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Cell, Vol. 122, 183–193, July 29, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.cell.2005.05.033
Structural Basis for the EBA-175
Erythrocyte Invasion Pathway
of the Malaria Parasite Plasmodium falciparum
Niraj H. Tolia,1,2 Eric J. Enemark,2 B. Kim Lee Sim,3,*
and Leemor Joshua-Tor1,2,*
1
Watson School of Biological Sciences
2
Keck Structural Biology Laboratory
Cold Spring Harbor Laboratory
1 Bungtown Road
Cold Spring Harbor, New York 11724
3
Protein Potential
9640 Medical Center Drive
Rockville, Maryland 20878
Summary
Erythrocyte binding antigen 175 (EBA-175) is a P. fal-
ciparum protein that binds the major glycoprotein
found on human erythrocytes, glycophorin A, during
invasion. Here we present the crystal structure of the
erythrocyte binding domain of EBA-175, RII, which
has been established as a vaccine candidate. Binding
sites for the heavily sialylated receptor glycophorin A
are proposed based on a complex of RII with a glycan
that contains the essential components required for
binding. The dimeric organization of RII displays two
prominent channels that contain four of the six ob-
served glycan binding sites. Each monomer consists
of two Duffy binding-like (DBL) domains (F1 and F2).
F2 more prominently lines the channels and makes
the majority of the glycan contacts, underscoring its
role in cytoadherence and in antigenic variation in
malaria. Our studies provide insight into the mecha-
nism of erythrocyte invasion by the malaria parasite
and aid in rational drug design and vaccines.
Introduction
Malaria causes an estimated 300–500 million clinical
cases and 1–3 million deaths annually (Breman et al.,
2001). More than 80% of deaths from malaria occur in
children in sub-Saharan Africa (Snow et al., 1999). The
disease is caused by parasites of the genus Plasmo-
dium, of which Plasmodium falciparum is the most
prevalent and is responsible for almost all the associ-
ated mortality. Sporozoite-stage malarial parasites in-
oculated by an infected female Anopheles mosquito
rapidly invade hepatocytes. After five to six days, the
parasites become mature liver-stage schizonts with
thousands of merozoites. When the hepatocyte rup-
tures, the merozoites are released into the blood
stream. Each of these merozoites can invade an eryth-
rocyte, initiating the erythrocytic stage of the parasite’s
life cycle (for review see Miller et al. [2002]).
All of the pathological and clinical manifestations of
the disease are caused by the asexual erythrocytic
stage. Thus, the invasion of erythrocytes by merozoites
marks a critical step that is a logical target for the de-
*Correspondence: leemor@cshl.edu (L.J.); ksim@protpot.com
(B.K.L.S.)
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velopment of interventions to control malaria. Several
specific ligand-receptor interactions have been iden-
tified for invasion and genetic disruption of any one in-
teraction results in a shift to using other pathways (Dur-
aisingh et al., 2003a, 2003b; Reed et al., 2000; Triglia et
al., 2005).
EBA-175 is a P. falciparum protein that binds its re-
ceptor glycophorin A (GpA) on human erythrocytes dur-
ing invasion (Adams et al., 1992; Camus and Hadley,
1985; Klotz et al., 1992; Orlandi et al., 1992; Sim et al.,
1990). GpA is the major glycoprotein found on human
erythrocytes and is heavily sialylated (Marchesi et al.,
1972). The EBA-175/GpA pathway is the dominant chy-
motrypsin-resistant invasion pathway (Duraisingh et al.,
2003a). Antibodies against EBA-175 inhibit binding to
GpA and block invasion of merozoites in vitro (Sim et
al., 1990, 2001). The receptor-ligand interaction in-
volves sialic acids on the mucin domain of GpA, prefer-
entially Neu5Ac(α2-)Gal(Neu5Ac(α-2,6)Gal) on O-linked tet-
rasaccharides. GpA, the major sialoglycoprotein found on
erythrocytes, is a 131 amino acid transmembrane dimer.
Each monomer spans the membrane once exposing its
N terminus extracellularly (MacKenzie et al., 1997). The
erythrocyte binding domain of EBA-175 is region II (RII)
(Figure 1). RII is a 616 amino acid fragment of EBA-175
that consists of two cysteine-rich regions—F1 and F2.
