mapping binding residues in the plasmodium vivax domain that binds duffy antigen during red cell...

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Molecular Microbiology (2005) 55 (5), 1423–1434 doi:10.1111/j.1365-2958.2005.04484.x © 2005 Blackwell Publishing Ltd oBlackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Publishing Ltd, 2004 ? 2004 55 514231434 Original Article Binding residues of P. vivax Duffy binding proteinD. Hans et al. Accepted 19 November, 2004. *For correspondence. E-mail [email protected]; Tel./Fax ( + 91) 112 618 7695. †Both authors contributed equally to this work. Mapping binding residues in the Plasmodium vivax domain that binds Duffy antigen during red cell invasion Dhiraj Hans, 1† Priyabrata Pattnaik, 1† Arindam Bhattacharyya, 1 Ahmad Rushdi Shakri, 1 Syed Shams Yazdani, 1 Monal Sharma, 1 Hyeryun Choe, 2 Michael Farzan 2 and Chetan E. Chitnis 1 * 1 Malaria Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110067, India. 2 Department of Medicine, Brigham and Women’s Hospital, Division of AIDS, Harvard Medical School, Boston, MA 02115, USA. Summary Plasmodium vivax depends on interaction with the Duffy antigen/receptor for chemokines (DARC) for invasion of human erythrocytes. The 140 kDa P. vivax Duffy-binding protein (PvDBP) mediates interaction with DARC. The receptor-binding domain of PvDBP maps to its N-terminal, cysteine-rich region, region II (PvRII), which contains approximately 300 amino acid residues including 12 conserved cysteines. Using surface plasmon resonance, we show that binding of PvRII to DARC is a high-affinity interaction with a binding constant ( K D ) of 8.7 nM. The minimal binding domain of PvRII has been previously mapped to a central 170-amino-acid stretch that includes cys- teines 5–8. Here, we have used site-directed mutagen- esis and quantitative binding assays to map amino acid residues within PvRII that make contact with DARC. Of the seven alanine replacement mutations that had an effect on binding, five were mutations in hydrophobic residues suggesting that hydrophobic interactions play a major role in the interaction of PvDBP with DARC. Genetic diversity studies have shown that six of the seven binding residues identi- fied in PvRII are conserved in P. vivax field isolates, which provides support for their role in interaction with DARC. Introduction Red cell invasion by malaria parasites requires specific receptor–ligand interactions (Chitnis, 2001). Plasmodium vivax and the related simian malaria parasite Plasmodium knowlesi are absolutely dependent on interaction with the Duffy antigen/receptor for chemokines (DARC) on human erythrocytes for invasion (Miller et al ., 1975; 1976; Horuk et al ., 1993). Duffy-negative human erythrocytes are com- pletely resistant to invasion by these parasite species. P. knowlesi invades rhesus erythrocytes by multiple path- ways using DARC as well as alternative receptors (Haynes et al ., 1988). Plasmodium falciparum , the most lethal of the human malaria parasites, primarily uses sialic acid residues on glycophorin A although invasion by other pathways is also commonly observed in both laboratory strains and field isolates (Miller et al ., 1977; Pasvol et al ., 1982; Friedman et al ., 1984; Mitchell et al ., 1986; Hadley et al ., 1987; Perkins and Holt, 1988; Dolan et al ., 1994; Okoyeh et al ., 1999). The parasite proteins that bind these erythrocyte recep- tors to mediate invasion belong to a family of erythrocyte- binding proteins (EBPs) that includes P. vivax and P. knowlesi Duffy-binding proteins (PvDBP and PkDaBP), P. knowlesi b and g proteins, which bind receptors other than DARC on rhesus erythrocytes, P. falciparum EBA-175, which binds sialic acid residues on glycophorin A, and EBA-175 paralogues, EBA-140, EBA-181 and EBL-1, which bind alternative receptors on human erythrocytes (Camus and Hadley, 1985; Haynes et al ., 1988; Werthe- imer and Barnwell, 1989; Adams et al ., 1990; 1992; Fang et al ., 1991; Sim et al ., 1994; Chitnis, 2001; Mayer et al ., 2001; 2002; 2004; Thompson et al ., 2001; Gilberger et al ., 2003; Maier et al ., 2003). Members of the EBP family share similar features in that they all contain two con- served cysteine-rich regions referred to as regions II and VI (Adams et al ., 1992; Chitnis, 2001). The receptor-bind- ing domains of EBPs have been mapped to their N-termi- nal cysteine-rich regions, regions II, which are also referred to as Duffy-binding-like (DBL) domains after the first binding domains identified from PvDBP and PkDBP (Chitnis and Miller, 1994; Sim et al ., 1994; Ranjan and Chitnis, 1999). Malaria parasites thus use the conserved, cysteine-rich, DBL domains to generate parasite ligands with diverse receptor-binding specificity. It is important to understand the structure-function bases for the interaction of DBL domains with their recep- tors. The binding sites for PvDBP and PkDBP have been mapped to a 35-amino-acid region of the N-terminal extra-

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Page 1: Mapping binding residues in the Plasmodium vivax domain that binds Duffy antigen during red cell invasion

Molecular Microbiology (2005)

55

(5), 1423–1434 doi:10.1111/j.1365-2958.2005.04484.x

© 2005 Blackwell Publishing Ltd

oBlackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Publishing Ltd, 2004

? 2004

55

514231434

Original Article

Binding residues of P. vivax Duffy binding proteinD. Hans

et al.

Accepted 19 November, 2004. *For correspondence. [email protected]; Tel./Fax (

+

91) 112 618 7695. †Both authorscontributed equally to this work.

Mapping binding residues in the

Plasmodium vivax

domain that binds Duffy antigen during red cell invasion

Dhiraj Hans,

1†

Priyabrata Pattnaik,

1†

Arindam Bhattacharyya,

1

Ahmad Rushdi Shakri,

1

Syed Shams Yazdani,

1

Monal Sharma,

1

Hyeryun Choe,

2

Michael Farzan

2

and Chetan E. Chitnis

1

*

1

Malaria Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110067, India.

2

Department of Medicine, Brigham and Women’s Hospital, Division of AIDS, Harvard Medical School, Boston, MA 02115, USA.

