From the Structural Biology Section, Laboratory of
Immunogenetics and ¶ Malaria Vaccine Development Unit, Laboratory
of Parasitic Diseases, NIAID, National Institutes of Health, Rockville,
Maryland 20852
Received for publication, October 18, 2002
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ABSTRACT |
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The protozoan parasite Plasmodium
causes malaria, with hundreds of millions of cases recorded annually.
Protection against malaria infection can be conferred by antibodies
against merozoite surface protein (MSP)-1, making it an attractive
vaccine candidate. Here we present the structure of the C-terminal
domains of MSP-1 (known as MSP-119) from Plasmodium
knowlesi. The structure reveals two tightly packed epidermal
growth factor-like domains oriented head to tail. In domain 1, the
molecule displays a histidine binding site formed primarily by a highly
conserved tryptophan. The protein carries a pronounced overall negative
charge primarily due to the large number of acidic groups in domain 2. To map protein binding surfaces on MSP-119, we have
analyzed the crystal contacts in five different crystal environments,
revealing that domain 1 is highly preferred in protein-protein
interactions. A comparison of MSP-119 structures from
P. knowlesi, P. cynomolgi, and P. falciparum shows that, although the overall protein folds are
similar, the molecules show significant differences in charge
distribution. We propose the histidine binding site in domain 1 as a target for inhibitors of protein binding to MSP-1, which might
prevent invasion of the merozoite into red blood cells.
A necessary step in the life cycle of the malaria parasite is its
entry into the red blood cell of a mammalian host. This process
involves many surface proteins specifically expressed at the merozoite
stage of the parasite, including merozoite surface protein
(MSP)1-1 (also known as
merozoite surface antigen-1 or MSA-1). MSP-1, a required component of
the merozoite invasion machinery, associates noncovalently with other
parasite proteins, including the C-terminal portion of MSP-6 (1). MSP-1
is synthesized as a 180-225-kDa polypeptide capable of binding sialic
acid (2, 3). As a requirement for merozoite entry into a red cell,
MSP-1 undergoes two processing steps, the first at merozoite release
from an infected cell and the second during invasion of a red cell (4)
(Fig. 1a). In the first processing step, the precursor
polypeptide is cleaved into four chains (with apparent molecular
masses of 83, 30, 38, and 42 kDa) held together by noncovalent
contacts (4-6). In the second processing step, the 42-kDa segment
(MSP-142) is cleaved into two fragments with apparent
molecular masses of 33 kDa (MSP-133) and 19 kDa
(MSP-119 or MSP-1inv) (4). MSP-133 sheds from the surface, whereas MSP-119 remains anchored to
the merozoite membrane by a glycosylphosphatidylinositol tail attached to the C-terminal residue (7-10).
Because high antibody titer against the C-terminal region of MSP-1
protects against severe malaria, this molecule represents an attractive
vaccine candidate (11-13). Antibodies against MSP-119 correlate with clinical immunity (14, 15); however, recombinant MSP-119 is poorly immunogenic, presumably due to poor
processing of the highly disulfide-bonded protein (16, 17). Antibodies to the C-terminal region of MSP-1 fall into two categories: protective antibodies that inhibit merozoite invasion of red cells, and blocking antibodies that enhance invasion by interfering with the binding of
protective antibodies. Protective antibodies include those that
recognize the native conformation of MSP-119 (7, 18-20), those that block proteolytic processing of MSP-142 (21),
and certain antibodies to the 83-kDa N-terminal fragment of intact MSP-1 (22). Blocking antibodies bind to the first domain of MSP-119, near the N terminus introduced by the processing
of MSP-142 (22, 23).
The extremely stable MSP-119 contains two consecutive
epidermal growth factor (EGF)-like domains, a small disulfide-rich fold also found in diverse proteins including Factor Xa (24), E-selectin (25), cyclooxygenase-2 (26), and integrin
To better understand the process of invasion of the
Plasmodium merozoite into a red blood cell, we have solved
the structure of the C-terminal domains of MSP-1 from P. knowlesi by x-ray crystallography. We have refined the structure
to an R-factor of 23.4% and R-free of 26.4% to
2.4 Å resolution (Table I). Our
structure reveals features of MSP-1 novel to P. knowlesi
and provides new insight into MSP-1 from other
Plasmodium species. With four copies in the crystallographic
asymmetric unit, we observe MSP-1 in four different environments,
allowing us to infer novel properties of the molecule.