F1 and F2 are homologous to the Duffy binding protein
of P. vivax and are therefore called Duffy binding-like
(DBL) domains. The presence of one or two DBL do-
mains, a C-terminal cysteine-rich region (region VI), and
a type I transmembrane-like domain define EBA-175 as
a member of the erythrocyte binding-like (EBL) super-
family (Adams et al., 1992; Figure 1). The EBL super-
family includes several other proteins believed to be
involved in erythrocyte invasion, such as the following:
Duffy binding protein, BAEBL/EBA-140/EBP2 (Mayer et
al., 2001; Narum et al., 2002; Thompson et al., 2001),
EBA-181/JESEBL (Gilberger et al., 2003b), and MAEBL
(Blair et al., 2002), and other candidate genes where
the presence of protein was not identified (Triglia et al.,
2001). Also included are the var genes, which are a
large family of genes regulated by chromatin modifica-
tion (Duraisingh et al., 2005; Freitas-Junior et al., 2005)
that encode antigenically variant proteins including
PfEMP-1 (Baruch et al., 1995; Smith et al., 1995; Su
et al., 1995). PfEMP-1 contains several copies of DBL
domains and is responsible for cytoadherence of para-
sitized erythrocytes to endothelium causing the most
fatal form of malaria. The signature DBL domains of the
EBL family have been found to be important for recep-
tor binding (Mayor et al., 2004) and it appears that these
domains are unique to Plasmodium and thus constitute
a good drug target (Aravind et al., 2003).
RII is highly conserved among more than 30 labora-
tory and field isolates (Liang and Sim, 1997) and has
been established as a vaccine candidate (Jones et al.,
2001; Sim et al., 2001). Toward the understanding of
receptor-ligand interactions critical for erythrocyte in-
vasion by P. falciparum, we have determined the crystal
Cell
184

Figure 1. DBL-Containing Proteins from Plasmodium Species

(A) The ebl family shares a conserved gene structure that encodes either one or two DBL domains in region II. The var superfamily contains
genes that encode multiple repeats of DBL domains. MAEBL is a unique member of the EBL family. It has the conserved domain structure,
but its region II does not contain DBL domains. Instead, the MAEBL ligand domains are related to another merozoite protein, apical membrane
antigen 1 (AMA-1). PvDBP—Plasmodium vivax Duffy binding protein; PkDBP—Plasmodium knowlesi DBP; PfEMP-1—Plasmodium falciparum
erythrocyte membrane protein 1.
(B) Alignment and structural depiction of DBL-containing proteins. Light blue indicates conserved residues, and deep blue indicates invariant
residues. Cysteines are shown in red. Secondary structure is shown above the sequence alignment with F1 in green, linker regions in gold,
and F2 in purple. Dashed lines indicate regions not observed in the structure of RII. Blue and red dots indicate residues involved in dimeriza-
tion and glycan binding, respectively.
Crystal Structure of a Malaria Vaccine Candidate
185

structure of EBA-175 RII of P. falciparum strain 3D7 to

2.3 Å resolution.
The structure shows a dimeric form of RII, with exten-
sive interactions between the elongated monomers.
Two channels are prominent in the center of the dimer.
The F1 and F2 domains are mostly helical and are sim-
ilar in structure. To identify possible receptor binding
sites, we cocrystallized RII with α-2,3-sialyllactose. The
glycan binding sites suggest a possible interaction sur-
face for the receptor. Our structural studies facilitate
the elucidation of the mechanism of erythrocyte inva-
sion and allow the rational design of receptor blockade
therapy and vaccines.

Results and Discussion

Overall Structure of RII

The unbound RII domain of EBA-175 of P. falciparum
crystallized in two distinct crystal forms. The structure
was determined by multiwavelength anomalous disper-
sion (MAD) (see Experimental Procedures). The overall
organization of RII in both crystal forms is conserved,
with an rmsd for all Cα atoms of 0.9 Å.
The crystal structure of the RII monomer reveals an
elongated molecule, composed primarily of α helices,
with two antiparallel β hairpins and several bound sul-
fate molecules (Figure 2A). The two DBLs, F1 (residues
8–282) and F2 (residues 297–603), have a very similar
structure with an rmsd of 1.96 Å (calculated by LSQMAN
[Kleywegt, 1999] for 194 Cα atoms; Figure 2B). The two
domains are connected by a three helix linker (283–
317). These domains possess a novel fold, based on
an analysis with DALI (Holm and Sander, 1993). Each
domain is composed of two subdomains. The topology
of RII, shown in Figure 2C, demonstrates the similarity
of the two repeats and depicts the two subdomains of
each repeat. All but one of the cysteines, C273, are in-
volved in disulfide bridges. C598 is not well ordered in
the structure of unbound RII but is in a clear disulfide
bridge with C513 as seen in the complex described be-
low. All disulfide bridges are contained within a given
subdomain (Figure 2C). The bridges superimpose nearly Figure 2. The RII Monomer
perfectly between the two domains, and stabilize the (A) Overall structure of the RII monomer. F1—green; F2—purple;
RII cysteine-rich repeat fold (F1/F2). linker—gold; bound sulfates—CPK spheres.