Summary

Plasmodium vivax

depends on interaction with theDuffy antigen/receptor for chemokines (DARC) forinvasion of human erythrocytes. The 140 kDa

P. vivax

Duffy-binding protein (PvDBP) mediates interactionwith DARC. The receptor-binding domain of PvDBPmaps to its N-terminal, cysteine-rich region, region II(PvRII), which contains approximately 300 amino acidresidues including 12 conserved cysteines. Usingsurface plasmon resonance, we show that binding ofPvRII to DARC is a high-affinity interaction with abinding constant (

K

D

) of 8.7 nM. The minimal bindingdomain of PvRII has been previously mapped to acentral 170-amino-acid stretch that includes cys-teines 5–8. Here, we have used site-directed mutagen-esis and quantitative binding assays to map aminoacid residues within PvRII that make contact withDARC. Of the seven alanine replacement mutationsthat had an effect on binding, five were mutations inhydrophobic residues suggesting that hydrophobicinteractions play a major role in the interaction ofPvDBP with DARC. Genetic diversity studies haveshown that six of the seven binding residues identi-fied in PvRII are conserved in

P. vivax

field isolates,which provides support for their role in interactionwith DARC.

Introduction

Red cell invasion by malaria parasites requires specific

receptor–ligand interactions (Chitnis, 2001).

Plasmodiumvivax

and the related simian malaria parasite

Plasmodiumknowlesi

are absolutely dependent on interaction with theDuffy antigen/receptor for chemokines (DARC) on humanerythrocytes for invasion (Miller

et al

., 1975; 1976; Horuk

et al

., 1993). Duffy-negative human erythrocytes are com-pletely resistant to invasion by these parasite species.

P.knowlesi

invades rhesus erythrocytes by multiple path-ways using DARC as well as alternative receptors(Haynes

et al

., 1988).

Plasmodium falciparum

, the mostlethal of the human malaria parasites, primarily uses sialicacid residues on glycophorin A although invasion by otherpathways is also commonly observed in both laboratorystrains and field isolates (Miller

et al

., 1977; Pasvol

et al

.,1982; Friedman

et al

., 1984; Mitchell

et al

., 1986; Hadley

et al

., 1987; Perkins and Holt, 1988; Dolan

et al

., 1994;Okoyeh

et al

., 1999).The parasite proteins that bind these erythrocyte recep-

tors to mediate invasion belong to a family of erythrocyte-binding proteins (EBPs) that includes

P. vivax

and

P.knowlesi

Duffy-binding proteins (PvDBP and PkDaBP),

P.knowlesi

b

and

g

proteins, which bind receptors other thanDARC on rhesus erythrocytes,

P. falciparum

EBA-175,which binds sialic acid residues on glycophorin A, andEBA-175 paralogues, EBA-140, EBA-181 and EBL-1,which bind alternative receptors on human erythrocytes(Camus and Hadley, 1985; Haynes

et al

., 1988; Werthe-imer and Barnwell, 1989; Adams

et al

., 1990; 1992; Fang

et al

., 1991; Sim

et al

., 1994; Chitnis, 2001; Mayer

et al

.,2001; 2002; 2004; Thompson

et al

., 2001; Gilberger

et al

.,2003; Maier

et al

., 2003). Members of the EBP familyshare similar features in that they all contain two con-served cysteine-rich regions referred to as regions II andVI (Adams

et al

., 1992; Chitnis, 2001). The receptor-bind-ing domains of EBPs have been mapped to their N-termi-nal cysteine-rich regions, regions II, which are alsoreferred to as Duffy-binding-like (DBL) domains after thefirst binding domains identified from PvDBP and PkDBP(Chitnis and Miller, 1994; Sim

et al

., 1994; Ranjan andChitnis, 1999). Malaria parasites thus use the conserved,cysteine-rich, DBL domains to generate parasite ligandswith diverse receptor-binding specificity.

It is important to understand the structure-functionbases for the interaction of DBL domains with their recep-tors. The binding sites for PvDBP and PkDBP have beenmapped to a 35-amino-acid region of the N-terminal extra-

Page 2: Mapping binding residues in the Plasmodium vivax domain that binds Duffy antigen during red cell invasion

1424

D. Hans

et al.

© 2005 Blackwell Publishing Ltd,

Molecular Microbiology

,

55

, 1423–1434

cellular domain of DARC (Chitnis

et al

., 1996). In addition,sulphation of tyrosine 41 in the binding site of DARC hasbeen shown to be critical for binding of PvDBP and PkD-aBP (M. Farzan, unpublished data). The binding domainsof PvDBP and PkDBP have been mapped to region II,which are prototypical DBL domains with approximately300 amino acids including 12 conserved cysteines. Here,we have analysed the kinetics of the interaction of thebinding domain, region II, of PvDBP (PvRII) with DARCusing surface plasmon resonance (SPR). We confirm thecritical role of tyrosine 41 of DARC and demonstrate thatthe binding of PvRII with DARC is a high-affinity interac-tion with a binding constant (

K

D

) of 8.7 nM. We havepreviously shown that binding residues for DARC lie in thecentral 170-amino-acid stretch of PvRII, which includescysteines 5–8 (Ranjan and Chitnis, 1999; Singh

et al

.,2003). Here, we have used site-directed mutagenesis andquantitative binding assays to map residues in this centralregion of PvRII that mediate binding with DARC. Interest-ingly, mutagenesis of a number of conserved hydrophobicresidues that lie within the central stretch of PvRII had amajor effect on binding. Hydrophobic interactions thusappear to play an important role in the binding of PvDBPwith DARC.

Results

Recombinant PvRII binds Duffy-positive human erythrocytes with specificity

Recombinant PvRII with a C-terminal six-histidine (6-His)fusion was expressed in

Escherichia coli

, purified frominclusion bodies by metal affinity chromatography,refolded by rapid dilution and purified further to homoge-neity by ion-exchange chromatography and gel filtrationchromatography as described earlier (Singh

et al

., 2001).Refolded PvRII was characterized for purity, homo-geneity and functional activity as previously described(Singh

et al

., 2001). Refolded and purified PvRIImigrates on SDS-PAGE with the expected mobility of

ª

39 kDa following reduction with dithiothriotol (DTT) andmigrates faster on SDS-PAGE under non-reducing condi-tions indicating presence of disulphide linkages (Singh

et al

., 2001). Analysis by gel filtration chromatographyconfirms that refolded PvRII is monomeric (data notshown). Refolded PvRII elutes as a single symmetricpeak upon reverse phase chromatography on a C-8column (Singh

et al

., 2001) indicating that it is conforma-tionally homogenous. Analysis by reverse phasechromatography and densitometry scanning of SDS-PAGE gels confirms that purity of refolded and purifiedPvRII is greater than 98%.