Expression and Purification--
DNA encoding the C-terminal 92 amino acids of the of P. knowlesi (Malayan H strain) MSP-1
(GenBankTM accession code AAG24615) was inserted into the
vector YEpRPEU-3 and expressed in the Saccharomyces
cerevisiae VK1 cell line (36). The vector included an N-terminal
yeast Crystallization and X-ray Data Collection--
Purified
MSP-119 protein was concentrated to 10 mg/ml for
crystallization trials. Crystals were grown via vapor diffusion with a
solution containing 30% polyethylene glycol 6000, 100 mM HEPES, pH 7.0. Crystals were transferred into the same solution supplemented with 20% glycerol before cooling to 100 K in a nitrogen stream. X-ray data were collected on a Rigaku RU-200 rotating anode
generator equipped with nickel mirrors and a RAXIS 4 detector (Molecular Structure Corp., The Woodlands, TX). 180° of diffraction data were processed using DENZO (37), SCALEPACK (37), and TRUNCATE (38)
to a resolution limit of 2.4 Å.
Phasing and Refinement--
Molecular replacement was performed
with the AMoRe package (38) using coordinates from the C-terminal
domains of MSP-1 from P. cynomolgi (29). The P. cynomolgi model was rotated and translated against the 8-4 Å P. knowlesi diffraction amplitudes, using correlation coefficients to rank solutions. Four successive objects were placed in
the crystallographic asymmetric unit, whereas insertion of a fifth
object failed to improve the correlation coefficient. Inspection of the
packing showed no steric clashes in a unit cell with 49% solvent
content. Rigid body refinement in the programs AMoRe (38) and
Crystallography and NMR System (CNS) (39) was followed by model
building in the program O (40). Residue numbering of the P. knowlesi structure corresponds to the mature secreted protein and
begins at the first residue of the cleaved Calculations--
Least squares superpositions were performed
using the program LSQMAN (41) with a distance cutoff of 3.8 Å, and
coordinate transformations were applied using the program MOLEMAN2
(41). Structural comparisons with the NMR structure of P. falciparum MSP-119 (28) use the most representative
member of the ensemble. Electrostatic calculations include only the
native MSP-119 sequence, without the secretory signal and
purification epitope. Molecular figures were prepared using the
programs MOLSCRIPT (42), BOBSCRIPT (43), and GRASP (44).
Overall Description of the Structure--
The C-terminal region of
P. knowlesi MSP-1 contains two successive EGF-like domains
packed together tightly and related by a rotation of ~170° (Fig.
1b). The molecule is roughly
square-shaped, with dimensions of about 35 Å × 35 Å × 15 Å. The
angle between domains 1 and 2 leads to close approach of the N terminus
of domain 1 and the C terminus of domain 2. Thus, as domain 2 is
anchored in the membrane by a glycosylphosphatidylinositol linkage
attached to the C-terminal residue, the N-terminal residue of domain 1 (i.e. the cleavage site in MSP-142) also points
toward the membrane, in an appropriate orientation for processing by
proteases attached to the merozoite membrane (28).
Each EGF-like domain in P. knowlesi MSP-1 contains a segment
of random coil followed by four antiparallel
The crystals of P. knowlesi MSP-119 contain four
molecules in the asymmetric unit, revealing the protein in four
different environments. The four molecules are highly similar, with
r.m.s. deviations of 0.4-0.6 Å for 77-81 C
In P. knowlesi MSP-119, the extensive interface
between the two domains buries 26 residues and 1090 Å2 of
surface area. This produces a rigid interface with little variation in
the interdomain angle among the four copies in the asymmetric unit
(Fig. 1c). The residues in the interface are among the most
conserved in the MSP-119 protein: 23 of 26 residues are identical between P. knowlesi and P. cynomolgi,
with three conservative substitutions; and 14 of 26 residues are
identical between P. knowlesi and P. falciparum,
with seven conservative substitutions. The highly conserved domain 2 sequence
Asn57-Asn58-Gly59-Gly60
packs in the interface between domains 1 and 2, forming a short stretch of left-handed helix in P. knowlesi and P. cynomolgi and a tight turn in P. falciparum. P. knowlesi domain 1 superimposes on domain 2 with an r.m.s.
deviation of 1.2-1.6 Å for the four independent copies in the
asymmetric unit (Table II).
Histidine Binding Site--
In all four copies of
MSP-119 in the crystallographic asymmetric unit, electron
density appears in a shallow depression on the surface (Fig.
2a). In each copy, the density
traces back to a histidine from the hexahistidine tag of a
symmetry-related molecule. For each of the four bound histidines, the
plane of the imidazole ring stacks roughly parallel to the plane of the
indole ring of Trp34, and the carboxylic acid of
Glu42 pairs with the bound (and presumably protonated)
histidine. In the histidine-binding pocket, Trp34 forms the
floor, and Glu33 and Arg35 form the walls (Fig.
2b). The hydrophobic tryptophan and the bound histidine
appear in the middle of a region of negative electrostatic potential
(Fig. 2c). Although the conformation of the C-terminal hexahistidine tail differs in the four copies in the asymmetric unit,
one histidine from the tail fills a histidine-binding pocket of a
symmetry-related molecule. In the four copies in the asymmetric unit,
each histidine binding buries between 167 and 206 Å2 of
accessible surface area. This same pocket can bind other positively charged moieties: in the crystal of MSP-119 from P. cynomolgi (29), a lysine from a symmetry-related molecule inserts
into this pocket.