Alignment of EBA-175 RII with other RIIs from the (B) Overlay of RII subdomains F1 (green) and F2 (purple). Red
bonds and yellow bonds indicate disulfide bridges in F1 and F2, re-
EBL family of proteins (Figure 1B) reveals 34 invariant spectively.
residues. With the exception of Q43, W330, W450, W458 (C) Topology diagram of RII denoting similarity between F1 (green)
and W528, all invariant residues are involved in fold sta- and F2 (purple). Disulfide bridges (red) are all within subdomains of
bilization, further contributing to the stability of the DBL F1 and F2. The dotted line indicates portions of the structure or-
domain fold. As with the disulfide bridges, a large num- dered in the complex but not in unbound RII.
ber of the invariant residues interact almost exclusively
with other residues in the same subdomain. Exceptions
are a salt bridge between R349 and E488, hydrogen with the F2 domain of the second monomer and vice-
bonds (H bonds) between D414 and Q481, and hy- versa. This dimeric interaction is conserved in all crys-
drophobic interactions between W330 and W489 and tal forms of RII. The buried surface area is 1480 Å2 per
between R349 and W485, all of which are involved in dimer, consistent with the expected size of a total bur-
interactions between the two subdomains of F2. ied area of 1600 ± 400 Å2 for noncovalently interacting
proteins (Lo Conte et al., 1999). Dynamic light scatter-
The RII Dimer—a Handshake Interaction ing (DLS) experiments show that RII exists as a dimer
RII crystallizes as a dimer where two symmetrically re- with a Stoke’s radius of 4.29 ± 0.87 nm and an apparent
lated RII molecules interact extensively with each other molecular weight of 160 kDa at high concentrations
in an antiparallel fashion resembling a handshake (Fig- (w2 mg/ml; data not shown). However, analytical ultra-
ure 3A). The F1 domain of one monomer interacts mostly centrifugation experiments at lower concentrations (0.4
Cell
186
mg/ml) indicate RII is predominantly monomeric with a
small percentage of dimers (data not shown). In addi-
tion, crosslinking studies with bis-sulfosuccinimidyl
show that RII exists in both monomeric and dimeric
forms (data not shown). Thus, we observe a mixture
of both species, monomers and dimers, in solution. A
surface representation of the RII dimer (Figure 3B)
shows a highly basic protein surface (the calculated pI
is 8.8) and reveals the presence of two channels that
span the dimer. The channel surfaces consist of resi-
dues from both monomers. Most of these residues,
about two-thirds, come from the two F2 domains of
the dimer.
The two β hairpins of F1 and F2 form structural mod-
Figure 3. The RII Dimer
(A) Stereo ribbon representation of the RII di-
mer. Monomers are shaded in different inten-
sities (light/dark) for clarity. F1 domains are
green or light green; F2 domains are purple
or light purple, and the linker regions are
gold or light gold. Sulfates are depicted in
ball and stick (sulfurs in yellow, oxygens in
red).
(B) Two views of the electrostatic surface po-
tential of the RII dimer calculated by GRASP
at an ionic strength of 0.1 M. Surface poten-
tial is colored is from −10 kT (red) to +10 kT
(blue). Sulfates can be clearly seen on the
“bottom” surface on the left and in the
channels.
(C) A close up view of the β finger of F1
(green) inserted into a cavity of F2 (purple).
(D) A close up view of the β finger of F2 (pur-
ple) and the cavity of F1 (green).
(E) Interactions between the two-stranded
antiparallel β sheet of the two F2s. Coloring
scheme as in (A). Dashed lines indicate hy-
drogen bonds and/or electrostatic interac-
tions.
ules we termed “β fingers” that mediate intermolecular
interactions. Residues of the F1 β finger insert into a
cavity in F2 (Figure 3C), and residues of the F2 β finger
insert into a cavity in F1 (Figure 3D). Some key interac-
tions are a salt bridge between D30 (F1 β finger) and
R446 (F2 cavity) and van der Waals interactions be-
tween T114 (F1 cavity) and L338 and T340 (F2 β finger).
The dimer is further stabilized by H bonds between the
N118 side chain (F1 cavity) and the backbone carbonyl
of S339 of the F2 β finger and between the carbonyl of
T340 (F2 β finger) and the amide group of Q121 (F1
cavity). The third intermolecular interaction is the for-
mation of a two strand antiparallel β sheet between
identical F2 residues (N433-H436—β5) of each mono-
Crystal Structure of a Malaria Vaccine Candidate
187
mer (Figure 3E). This β sheet is found in the center of
the dimer, and separates the two channels.
Several well-ordered sulfate molecules are observed
at the same positions in both crystal forms. Interest-
ingly, these are bound exclusively in the channels and
on one face of the dimer (Figure 3B), which for our dis-
cussion we call the “bottom” surface.
Receptor Binding Sites
In order to locate receptor binding surfaces and study
the binding mode of RII to the GpA receptor, we cocrys-
tallized RII with the glycan α-2,3-sialyllactose. This gly-
can contains the essential component, Neu5Ac(α2,3)-
Gal, of the receptor glycan that is required for binding
(Neu5Ac denotes sialic acid). The complex crystallized
in a third, related crystal form, with one dimer in the
asymmetric unit (see Experimental Procedures). The
protein structure is largely unchanged (rmsd 0.39 Å),
though there is a slight reduction in the hinge angle
between F1 and F2 in the complex. In addition, resi-
dues 597–601 that were disordered in the unbound
structure are ordered in the complex. This segment in-
cludes C598, which is now clearly seen in a disulfide
bond with C513. All but two of the sulfates are ob-
served here as well.