Refolded PvRII has been previously shown to bindDuffy-positive human erythrocytes but not Duffy-negative

human erythrocytes (Singh

et al

., 2001). Here, we havestudied the interaction of recombinant PvRII with DARCon human erythrocytes using SPR. Recombinant PvRIIwith a C-terminal 6-His tag was immobilized on a BiacoreNTA sensor chip coated with Ni and tested for binding toDuffy-positive human erythrocytes and chymotrypsin-treated erythrocytes that have lost DARC. Duffy-positivehuman erythrocytes yield a significant response differ-ence that is dependent on the haematocrit (Fig. 1). Chy-motrypsin-treated human erythrocytes do not yield asignificant response difference (Fig. 1) indicating that thebinding of PvRII to Duffy-positive human erythrocytes isspecific.

Recombinant PvRII binds DARC with high affinity

Recombinant PvRII has been shown to bind the N-terminal extracellular region of DARC (Chitnis

et al

.,1996; Choe

et al.

, 2005). The N-terminal 60-amino-acidextracellular domain of DARC fused with the Fc regionof human IgG1 at the C-terminus (nDARC-Ig) wasexpressed as a secreted protein in mammalian 293T cells.Sulphation of tyrosine 41 of DARC has been shown to becritical for binding of PvDBP (Choe

et al.

, 2005). Mamma-lian 293T cells were thus co-transfected with constructsfor expression of nDARC-Ig and a sulphotransferase toensure sulphation of recombinant nDARC-Ig. Recombi-nant nDARC-Ig was purified from culture supernatants byaffinity chromatography using Protein A. Analysis by SDS-PAGE revealed that under non-reducing conditionsnDARC-Ig contains two bands with apparent molecularweights of 110 kDa and 55 kDa (Fig. 2). Upon reduction,recombinant nDARC-Ig migrates as a single band ofapparent molecular weight of 55 kDa suggesting that puri-fied nDARC-Ig contains monomers (55 kDa) as well asdisulphide-linked dimers (110 kDa). nDARC-Ig monomersand dimers were separated by gel filtration chromatogra-phy (Fig. 2). nDARC-Ig monomers form dimers spontane-ously upon storage, whereas dimeric nDARC-Ig is stable.Purified, disulphide-linked, nDARC-Ig dimers were thusused for quantitative binding studies with PvRII usingSPR. A mutant nDARC construct (YF-nDARC-Ig) in whichtyrosine 41 was altered to phenylalanine and the N-terminal extracellular domain of the chemokine receptorCCR5 (nCCR5-Ig) were also expressed with C-terminalFc fusions and used as controls.

We initially attempted to immobilize nDARC-Ig onCM5 sensor chips, which contain a carboxymethylateddextran matrix, and inject PvRII as an analyte to studythe binding interaction. However, PvRII bound to blankflow cells on CM5 sensor chips and could thus not beused as an analyte in the binding assays. Other sensorchips such as B1, which are coated with lower levels ofshorter carboxymethylated dextran chains, were also

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Binding residues of

P. vivax

Duffy binding protein

1425

© 2005 Blackwell Publishing Ltd,

Molecular Microbiology

,

55

, 1423–1434

tested but PvRII bound to blank flow cells of these sen-sor chips as well. Because of this problem, PvRII wasimmobilized in flow cells of Biacore CM5 sensor chipsby amine coupling to the dextran matrix and recombi-nant nDARC-Ig, YF-nDARC-Ig and nCCR5-Ig wereinjected as analytes to test binding. Recombinant PfF2,the binding domain of EBA-175, which binds sialic acidresidues on glycophorin A, was produced in its correctlyfolded conformation as described previously (Pandey

et al

., 2002) and immobilized in control flow cells. Ablank flow cell was used to estimate bulk refractiveindex changes caused by flow of analyte in the flowcells. Injection of nDARC-Ig resulted in a measurableconcentration-dependent response difference in flowcells with immobilized PvRII (Fig. 3). No response differ-ence was detected when nDARC-Ig was injected ineither blank flow cells or flow cells coated with recombi-nant PfF2 (Fig. 3). YF-nDARC-Ig and nCCR5-Ig did notyield any response difference in flow cells immobilizedwith PfF2 (data not shown) or PvRII (Fig. 3). The obser-vation that nDARC-Ig does not bind PfF2 and nCCR5-Igdoes not bind PvRII indicates that the binding ofnDARC-Ig with PvRII results from a specific interaction.Importantly, YF-nDARC-Ig does not bind PvRII confirm-

ing the critical role played by tyrosine 41 of DARC in thebinding interaction.

The response curves obtained with different concentra-tions of nDARC-Ig flowing in cells with immobilized PvRIIwere used to estimate kinetic rate and binding constants(Fig. 3). As dimeric nDARC-Ig was used as analyte, abivalent interaction model in which one nDARC-Ig mole-cule can bind two PvRII molecules was used to fit thebinding data. The calculated binding constant,

K

D

, for thePvRII–nDARC-Ig interaction was estimated to be 8.7 nMwith association rate constants,

k

a1

and

k

a2

, of 1.5

¥

10

5

M

-

1

s

-

1

and 3.2

¥

10

-

2

RU

-

1

s

-

1

, respectively, and dissoci-ation rate constants,

k

d1

and

k

d2

, of 1.3

¥

10

-

3

s

-

1

and1.8

¥

10

-

2

s

-

1

respectively (the chi-square value for the fitis 0.186). The binding of PvRII with DARC is thus a spe-cific, high-affinity interaction.

Mapping the binding residues for DARC within PvRII, the receptor-binding domain of PvDBP

PvRII, the binding domain of PvDBP, contains approxi-mately 300 amino acid residues with 12 conserved cys-teines. We have previously demonstrated that criticalbinding residues for DARC lie in the central 170-amino-

0.025% RBC

0.05% RBC

0.05% chymotrypsin-treated RBC

0.0125% RBC

–30

40

110

180

250

320

390

460

530

600A

B

–30 10 50 130 210 290 370 450 530

Time (s)

Res

po

nse

dif

fere

nce

(R

U)

–50

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100

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–50 0 50 100 150 200 250 300 350 400 450

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dif

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PvRIIN291A I376A

Fig. 1.

