfavre, D.D., McNally, E., Rothlein, R., and Bormann, B.J. (1995) The native structure of intercellular adhesion molecule-1 (ICAM-1) is a dimer. Correlation with binding to LFA-1. J Immunol 155: 529–532. Robinson, B.A., Welch, T.L., and Smith, J.D. (2003) Widespread functional specialization of Plasmodium falciparum erythrocyte membrane protein 1 family members to bind CD36 analysed across a parasite genome. Mol Microbiol 47: 1265–1278. Sayle, R.A., and Milner-White, E.J. (1995) RASMOL: biomolecular graphics for all. Trends Biochem Sci 20: 374. Schwede, T., Kopp, J., Guex, N., and Peitsch, M.C. (2003) SWISS-MODEL: an automated protein homologymodeling server. Nucleic Acids Res 31: 3381–3385. Singh, S.K., Hora, R., Belrhali, H., Chitnis, C.E., and Sharma, A. (2006) Structural basis for Duffy recognition by the malaria parasite Duffy-binding-like domain. Nature 439: 741–744. Smith, J.D., Craig, A.G., Kriek, N., Hudson-Taylor, D., Kyes, S., Fagen, T., et al. (2000a) Identification of a Plasmodium falciparum intercellular adhesion molecule-1 binding domain: a parasite adhesion trait implicated in cerebral malaria. Proc Natl Acad Sci USA 97: 1766–1771. Smith, J.D., Subramanian, G., Gamain, B., Baruch, D.I., and Miller, L.H. (2000b) Classification of adhesive domains in the Plasmodium falciparum erythrocyte membrane protein 1 family. Mol Biochem Parasitol 110: 293–310. Springer, A.L., Smith, L.M., Mackay, D.Q., Nelson, S.O., and Smith, J.D. (2004) Functional interdependence of the DBLbeta domain and c2 region for binding of the Plasmo-
dium falciparum variant antigen to ICAM-1. Mol Biochem Parasitol 137: 55–64. Tolia, N.H., Enemark, E.J., Sim, B.K., and Joshua-Tor, L. (2005) Structural basis for the EBA-175 erythrocyte invasion pathway of the malaria parasite Plasmodium falciparum. Cell 122: 183–193. Tse, M.T., Chakrabarti, K., Gray, C., Chitnis, C.E., and Craig, A. (2004) Divergent binding sites on intercellular adhesion molecule-1 (ICAM-1) for variant Plasmodium falciparum isolates. Mol Microbiol 51: 1039–1049. Turner, G.D., Morrison, H., Jones, M., Davis, T.M., Looareesuwan, S., Buley, I.D., et al. (1994) An immunohistochemical study of the pathology of fatal malaria. Evidence for widespread endothelial activation and a potential role for intercellular adhesion molecule-1 in cerebral sequestration. Am J Pathol 145: 1057–1069. Wallace, I.M., O’Sullivan, O., Higgins, D.G., and Notredame, C. (2006) M-Coffee: combining multiple sequence alignment methods with T-Coffee. Nucleic Acids Res 34: 1692– 1699.
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