Six glycan binding sites are observed in the RII dimer,
all at the dimer interface (Figure 4 and see Figure S1 in
the Supplemental Data available with this article on-
line). Four of these, 1–4, are inside the channels. The
other two, 5 and 6, are exposed to the top surface and
are separated from the channel by the β fingers and by
glycans 1 and 2. There is also a sulfate between glycan
positions 1 and 5 and between 2 and 6. Glycan posi-
tions 1 and 2, 3 and 4, and 5 and 6 are related by an
approximate 2-fold axis. Though it is possible to model
sialic acid moieties for all glycans, and even an addi-
tional galactose moiety for glycan positions 5 and 6
(Figure S1), it was not possible to model these accu-
rately and unambiguously due to somewhat limited res-
olution (2.25 Å) combined with partial occupancy (0.4–
0.6) for the glycans, resulting in a mixture of bound and
unbound conformations for some of the side chains in
the vicinity of these glycans.
All six glycans contact both monomers, implicating
dimerization as important for receptor binding (Figures
4B and S1D; Table S1). Glycans 1 and 2 contact resi-
dues N417, R422, N429, K439, and D442 of one mono-
mer and K28 of the other monomer. It should be noted
that Y55 from a neighboring complex also contacts gly-
cans 1 and 2; however, since this is a lattice interaction,
its significance is not clear. Glycans 3 and 4 contact
residues N550, N551, Y552, K553, and M554 from one
monomer and N33 from the other. Glycans 5 and 6 con-
tact residues T340, K341, D342, V343, Y415, Q542, and
Y546 of one monomer and residues K28, N29, R31, and
S32 of the second monomer. We modeled the full
O-glycan of GpA, Neu5Ac(α2-3)Gal (NeuAc(α-2,6)Gal),
at each of the glycan binding sites observed in the
complex to test whether it would fit. Indeed these
studies indicate that the full O-glycan could be accom-
modated in all of these sites with good geometry (Fig-
ure S2).
Functional Analysis of RII Mutants
To test the relevance of the dimeric state and glycan
binding sites to RII erythrocyte binding, we performed
a functional analysis on selected residues involved in
these characteristics of RII. RII single point mutants
were individually expressed on the surface of COS cells
and tested for their ability to bind normal human eryth-
rocytes (Table 1; Figure S3; Sim et al., 1994).
Residues involved in the dimeric interaction include
D30 and R446, which form a direct salt bridge as well
as a water-mediated interaction between the mono-
mers (Figure 3C); T114, which forms van der Waals in-
teractions with side chains of residues L338 and T340
(Figure 3D); and backbone atoms in N433-H436 of both
monomers that form an antiparallel β sheet (Figure 3E).
To disrupt the D30/R446 salt bridge, R446 was mutated
to a glutamate (R446E) and an aspartate (R446D). Both
mutants demonstrated reduced binding to erythrocytes
(Table 1). Steric disruption of interactions with T114 by
mutation to phenylalanine (T114F) resulted in a reduc-
tion in binding efficiency (Table 1). Since the interac-
tions that form the antiparallel β sheet in the center of
the dimer are all backbone interactions, V435 was mu-
tated to aspartate (V435D) to introduce charge repul-
sion. Again, a reduction in binding is observed (Table
1). Therefore, we conclude that RII dimerization is im-
portant for erythrocyte binding.
Several residues involved in glycan binding were also
selected for mutagenesis. N417, R422, and K439 are
involved in interactions with glycans 1 and 2. Mutation
of these residues result in reduced binding (Table 1).
Note that, unlike the others, R422 is a severe mutation
to glutamate, which may explain its stronger effect.
N33, N551, Y552, and K553, which are all involved in
interactions with glycans 3 and 4, were mutated to ala-
nine. Each reduced binding to different extents. Finally,
K28, R31, and K341 interact with glycans 5 and 6, and
mutation of these residues to alanine also resulted in
varying degrees of reduced binding. The relative levels
of reduction in binding roughly correlate with the num-
ber of glycan contacts made by each residue (Tables 1
and S1; Figure S1D).
This analysis suggests that all six glycan binding
sites are important for RII binding to GpA, as mutations
that are predicted to disrupt binding to the glycans ob-
served in the structure of the complex prevent binding
of RII to red blood cells. The reduction or lack of bind-
ing by these mutants was not due to inappropriate ex-
pression, as expression levels were comparable to wild-
type RII as shown by immunofluorescence (Figure S3). As
a negative control, two residues on the outer surface of
the RII dimer that are not predicted to be involved in
dimerization or glycan binding were changed to ala-
nine, and these show full binding activity (Table 1).