Interaction of Duffy-positive human erythrocytes with wild-type and mutant PvRII analysed by surface plasmon resonance.A. Binding of wild-type PvRII to human red cells. Recombinant PvRII with a C-terminal 6-His fusion was immobilized in flow cells of Bia-core NTA sensor chip coated with Ni. Normal human Duffy-positive human erythrocytes as well as chymotrypsin-treated human erythro-cytes were injected at different haematocrits (0.0125%, 0.025% and 0.05%). Sensograms recorded during both association and dissocia-tion phases are shown. Duffy-positive human erythrocytes bind PvRII and yield a binding response that is dependent on haematocrit, whereas chymotrypsin-treated erythrocytes that have lost DARC fail to bind PvRII and do not yield a significant binding response. RBC, red blood cell.B. Binding of wild-type and mutant PvRII to Duffy-positive human red cells. Wild-type PvRII and mutants N291A and I376A were immobi-lized on Biacore NTA sensor chips coated with Ni and tested for binding to Duffy-positive human erythrocytes at 0.025% haematocrit.

Page 4: Mapping binding residues in the Plasmodium vivax domain that binds Duffy antigen during red cell invasion

1426

D. Hans

et al.

© 2005 Blackwell Publishing Ltd,

Molecular Microbiology

,

55

, 1423–1434

acid stretch of PvRII that spans amino acids 258–429 andincludes cysteines 5–8 (Ranjan and Chitnis, 1999; Singh

et al

., 2003). Here, we have mapped the residues in thiscentral region of PvRII that make contact with DARC. Inorder to identify potential binding residues of PvRII thatmay be involved in interaction with DARC, we performeda multiple sequence alignment to compare PvRII withreceptor-binding DBL domains of EBP orthologues

derived from

P. knowlesi

and

P. falciparum

(Fig. 4). Of the

P. knowlesi

EBPs used for the multiple sequence align-ment, PkDBP binds DARC on human and rhesus eryth-rocytes (Haynes

et al

., 1988; Chitnis and Miller, 1994),

P.knowlesi

b

binds sialic acid residues on an unidentifiedreceptor on rhesus erythrocytes and

P. knowlesi

g

bindsan unidentified receptor other than DARC or sialic acidresidues on rhesus erythrocytes (Ranjan and Chitnis,

Fig. 2.

Recombinant nDARC-Ig used for binding assays.A. Gel filtration profile of recombinant nDARC-Ig. Recombinant nDARC-Ig, which comprises of the N-terminal 60 amino acids of DARC fused to Fc region of human IgG, was expressed as a secreted protein in 293T mammalian cells and purified by affinity chromatography using Protein A and gel filtration chromatography using Superdex 200. The gel filtration profile of nDARC-Ig following purification using Protein A is shown. Proteins eluting in peaks A and B were collected and analysed by SDS-PAGE.B. SDS-PAGE analysis of recombinant nDARC-Ig. Recombinant nDARC-Ig was analysed by SDS-PAGE before and after reduction with dithiothreitol (

+

DTT, –DTT). Lane A, nDARC-Ig eluting in peak A during purification by gel filtration chromatography; lane B, nDARC-Ig eluting in peak B during purification by gel filtration chromatography; PL, preload, nDARC-Ig purified by affinity chromatography using Protein A that was used for gel filtration chromatography. Molecular weights are shown in kilodaltons (kDa).

+ DTT - DTT

116

66

45

35

kDa PL A B

110 kDa

55 kDa

0.0

5.0

10.0

15.0

20.0

5.0 10.0 15.0 20.0 25.0

Volume (ml)

B

A

Abs

orba

nce

(mA

U)

B

A

PL A B

Page 5: Mapping binding residues in the Plasmodium vivax domain that binds Duffy antigen during red cell invasion

Binding residues of

P. vivax Duffy binding protein 1427

© 2005 Blackwell Publishing Ltd, Molecular Microbiology, 55, 1423–1434

1999). The binding domain of P. falciparum EBA-175,which binds sialic acid residues on glycophorin A, mapsto its DBL domain, PfF2 (Sim et al., 1994). The multiplesequence alignment was used to identify amino acid res-idues in the central region of PvRII that were conservedin the binding domain of PkDBP (PkaRII) but had switchedto other residues or were missing in the binding domainsfrom P. knowlesi b and g proteins (PkbRII and PkgRII) andregion PfF2 of P. falciparum EBA-175, which bind recep-tors other than DARC (Fig. 4). Such amino acid residuesof PvRII that are shared with PkaRII and have been sub-stituted or are absent in the binding domains of other EBPorthologues were identified as potential binding residuesfor DARC (Fig. 4) and were targeted for alanine replace-ment mutagenesis.

The construct designed to express the wild-type PvRIIsequence from P. vivax Salvador I strain in E. coli wassubjected to site-directed mutagenesis to replace thecodons encoding potential binding residues with a codonfor alanine. The constructs generated by site-directedmutagenesis were used to express the alanine replace-ment mutants of PvRII with C-terminal 6-His tags in E. coliby methods similar to those described previously for wild-

type PvRII (Singh et al., 2001). The PvRII mutants werepurified by metal affinity chromatography from inclusionbodies, refolded by rapid dilution and purified further tohomogeneity by ion-exchange chromatography and gelfiltration chromatography as described previously for wild-type PvRII (Singh et al., 2001).

The recombinant PvRII mutants were analysed by SDS-PAGE before and after reduction with DTT. Each of themutants migrated with the expected mobility correspond-ing to molecular weight of ª39 kDa and migrated fasterunder non-reducing conditions indicating presence of dis-ulphide linkages (data not shown). Densitometry scanningof the Coomasie-stained SDS-PAGE gels confirmed thatthe purity of each mutant was greater than 90%. Twoconformationally sensitive monoclonal antibodies (mAbs),2H2 and 2H10, raised against PvRII were used to confirmby ELISA that the alanine replacement mutants are cor-rectly folded. Wild-type PvRII and alanine replacementPvRII mutants were coated on ELISA plates and detectedby polyclonal mouse antisera raised against PvRII as wellas mAbs 2H2 and 2H10. Each of the PvRII mutants wasrecognized by both mAbs 2H2 and 2H10 yielding OD490

values that were 70–100% of values obtained for wild-type

B

A

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60

–65 0 130 260 390 520 650

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po

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800 nM400 nM200 nM100 nM50 nM25 nM12.5 nM

–5

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400 nM nDARC-Ig

400 nM PfF2

400 nM YF-nDARC-Ig

400 nM nCCR5-Ig

Fig. 3. Analysis of PvRII–DARC interaction by surface plasmon resonance.A. Binding of PvRII to nDARC-Ig is specific. Recombinant PvRII was immobilized on Bia-core CM5 sensor chip surface and tested for binding to nDARC-Ig, nCCR5-Ig and YF-nDARC-Ig. Binding response was observed only in case of nDARC-Ig. nCCR5-Ig and YF-nDARC-Ig do not yield any binding response with immobilized PvRII indicating that the bind-ing of PvRII with nDARC-Ig is a specific inter-action. nDARC-Ig does not bind immobilized recombinant PfF2, the binding domain of EBA-175.B. Kinetic analysis of PvRII–DARC interaction. Different concentrations of nDARC-Ig ranging from 12.5 to 800 nM were injected over flow cells with immobilized PvRII and blank flow cells. Data points in grey are the resonance units obtained after subtraction of response recorded in blank flow cells. The black solid line represents the best fit of the experimental data after integrating all the curves simultaneously using a 1:2 binding model. The best fits were calculated using BIAEVALUATION 3.2RC1 soft-ware (Biacore). The calculated fits overlap with experimental data with random distribution of residuals having chi-square value of 0.186. The calculated binding constant (KD) for the PvRII–DARC interaction was 8.7 nM.