A Model for Erythrocyte Binding
GpA exists as an erythrocyte transmembrane dimer
with two heavily glycosylated extracellular domains per
dimer. The crystal structure of RII, the subject of this
study, reveals a dimer with two channels. In addition,
we identified six glycan binding sites per dimer of RII,
four of which (1–4) are accessible from the channels
and the other two (5 and 6) are accessible through a
cavity on the “top” surface of RII.
Cell
188

Figure 4. Crystal Structure of RII with Sialyllactose

(A) Ribbon diagram of RII with the position of the glycans indicated by the Fo − Fc electron density map contoured at 2.5 σ in red.
(B) Close-up view of three of the glycan binding sites, glycans 1, 3, and 5 are shown from left to right. All glycans are contacted by residues
from both monomers. Color scheme as in Figure 3.

The extracellular domain of GpA inhibits RII binding suggests a mode for GpA binding (Figure 5). Since the
to erythrocytes, implicating this region of GpA as the glycans are bound at the dimer interface and make
receptor for RII (Sim et al., 1994). The presence of two contacts to both monomers, we reason that the RII di-
channels in the center of the RII dimer along with the mer might assemble around the dimeric GpA extracel-
location of the glycans in the sialyllactose complex lular domain and that dimerization of RII may occur
upon ligand-receptor binding at the erythrocyte sur-
face during the invasion process. Moreover, binding-
Table 1. Binding Efficiency for RII and Mutants induced dimerization is consistent with our observation
that glycan 5/6 is bound in a deep elongated pocket
Binding No. Glycan
Mutant Class Mutation Ability Contacts
created by both monomers. Although this pocket is ac-
cessible through a cavity on the top surface of the di-
Wildtype RII ++++ N/A
mer, the glycan is too bulky to enter without some
Dimer R446E — N/A
Dimer R446D — N/A
movement of the protein. On the other hand, GpA bind-
Dimer T114F + N/A ing is straightforward if monomers would assemble
Dimer F2 V435D ++ N/A around it. Thus, any preexisting dimers would dissoci-
Glycan 1/2 N417A ++ 2 ate to allow for receptor binding. This scenario is con-
Glycan 1/2 R422E — 3 sistent with both analytical ultracentrifugation and
Glycan 1/2 K439A + 2 crosslinking studies, mentioned above, that indicate
Glycan 3/4 N33A + 1
the predominance of monomers in solution. In addition,
Glycan 3/4 N551A +++ 1
Glycan 3/4 Y552A + 2
we should note that in a separate study, recombinant
Glycan 3/4 K553A ++ 2 non-N-glycosylated RII in phosphate buffered saline at
Glycan 5/6 K28A +++ 2 pH 7.4 manufactured for clinical use is a stable mono-
Glycan 5/6 R31A ++ 1 mer (B.K.L.S., unpublished data). The remaining do-
Glycan 5/6 K341A + 3 mains of EBA-175 may also influence the assembly of
Surface control E153A ++++ N/A RII around GpA, impacting a conversion from monomer
Surface control K548A ++++ N/A
to dimer. Invasion involves dramatic changes in the
Crystal Structure of a Malaria Vaccine Candidate
189
parasite that would require signaling within the merozo-
ite. It has been shown that the cytoplasmic C-terminal
domain of EBA-175 is essential for invasion but not for
trafficking of the protein (Gilberger et al., 2003a). This
suggests that signaling occurs through the cytoplasmic
domain during the invasion process and that this sig-
naling might be triggered following dimerization upon
ligand binding.
The channels are 15 Å in diameter at their narrowest;
therefore, this region of the channel would be able to
accommodate an unglycosylated segment of GpA,
such as residues 5–9, 16–21, 27–36, or 38–43. The gly-
cosylated regions of GpA would be bound to the outer
segments of the channels and the top face of RII, while
an unglycosylated region of GpA would be bound at
the center, the narrowest part, of the channel (Figure 5).
Alternatively, the GpA monomers may dock on the
outer surface of RII, feeding glycans into the channels
(Figure 5). Since the structure indicates that the glycan
binding sites are formed by both monomers, dimeriza-
tion of RII would greatly enhance binding to GpA. In-
deed, mutations of residues involved in dimerization or
in putative glycan binding impair the ability of RII to
bind erythrocytes (Table 1).
It was shown previously that F2 alone can bind eryth-
rocytes, but that F1 is insufficient (Sim et al., 1994). F2
contributes about two-thirds of the residues that line
the channels (see Figures 3A and S2). In addition, there
are dimeric interactions exclusively between F2 mono-
mers, but F1 depends on F2 for dimerization (Figures
3E and S2). Thus, we speculate that F2 alone can form
a weak dimer or can be induced to dimerize by the re-
ceptor. Moreover, F2 also contributes 75% of the con-
tacts to the glycans observed in the complex with sial-
yllactose described above. Thus it appears to provide
most of the interaction sites for the receptor.
Interestingly, two peptides (355–375 and 435–455)
generated from RII that were shown to bind erythro-
cytes and inhibit in vitro merozoite invasion (Rodriguez
et al., 2000) mapped to residues lining the channels
(Figure S4). Peptide 435–455 includes part of the dimer-
Figure 5. Model for P. falciparum EBA-175-
RII Binding to the Red Blood Cell Receptor
Glycophorin A (GpA)
The P. falciparum membrane is shown on the
top and the erythrocyte membrane on the
bottom. The receptor binding domain of
EBA-175, RII, is shown as a surface repre-
sentation with F1 in green and F2 in purple
and the linker in gray. Blue lines represent
portions of EBA-175 backbone not included
in the crystal structure. GpA is shown in red
with the membrane-spanning region in detail
using the NMR structure and the extracellu-
lar domain drawn as a schematic flexible
line. The modeled O-glycans of GpA (see
Figure S2) are shown as space-filling models
in gold. In the left panel, the RII dimer as-
sembles around the GpA dimer, with GpA
binding within the channels (see text). An al-
ternative model is shown on the right, where
the GpA monomers dock on the outer sur-
face of the protein, feeding glycans into the
channels.
ization interface of the cavity of F2 and includes R446,
which we show here to be required for binding to eryth-
rocytes. This peptide also includes residues that line
the channels of the dimer and are involved in glycan
interactions. Thus, peptide 435–455 may function either
by binding to GpA directly, thereby capping GpA on
erythrocytes and blocking merozoite invasion, or by
binding to EBA-175 RII preventing dimerization. Muta-
genesis of this peptide also led to the generation of
more immunogenic peptides that protect from chal-
lenge with P. falciparum (Guzman et al., 2002).
Implications for the DBL Superfamily
Several erythrocyte invasion pathways have been iden-
tified in P. falciparum. Some pathways involve DBL-
containing proteins, in particular those of the EBL fam-
ily, with the EBA-175/GpA pathway being the dominant
chymotrypsin-resistant invasion pathway (Duraisingh et
al., 2003a). Other pathways do not involve DBL-con-
taining proteins, such as the reticulocyte binding-like
family (Duraisingh et al., 2003b; Kaneko et al., 2002;
Rayner et al., 2000; Rayner et al., 2001; Taylor et al.,
2002; Triglia et al., 2005). Apart from the cysteine-rich
binding domain proteins of P. falciparum, P. vivax, and
P. knowlesi, involved in erythrocyte invasion, the DBL
superfamily also includes the highly polymorphic
multicopied var gene products adapted for functions
including adhesion and receptor recognition. The struc-
ture of EBA-175 RII now allows modeling of these DBL-
containing proteins and predictions about the alternate
DBL-dependent pathways of invasion. Furthermore,
models of the DBL domains of PfEMP-1 could reveal
conserved regions in the variant PfEMP-1 DBL domains
and/or lead to an understanding of PfEMP-1 receptor/
ligand interactions. Figure 1B shows a sequence align-
ment of the double DBL domain containing RIIs of
EBA-175, BAEBL, EBL-1, and JESEBL. Interestingly,
the sequence conservation is higher in F2 than in F1,
suggesting a greater selective pressure on F2. This is
in accordance with the observation that binding is de-
pendent on F2 to a greater extent than F1. Most invari-
Cell
190
ant residues including the cysteines are involved in
interactions that stabilize the DBL domain fold. In addi-
tion, a large number of residues that are involved in
Neu5Ac(α2-3)Gal binding are conserved among five
RIIs of the EBL family.
It is interesting that F1 is similar to the single DBL
domain of P. vivax and P. knowlesi Duffy binding pro-
teins that have been shown to bind erythrocytes during
invasion (Adams et al., 1992; Chitnis and Miller, 1994).
There are also similarities in amino acid signatures be-
tween F1 and the DBL and CIDR domain of the var
gene family that encode proteins responsible for cy-
toadherence and antigenic variation. Thus, the three-
dimensional structure of the RII DBL domain should be
conserved throughout the DBL protein family. The ob-
served amino acid differences may be driven by pres-
sure for immune evasion. Indeed, in a previous study
(Liang and Sim, 1997), a strong conservation of both
the structure and function of EBA-175 RII was found
across laboratory strains and field isolates with only 16
point mutations leading to radical amino acid changes
that were few and scattered. Localization of the amino
acids that were involved in the point mutations revealed
that 75% of them were on the surface of RII arguing
that these mutations might have arisen from immune
pressure.
Regions of the EBL family that are surface exposed
could be used for targeted vaccine design. Loop re-
gions are often good targets for use in immunization
due to their inherent flexibility and conformability. Fur-
thermore, the use of residues involved in dimerization
or in glycan binding may prove fruitful for targeted vac-
cine design as described for peptide 435–455. Design
of small molecule inhibitors using the glycan binding
sites could prove useful as well. Though protein-protein
interactions including dimerization have not been tradi-
tional targets for drug intervention, using a small mole-
cule targeted to a well-formed cavity as seen in a crys-
tal structure has proven successful, with disruption of
the p53-MDM2 interaction as a recent example (Vassi-
lev et al., 2004). The F2 cavity, required for dimerization,
which in turn is necessary for EBA-175 function, could
serve as such a target.