Page 6: Mapping binding residues in the Plasmodium vivax domain that binds Duffy antigen during red cell invasion

1428 D. Hans et al.

© 2005 Blackwell Publishing Ltd, Molecular Microbiology, 55, 1423–1434

PvRII confirming that the PvRII mutants are correctlyfolded. PvRII mutants were also detected by ELISA usingpolyclonal anti-PvRII mouse sera. The OD490 values forthe PvRII mutants were 75–100% of OD490 valuesobtained for wild-type PvRII. The ELISA values obtainedwith polyclonal anti-PvRII sera were used to correct forthe amount of protein used for coating wells used for theELISA-based binding assays with nDARC-Ig describedbelow.

As the Biacore assay required immobilization of PvRIIin flow cells, it was not possible to use this assay in a high-throughput format. We therefore developed an ELISA-based binding assay in which binding of wild-type PvRIIand alanine replacement PvRII mutants was tested tonDARC-Ig immobilized in ELISA wells. In parallel controlexperiments, YF-nDARC-Ig and nCCR5-Ig were coated inELISA plate wells and tested for binding to wild-typePvRII. Bound PvRII was detected using polyclonal anti-PvRII mouse sera (Fig. 5). A concentration-dependentincrease in bound PvRII was detected (Fig. 5) indicatingthat the ELISA capture assay can be used as a semi-quantitative assay to compare binding of wild-type PvRIIand PvRII mutants. Wild-type PvRII did not bind YF-nDARC-Ig or nCCR5-Ig in parallel experiments indicatingthat the binding of PvRII observed in nDARC-Ig-coatedwells was specific (Fig. 5).

The alanine replacement PvRII mutants were tested forbinding to nDARC-Ig in the ELISA-based binding assay.Binding of the PvRII mutants to nDARC-Ig is reported asa percentage of binding of wild-type PvRII to nDARC-Ig,which was taken as a reference (Fig. 5). The alaninereplacement PvRII mutants have been divided into fourcategories based on their level of binding with referenceto wild-type PvRII (Fig. 5 and Table 1). Interestingly themutations that have the most severe effect on binding(<30% reference binding) involve hydrophobic amino acidresidues (F299A, Y363A, F373A and I376A). Three ofthese mutations, namely, Y363A, F373A and I376A, clus-ter together adjacent to cysteine 6. F299A lies in anothercluster of mutations adjacent to cysteine 5 that affects

Fig. 4. Multiple sequence alignment of recep-tor-binding domains from EBPs. Multiple sequence alignment of receptor-binding DBL domains (region II) from P. vivax Duffy-binding protein (PvRII), P. knowlesi Duffy-binding pro-tein (PkaRII), P. knowlesi b protein (PkbRII), P. knowelsi g protein (PkgRII) and P. falciparum EBA-175 (PfF2). Amino acid residues in the central stretch of PvRII (amino acids 258–429) that were conserved in PkaRII but had switched or were missing in PkbRII, PkgRII and PfF2 were identified as potential binding residues for DARC and were targeted for alanine replace-ment mutagenesis. Amino acid residues for which replacement with alanine had no effect (>70–100% reference binding denoted by blank circles), minor effect (50–70% reference bind-ing denoted by filled triangle), moderate effect (30–50% reference binding denoted by filled circle) and major effect (<30% reference bind-ing denoted by exclamation marks) compared with reference binding of wild-type PvRII are shown.

Table 1. Summary of binding results.

Effect on binding % Reference bindinga Mutants

Major effect <30% reference binding Y363A, F373A,I376A, F299A

Moderate effect 30–50% reference binding N291AMinor effect 50–70% reference binding Y293A, D339ANo effect >70% reference binding R274A, E340A,

Q344A, Q348A,R398A

a. Binding of wild-type PvRII to nDARC-Ig was considered asreference.

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binding, namely, N291A, which has a moderate effect (30–50% reference binding), and Y293A, which has a minoreffect (50–70% reference binding). Mutation D339A,which lies between cysteines 5 and 6, also has a minoreffect on binding. Five mutations, R274A, E340A, Q344A,Q348A and R398A, have no effect on binding (0–30%reference binding). The residues that are critical for bind-ing thus appear to cluster primarily in two stretches of thecentral region of PvRII, one just upstream of cysteine 6and another just upstream of cysteine 5. All the mutations(F299A, Y363, F373 and I376) that have a major effect onbinding involve hydrophobic amino acid residues indicat-ing that hydrophobic interactions play an important role inbinding of PvDBP to DARC.

A subset of mutants, namely, I376A, N291A and Y293A,which have major (<30% reference binding), moderate(30–50% reference binding) and minor (50–70% refer-ence binding) effects on binding in the ELISA assay,respectively, were tested for binding with nDARC-Ig bySPR. The binding of nDARC-Ig to I376A in the SPR assaywas negligible and it was not possible to derive kinetic rateconstants for the interaction. The association and disso-

ciation rates (ka and kd) as well as binding constants (KD)derived from the response curves for interaction of N291Aand Y293A with nDARC-Ig are reported in Table 2. Theassociation rates constants for mutants N291A and Y293A(ka) are significantly lower than those for wild-type PvRII(Table 2). The binding constants (KD) for the mutants(Table 2) reflect the poor binding observed in the ELISA-based binding assays compared with wild-type PvRII.

A subset of PvRII mutants that had an effect on bindingto nDARC-Ig was also tested for binding to DARC onhuman red cells. Wild-type PvRII and mutants N291A andI376A were immobilized on Ni-NTA chips and tested forbinding to Duffy-positive human erythrocytes by SPR(Fig. 1B). Both mutants displayed reduced binding to

% R

efer

ence

bin

ding

0

10

20

30

40

50

60

70

80

90

100

PvRII R274A N291AY293A F299A D339A E340A Q344A Q348A Y363A F373A I376A R398A

PvRII mutants

B

A

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 0.05 0.1 0.15 0.2 0.25 0.3

PvRII concentration (µg ml–1)

OD

490

nDARC-Ig

nCCR5-Ig

YF-nDARC-Ig

Fig. 5. Binding of PvRII alanine replacement mutants to DARC.A. ELISA-based assay to test binding of PvRII with nDARC-Ig. Recombinant nDARC-Ig was coated in wells of ELISA plate and tested for binding to PvRII. Control wells were coated with BSA, nCCR5-Ig and YF-nDARC-Ig, in which tyrosine 41 of DARC is replaced with alanine. Bound PvRII was detected with anti-PvRII mouse polyclonal sera. PvRII binds nDARC-Ig but does not bind nCCR5-Ig or YF-nDARC-Ig indicating that the binding is specific.B. Binding of PvRII alanine replacement mutants to nDARC-Ig. Alanine replacement PvRII mutants were tested for binding to nDARC-Ig coated on ELISA. Binding of PvRII mutants to nDARC-Ig is reported as a percent-age of binding of wild-type PvRII.

Table 2. Kinetic analysis of binding of wild-type and mutant PvRIIwith DARC.

Ligand ka (M-1 s-1) kd (s-1) KD (M)

PvRII 1.5 ¥ 105 1.3 ¥ 10-3 8.7 ¥ 10-9

Y293A 2.5 ¥ 104 8.9 ¥ 10-4 3.6 ¥ 10-8

N291A 1.7 ¥ 104 8.0 ¥ 10-4 4.7 ¥ 10-8

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Duffy-positive human red cells compared with controlPvRII (Fig. 1B). The maximum response differencesrecorded for N291A and I376A were ª80% and ª20% ofthe maximum response difference recorded for wild-typePvRII. The reduction in binding of N291A and Y293A withnDARC-Ig observed in the ELISA and SPR assays is thusreflected in reduced binding to DARC on red cells.

Discussion

The EBP family of proteins plays a critical role in bindingred cell receptors to mediate invasion by Plasmodiummerozoites (Chitnis, 2001). The binding domains of EBPslie in N-terminal, conserved, cysteine-rich regions that arereferred to as DBL domains (Chitnis and Miller, 1994; Simet al., 1994; Ranjan and Chitnis, 1999; Chitnis, 2001;Prasad et al., 2003). Here, we have studied the interactionof PvRII, the binding domain of PvDBP, with its receptor,DARC, on erythrocytes. Recombinant PvRII with a C-terminal 6-His tag was expressed in E. coli, purified frominclusion bodies, refolded by rapid dilution and purified tohomogeneity as previously described (Singh et al., 2001).Refolded PvRII immobilized on a Biacore NTA chip coatedwith Ni bound red cells with specificity (Fig. 1A) indicatingthat it is functional.

The binding site for PvRII was previously mapped to theN-terminal extracellular region of DARC (Chitnis et al.,1996). Moreover, sulphation of tyrosine 41 in the N-termi-nal extracellular region of DARC has been shown to becritical for binding of PvRII (Choe et al., 2005). The 60-amino-acid N-terminal extracellular region of DARC wasexpressed with a C-terminal fusion with Fc portion ofhuman IgG (nDARC-Ig) as a secreted protein in mamma-lian 293T cells and purified by affinity chromatographyusing Protein A. Here, we have studied the binding kinet-ics of the interaction of PvRII with nDARC-Ig using SPR(Fig. 3). The association and dissociation responsecurves derived from PvRII-coated flow cells at differentconcentrations of analyte nDARC-Ig were analysed todetermine the kinetic rate constants for the PvRII–nDARC-Ig interaction. The binding constant (KD) for theinteraction of PvRII with nDARC-Ig is estimated to be8.7 nM, which indicates that it is a high-affinity, specificinteraction. Recombinant YF-nDARC-Ig did not yield anybinding response in PvRII-coated flow cells (Fig. 3), dem-onstrating the critical role of tyrosine 41 of DARC in bind-ing with PvRII.

It is important to understand the structure-functionbases for the interaction of PvRII with DARC. We havepreviously shown that the minimal binding domain ofPvRII maps to the central 170-amino-acid stretch (aminoacids 258–429) that includes cysteines 5–8 (Singh et al.,2003). Here, we have used site-directed mutagenesis andquantitative binding assays to map binding residues for

DARC within this central stretch of PvRII. In order toidentify potential binding residues, we performed a multi-ple sequence alignment of the binding domains of PvDBP(PvRII), PkDBP (PkaRII), P. knowlesi b (PkbRII) and g(PkgRII) proteins and P. falciparum EBA175 (PfF2)(Fig. 4). The alignment allowed the identification of aminoacid residues that were conserved in PvRII and PkaRII,which bind DARC, and either had radical substitutions orwere missing in PkbRII, PkgRII or PfF2, which bind recep-tors other than DARC. Twelve such potential binding res-idues identified in PvRII were replaced with alanine bysite-directed mutagenesis and recombinant alaninereplacement mutants were tested for binding to nDARC-Ig. The recombinant alanine replacement PvRII mutantswere produced using methods similar to those describedfor wild-type PvRII. Two conformation sensitive mAbs 2H2and 2H10 were used to confirm correct folding of thePvRII mutants. These assays indicate that the refoldedPvRII mutants are correctly folded, although the possibilitythat the proteins are misfolded cannot be completely ruledout based on reactivity with two mAbs. Of the 12 mutantstested, four had a major effect on binding with greater than70% reduction in binding compared with wild-type PvRII.Interestingly, all four mutations that had a major effect onbinding were in hydrophobic amino acid residues, namely,F299, Y363, F373 and I376. Of these, Y363, F373 andI376 cluster together adjacent to cysteine 6 suggestingthat this region forms part of the binding pocket for DARC.The other mutation that has a major effect, namely, F299,lies in another cluster of amino acid residues adjacent tocysteine 5 including N291 and Y293 that have moderate(30–50% reference binding) and minor (50–70% refer-ence binding) effects on binding respectively. The regionincluding N291, Y293 and F299 may also form part of thebinding pocket for DARC. Hydrophobic interactions areknown to play important roles in receptor–ligand bindinginteractions. For example, the binding site of the humangrowth hormone receptor comprises a hydrophobic patchincluding two critical tyrosine residues surrounded by ahydrophilic periphery (Clackson et al., 1998).