Here, we have solved the crystal structure of a
double-DBL domain from the EBL family of proteins un-
bound and in complex with α-2,3-sialyllactose. These
structures provide a glimpse into the mechanism of
erythrocyte invasion by the Plasmodium parasite. They
also provide insights into a large superfamily of pro-
teins critical for the pathogenesis and survival of the
parasite. Lastly, this study will help the rational design
of novel therapeutics that could aid in the desperately
needed treatment of malaria.
Experimental Procedures
Protein Expression and Purification
The native RII domain of EBA-175 is unglycosylated in P. falci-
parum due to the lack of posttranslational modification capabilities
for glycosylation (Gowda and Davidson, 1999); however, expres-
sion in Pichia pastoris resulted in a glycosylated protein that was
refractory to crystallization. A synthetic gene fragment encoding RII
with five putative glycosylation sites mutated (N3Q, S50A, S195A,
T206A, N400Q) was cloned and expressed in Pichia pastoris, a
methylotropic yeast. Fermentation was performed with defined
salt media.
Purification was initiated by expanded bed adsorption chroma-
tography, i.e., sulphopropyl cation exchange chromatography (Am-
ersham Pharmacia Biotech), followed by additional ionic exchange
chromatographic steps (SP chromatography Streamline SP XL
resin and Q FF [Pharmacia]). The final steps of the purification in-
cluded a Streamline SP/XL cation exchange polishing step and an
ultrafiltration diafiltration concentration step.
Protein Crystallization and Derivatization
Form 1 crystals were grown by vapor diffusion by mixing 2 ␮l of
the protein solution (15 mg/ml in 10mM Tris-HCl [pH 7.4], 100mM
NaCl) with 2 ␮l of a reservoir solution containing 0.265 M ammo-
nium sulfate, 0.1 M sodium cacodylate (pH 6.5), 29% polyethylene
glycol 8000. Form 2 crystals were grown similarly but with a reser-
voir containing 2.6–2.8 M ammonium sulfate, 0.1 M sodium caco-
dylate (pH 6.5), 0.05%–2% polyethylene glycol 750 monomethyl
ether, and a protein concentration of 12 mg/ml. Derivatized crystals
were obtained by soaking form 1 or form 2 crystals in saturated
lithium sulfate containing either 1 mM sodium dicyanoaureate or
1 mM potassium tetrachloroplatinate for 2 hr.
Complex crystals were grown by mixing 2 ␮l of the protein solu-
tion (20–30 mg/ml in 10 mM Tris-HCl [pH 7.4], 100 mM NaCl) with
0.4–1.0 ␮l of a 10 mM α-2,3-sialyllactose (Sigma) solution and 2 ␮l
of a reservoir solution containing 2.7 M ammonium sulfate, 0.1 M
HEPES (pH 7.5), and 0.1% polyethylene glycol 750 monomethyl
ether.
Data Collection
Native and complex crystals were cryoprotected by transfer to a
solution of saturated lithium sulfate and flash frozen in liquid nitro-
gen. Data were collected at beamlines X26C and X25 at the Na-
tional Synchrotron Light Source (NSLS) at Brookhaven National
Laboratory (BNL). For derivatives, crystals were flash frozen di-
rectly from the soak solution prior to data collection. Multiwave-
length anomalous dispersion data were collected for the gold and
for the platinum derivatives. Data collection statistics are shown in
Tables S2 (form 1) and S3 (form 2).
Data reduction statistics for the complex indicated a loss of a
crystallographic 2-fold had occurred upon ligand binding. This re-
sulted in a conversion from a tetragonal P4122 to an orthorhombic
C2221 space group (see Table S4). The abolished 2-fold relates the
two monomers of the dimer, which is a noncrystallographic 2-fold
in the complex.
Structure Solution and Analysis
Four gold sites were identified by SnB (Weeks and Miller, 1999) and
confirmed by SHELXS (Sheldrick et al., 1993) with data collected
at the absorption peak for a form 1 gold derivative. Following phase
refinement with SHARP (de la Fortelle and Bricogne, 1997), plati-
num sites in form 1 were determined by crossphasing with CNS
(Brünger et al., 1998). Phase refinement with the form 1 native and
derivative data (4 Au wavelengths, 3 Pt wavelengths with SHARP)
allowed the building of a polyalanine model comprising w60% of
the backbone in O (Jones and Kjeldgaard, 1997).
This model was separated into two halves and used to solve the
structure of form 2 by molecular replacement (AMoRe [Navaza and
Saludjian, 1997]). Four low-occupancy gold sites were then iden-
tified by difference Fourier for a form 2 gold derivative. Simulta-
neous cross crystal averaging, density modification, and phase ex-
tension (DMMULTI–CCP4 [CCP4, 1994]) using both form 1 and 2
native data and form 1 and 2 initial phases improved the density to
allow w90% of the backbone and 60% of sequence to be assigned
unambiguously. Iterative SIGMAA (CCP4) weighting, RESOLVE (Ter-
willliger, 2000) prime-and-switch density modification, and model
building (O) allowed unambiguous assignment of 95% of the se-
quence. Phasing statistics are shown in Tables S2 and S3.