PvRII is a prototype DBL domain that shares homologywith receptor-binding domains of other EBPs that bindreceptors other than DARC as well as DBL domains fromthe PfEMP-1 family of P. falciparum variant surface anti-gens that bind diverse receptors to mediate cytoadher-ence (Chitnis et al., 1999). Receptor-binding residueshave been mapped to the central regions of diverse DBLdomains from EBPs and PfEMP-1 that bind receptors asdiverse as sialic acid residues on glycophorin A, comple-ment receptor-1 and chondroitin sulphate A (Mayor et al.,2005). It remains to be seen whether hydrophobic resi-dues also play a major role in the interaction of these DBLdomains with their receptors. The mutation D339A inPvRII, which has a moderate effect on binding to DARC,

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lies between cysteines 5 and 6. Sequence polymorphismsstudies on PvDBP from P. vivax field isolates from PapuaNew Guinea and South America reveal that D339 is fre-quently replaced with glycine (Tsuboi et al., 1994; Ampu-dia et al., 1996; Xainli et al., 2000), suggesting that therole of D339 may not be critical and can be substituted byother amino acid residues in the variants. No polymor-phisms have been reported in any of the hydrophobicamino acid residues that that have been identified asbinding residues providing support for their role in bindingDARC.

PvRII has a predicted pI of 8.9, with a number of posi-tively charged residues such as arginine and lysine. TheN-terminal extracellular region of DARC, nDARC, is acidicwith a predicted pI of 3.5. Moreover, nDARC is also glyc-osylated and is therefore negatively charged. Althoughhydrophobic interactions appear to be critical for the inter-action of PvRII with DARC, the role of electrostatic inter-actions between oppositely charged residues on PvRIIand DARC is not completely ruled out. Mutagenesis ofR274 and R398 had a marginal effect on binding (20%and 30% reference binding respectively). It is possible thatmutagenesis of multiple positively charged residuestogether may show a more significant effect on binding.Positively charged residues in PvRII may play a role in theinitial recognition of DARC through electrostatic interac-tions. However, as demonstrated here, hydrophobic inter-actions appear to be primarily responsible for the highaffinity and specificity of binding. The three-dimensionalstructure of PvRII is needed to understand how the bind-ing residues identified here form the binding pocket inPvRII. Understanding the structural basis of the interac-tion of PvDBP and other EBPs with their receptors onerythrocytes may enable the development of novel mole-cules that block these interactions and inhibit erythrocyteinvasion to prevent malaria.

Experimental procedures

Mutagenesis of PvRII and expression of wild-type and mutant PvRII

A synthetic gene was designed to encode the region IIsequence of PvDBP (PvRII) from P. vivax Salvador I strainfused to a 6-His tag using codons optimized for expressionin E. coli. The synthetic gene for PvRII was cloned in the E.coli expression vector pET28a+ and the expression con-structs were transformed in E. coli BLR(DE3) plysS cells. Aseparate manuscript describing the synthetic gene for PvRIIand improvement in yield of recombinant PvRII is currentlyunder review. Single-amino-acid mutations were introducedinto the synthetic gene for PvRII cloned in pET28a+ by poly-merase chain reaction (PCR) by incorporation of mismatchedmutagenic oligonucleotide primers (QuickChange, Strat-agene). Mutagenesis was attempted on N296A and K297Ain addition to all the residues marked in Fig. 4. Despite

repeated attempts, it was not possible to produce mutantsN296A and K297A. DNA minipreps (Qiagen) of mutagenizedgenes were sequenced to screen for the correct mutationsand lack of other, PCR-induced, non-specific mutations.

Expression, refolding and purification of PvRII and alaninereplacement PvRII mutants were carried out essentially asdescribed previously for expression of PvRII using the nativegene (Singh et al., 2001). In brief, expression of recombinantwild-type PvRII and alanine replacement PvRII mutants wasinduced with IPTG in E. coli shake flask cultures. Wild-typeand mutant PvRII was purified by metal affinity chromatogra-phy using Ni-NTA matrix (Qiagen) from inclusion bodies thathad been solubilized with 6 M guanidine hydrochloride (Gdn-HCl). Recombinant proteins purified under denaturing condi-tions were refolded by rapid dilution and purified to homoge-neity by ion-exchange chromatography using Toyopearl-SP(Sigma) and gel filtration chromatography using Superdex 75(Pharmacia).

The wild-type and mutant PvRII proteins were analysed byseparation on SDS-PAGE gels before and after reduction withDTT. The recombinant proteins were also analysed by ELISAfor structural integrity using two mouse mAbs, 2H2 and 2H10,which recognize conformation-sensitive epitopes on PvRII.Wild-type and mutant PvRII were coated on ELISA plates ata concentration of 5 mg ml-1 and detected with mAbs 2H2 and2H10 used at dilutions of 1:1000 and 1:6000 respectively.Secondary anti-mouse IgG goat antibodies coupled to horse-radish peroxidase (HRP) (Sigma) were used at a dilution of1:3000. PvRII concentration of 5 mg ml-1 was within thedynamic range for detecting differences in concentrations ofcorrectly folded PvRII under these conditions. Mouse poly-clonal serum raised against PvRII, which detects bothreduced and non-reduced PvRII, was used at a dilution of1:32 000 in combination with HRP-coupled secondary anti-mouse IgG goat antibodies at a dilution of 1:3000 to detecttotal PvRII. PvRII concentration of 5 mg ml-1 is within thedynamic range for detecting differences in concentrations oftotal PvRII with the mouse polyclonal sera under theseconditions.

Expression and purification of nDARC-Ig, YF-nDARC-Ig and nCCR5-Ig

A plasmid encoding nDARC-Ig was generated by ligating thefirst 60 codons of human DARC (FyB) to sequences encod-ing Fc region of human IgG (nDARC-Ig) in a mammalianexpression vector (Choe et al., 2005). A plasmid encodingthe nDARC variant YF-nDARC-Ig, in which the codon fortyrosine 41 was altered to a codon for phenylalanine, wasgenerated by site-directed mutagenesis using the Quick-Change method (Stratagene). Coding regions for DARC andits variants were sequenced to confirm the presence of thecorrect mutations and the lack of other, PCR-induced, non-specific mutations. The plasmid encoding nCCR5-Ig was pre-viously described (Farzan et al., 1999).

Recombinant nDARC-Ig, YF-nDARC-Ig and nCCR5-Igwere produced by co-transfecting 293T cells with plasmidsencoding these fusion proteins and a plasmid encodinghuman tyrosyl protein sulphotransferase 2. The recombinantproteins were purified from cell culture supernatants by affin-ity chromatography using Protein A. nDARC-Ig dimers were

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further purified by gel filtration chromatography using Super-dex 200 (Amersham Biosciences) in phosphate-bufferedsaline (PBS) with 300 mM NaCl. The molecular weight ofnDARC-Ig dimers is 110 kDa. The concentration of purifiednDARC-Ig dimers was determined spectrophotometrically bymeasuring absorbance at 280 nm and using the theoreticalmolar extinction coefficient for nDARC-Ig (1.027 cm2 mg-1).

Surface plasmon resonance analysis of the interaction of PvRII with DARC on erythrocytes

Recombinant PvRII was immobilized on a Biacore NTA chipcoated with Ni and tested for binding to DARC on humanerythrocytes at 25∞C using a Biacore 2000 instrument asdescribed below. A NTA chip (Biacore AB) was coated withNi by injection of 40 ml of 500 mM NiCl2 at a flow rate of20 ml min-1. Recombinant wild-type PvRII with a C-terminal6-His tag was captured on the Ni-coated chip by injectingPvRII (10 mg ml-1) at a flow rate of 10 ml min-1 for 1 min.Around 100 RU of PvRII was immobilized on the Ni surface.Blood was collected in 10% citrate phosphate dextrose,stored at 4∞C for up to 4 weeks and washed with incompleteRPMI 1640 (Life Technologies) before use. Duffy phenotypesof blood samples were confirmed by standard blood typingmethods using two antisera (anti-Fya and anti-Fyb) (Ortho-clinical Diagnostics). As Duffy-negative human blood was notavailable, Duffy-positive human erythrocytes were treatedwith chymotrypsin to remove DARC as described earlier.Normal and chymotrypsin-treated human erythrocytes sus-pended at different haematocrits (0.0125%, 0.025% and0.05%) in PBS pH 7.2 containing 0.3 mg ml-1 of bovine serumalbumin (BSA) were injected for 4 min in flow cells coatedwith PvRII at a flow rate of 3 ml min-1. Dissociation was mon-itored for 300 s during which PBS containing BSA wasinjected at a flow rate of 3 ml min-1. The surface was regen-erated by injecting 40 ml of regeneration solution (350 mMEDTA, 10 mM Hepes, 150 mM NaCl, 0.005% surfactant P20,pH 8.3) at a flow rate of 20 ml min-1. A blank flow cell that wasnot coated with Ni or PvRII was used as control. The bindingresponse obtained in the control flow cell was deducted fromthe experimental flow cell to obtain the specific bindingresponse.

Surface plasmon resonance analysis of the interaction of PvRII with soluble nDARC-Ig peptide

We used a Biacore 2000 instrument to study the kinetics ofthe binding of PvRII with nDARC-Ig peptide. RecombinantPvRII was immobilized by amine coupling to the dextranmatrix on the surface of Biacore sensor chip CM5 as follows.The carboxymethylated dextran surface was activated byinjection of 1:1 mixture of N-hydroxysuccinimide (NHS) (Bia-core AB) and 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiim-ide hydrochloride (EDC) (Biacore AB) for 7 min at a flow rateof 10 ml min-1. Recombinant PvRII (10 mg ml-1) in 10 mMsodium acetate buffer, pH 4.5 was injected over the activatedsurface at a flow rate of 10 ml min-1 so as to get an immobi-lization of ª250 RU. A blank flow cell was activated by injec-tion of 70 ml of 1:1 mixture of NHS (Biacore AB) and EDC

(Biacore AB) at a flow rate of 10 ml min-1 and subsequentlyblocked by injection of ethanolamine (Biacore AB) at a flowrate of 10 ml min-1 for 5 min. The blank flow cell was used asa negative control.

Kinetic binding analysis for interaction of nDARC-Ig toPvRII was carried out by injecting nDARC-Ig at different con-centrations (12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM,400 nM, 800 nM) in running buffer (10 mM Hepes, pH 7.4,150 mM NaCl, 3 mM EDTA and 0.005% P20 surfactant) at aflow rate of 30 ml min-1 for 5 min. Dissociation was monitoredfor 300 min in the presence of running buffer at the same flowrate. The experimental surface was regenerated by injectingregeneration solution (10 mM Hepes, 3 M NaCl, 3 mM EDTAand 0.05% P20 surfactant, pH 7.4) at a flow rate of 60 mlmin-1. To correct for refractive index changes caused by run-ning buffer and instrument noise, running buffer was injectedat a flow rate of 30 ml min-1 in blank flow cells and theobserved response difference was subtracted from the exper-imental data. The flow rate of nDARC-Ig was varied from10 ml min-1 to 75 ml min-1 in order to assess whether the bind-ing interactions were limited by mass transfer. There was nochange in the observed response differences in wells coatedwith PvRII upon injection of nDARC-Ig at different flow ratesin this range, confirming that binding interactions were notmass transfer limited.

Given the bivalent (dimeric) nature of the analyte (A),nDARC-Ig, the kinetic rate constants for interaction with theligand (B), PvRII, were determined by fitting the correctedresponse data to a simple bimolecular (1:2) interactionmodel, A + B = AB; AB + B = AB2. The rate equations werenumerically integrated and the results were simultaneouslyfitted to association and dissociation phase response datausing the non-linear least square data analysis program BIAE-

VALUATION 3.2RC1 (Biacore AB). The binding constant, KD,was calculated as kd1/ka1.

ELISA-based binding assay to study interaction of PvRII and alanine replacement PvRII mutants with DARC

Recombinant nDARC-Ig, YF-nDARG-Ig and nCCR5-Ig werecoated in sodium bicarbonate buffer pH 9.6 overnight at 4∞Cat a concentration of 1 mg ml-1 in ELISA plate wells. Recom-binant wild-type and mutant PvRII proteins (0.05 mg ml-1)were added to the wells to allow binding. Bound PvRII wasdetected with mouse polyclonal anti-PvRII sera (1:2500 dilu-tion) and HRP-conjugated anti-mouse IgG goat antibodies(1:5000 dilution). Small variations in concentrations of PvRIImutants were corrected for using the results of ELISA per-formed with polyclonal anti-PvRII mouse sera to detect totalPvRII.

Acknowledgements

We thank Amit Sharma, Malaria Group, ICGEB, New Delhifor helpful discussions during the course of this work and forcomments on the manuscript. C.E.C. is an InternationalResearch Scholar of the Howard Hughes Medical Institute,USA and International Senior Research Fellow of TheWellcome Trust, UK.

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