Refinement of the model and rebuilding was performed with
CNS, Reduce and Probe (Word et al., 1999), MolProbity (Lovell et
al., 2003), and O. Final refinement statistics are shown in Table S4.
The final model contains residues 8–163, 166–508, and 513–596 of
Crystal Structure of a Malaria Vaccine Candidate
191
RII, 326 water molecules, 9 sulfates, and one chloride ion with an
Rwork/Rfree of 0.232/0.280.
The sialyllactose complex was solved by molecular replacement
(AMoRe) using the refined form 2 structure without the solvent
molecules as a search model. Two monomers were found in the
asymmetric unit. Initially, TLS and tightly restrained NCS refinement
with REFMAC5, was followed by model rebuilding and the addition
of solvent molecules with O. The locations of the six glycans were
identified by visual inspection of Fo − Fc electron density maps,
calculated with CNS and shown in Figure S1A. Final refinement
statistics are shown in Table S4. This protocol was chosen based
on careful monitoring of the Rfree. The final model contains two
monomers of RII each composed of residues 8–163, 166–508, and
513–601, 599 water molecules, 16 sulfates, and two chlorides with
an Rwork/Rfree of 0.216/0.232. A possible model for the glycans was
also built and the resulting 2Fo − Fc electron density maps for the
glycans are shown in Figure S1B. In addition, a simulated-annealed
Fo − Fc omit map (Figure S1C) was calculated by omitting the gly-
cans as well as a 2 Å sphere around them. Though the general
placement of these glycans was obvious, a precise atomic model
was not possible due to the relatively limited resolution. In addition,
due to only partial occupancy of the glycans (0.4–0.6), the protein
had to be modeled using alternate conformations for side chains
in bound and unbound states. Less-than-ideal density is not un-
usual for carbohydrates at this resolution.
Mutagenesis and Red Blood Cell Binding (Rosette) Assays
Single amino acid mutations were introduced in RII, previously
cloned into plasmid pRE4, by the Quickchange method (Strata-
gene). Fresh monolayers of COS-7 cells cultured on coverslips in
3.5 cm-diameter wells, were transfected with 2 ␮g of Qiagen-puri-
fied plasmids with the use of Fugene-6 reagent (Roche). Forty-eight
hours after transfection, one coverslip was removed from each well
for immunofluorescence assays to assess expression and control
of transfection efficiency. Cells were fixed with 2% formaldehyde
in phosphate buffered saline, and immunoflourescence observed
by standard methods (Sim et al., 1990). Rosetting assays were pre-
formed on the remaining coverslips in the 3.5 cm-diameter wells.
Two hundred microliters of 10% hematocrit erythrocytes in media
were added to each well, mixed, and incubated for 2 hr at 37°C.
The COS cells were washed three times with phosphate buffered
saline to remove nonadherent cells prior to visualization and
scoring.
Figures
Figures 2A, 2B, 3A, 3C–3E, and 4 were prepared with MolScript
(Kraulis, 1991), Raster 3D (Bacon and Anderson, 1988; Merritt and
Murphy, 1994) and POVScript+ (Fenn et al., 2003). Figure 3B was
prepared with GRASP (Nicholls et al., 1991) and POVScript+.
Supplemental Data
Supplemental Data include four figures and four tables and can be
found with this article online at http://www.cell.com/cgi/content/
full/122/2/183/DC1/.
Acknowledgments
Recombinant RII was produced by EntreMed, Inc. We thank Hong
Liang, Tom Chen, and the Molecular Biology and Pharmaceutical
Science teams for the production of purified recombinant RII, Mar-
garet Luk-Paszyc and Caroline Kisker for assistance with analytical
ultracentrifugation, Reinhard Brossmer for reagents and advice,
and Michael Becker (beamline X25) and Annie Heroux (beamline
X26C) for support with data collection at the National Synchrotron
Light Source (NSLS) at Brookhaven National Laboratory. Special
thanks to Stephen A. Johnston. The NSLS is supported by the US
Department of Energy, Division of Materials Sciences and Division
of Chemical Sciences. This work was supported by an SBIR grant
1R43 AI46209 (to B.K.L.S.), a Leslie C. Quick Jr. Predoctoral Fellow-
ship to N.H.T., and the Watson School of Biological Sciences and
the Louis Morin Charitable Trust (L.J.).
Received: March 2, 2005
Revised: April 22, 2005
Accepted: May 24, 2005
Published: July 28, 2005
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Crystal Structure of a Malaria Vaccine Candidate
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Accession Numbers

The atomic coordinates and structure factors have been deposited

in the Protein Data Bank (PDB entry codes 1ZRL and 1ZRO).