In all Plasmodium species except P. falciparum,
domain 1 of MSP-119 lacks the middle disulfide bond found
in canonical EGF-like domains. The replacement of a disulfide by a
small residue (valine, isoleucine, or threonine) and a tryptophan
produces a shelf on domain 1 capable of binding histidine. Tryptophan
is the residue most commonly found buried in protein-protein interfaces
(30) because burial of its large hydrophobic surface area yields
thermodynamic gain.
Protein Binding Surfaces--
The analysis of crystal contacts can
be used to map protein binding surfaces on a molecule to reveal
potential biologically important surfaces on the molecule (31, 32). We
searched crystal contacts in MSP-119 to identify the
favored sites for binding to proteins. Combining the four copies of
P. knowlesi MSP-119 in the crystallographic
asymmetric unit with the one copy of P. cynomolgi
MSP-119 (29), we have observations of MSP-119
in five different crystal environments. We counted the number of times each residue participates in crystal contacts and mapped that number
onto the surface of the protein (Fig.
3a). The P. knowlesi and P. cynomolgi crystal contacts correlate:
of the 9 residues involved in crystal contacts in all four P. knowlesi copies, 7 make contacts in the P. cynomolgi
crystals as well. The seven residues (Ile14,
Asp15, Leu28, Trp34,
Glu42, Ala50, and Ser51)
participate in crystal contacts in all five crystal packing environments. All of these except Ser51 are conserved
between P. knowlesi and P. cynomolgi,
suggesting protein binding sites conserved across species. All of these
except Leu28 differ between P. knowlesi and
P. falciparum, resulting in distinctly shaped
MSP-119 surfaces across these species.
The crystal contacts show that MSP-119 domain 1 is highly
favored for binding protein (Fig. 3a). Trp34,
the floor of the histidine binding site in the center of domain 1, falls in an ideal position to participate in protein-protein interactions on the surface of the merozoite. In P. falciparum MSP-119, domain 1 has been shown to be the
binding site of monoclonal antibody G17.12 (33).
Comparison across Species--
The high level of sequence identity
in MSP-119 across Plasmodium species results in
highly similar three-dimensional structures of the molecule (Figs.
3b and 4a). The
P. cynomolgi MSP-119 superimposes on the
P. knowlesi structure with an r.m.s. deviation of 1.3 Å for
78 C
MSP-119 from P. knowlesi exhibits a striking
charge distribution (Fig. 4b). The molecule carries a
pronounced negative potential, with an overall charge of
Hydrophobicity maps of the three Plasmodium
MSP-119 structures show a patch of nonpolar residues along
the interface between the two domains (Fig. 4, b
The arrangement of charged and hydrophobic residues on
MSP-119 may maintain specificity of the interactions
between MSP-119 and its binding partners such as the other
MSP-1 fragments and MSP-6. Complementary changes between residues in
MSP-119 and its binding partners may allow evolution of the
MSP-119 protein surface.
The region around Trp34 appears to be a site on
P. knowlesi MSP-119 well suited for
protein-protein interactions. First, all EGF-like domain structures
contain at least three pairs of disulfide bonds, with one exception:
the first domain of MSP-119. Of the 40 independent
EGF/laminin-like domain structures currently in the Protein Data Bank
(34), only Plasmodium MSP-119 has lost one of
its three canonical disulfide bonds. The loss of a structurally important and highly conserved disulfide bond is most
likely due to a functional requirement. Second, all of the
Plasmodium species (except P. falciparum) contain
a tryptophan in domain 1 of MSP-119 in place of the
canonical cysteine. The disulfide is replaced with a tryptophan and a
smaller residue; whereas the smaller residue may become a valine,
isoleucine, or threonine, the other member of the pair is always
tryptophan, suggesting functional selection. Third, tryptophan is the
residue most commonly found in protein-protein interfaces (30), so the
replacement of a strictly conserved disulfide-forming cysteine with a
tryptophan suggests the evolution of a protein binding site. Fourth,
there are no instances of crystals of MSP-119 with an empty
histidine-binding pocket. In all five independent crystallographic
environments, a histidine or lysine is found stacked above the
tryptophan ring. This pocket fills when the concentration of nearby
protein becomes sufficiently high. Fifth, the crystal contact map of
MSP-119 indicates that the tryptophan and nearby regions in
domain 1 are very active in participating in protein-protein interactions.
The sequence differences between P. falciparum and the other
Plasmodium species represent divergent evolution from a
common precursor, but it is unclear whether P. falciparum
has lost a binding site or the other Plasmodium species have
evolved new functionality. Alternatively, the difference between
P. falciparum and the other Plasmodia may be due
to the strong bias against tryptophan in P. falciparum (35).
The virulent P. falciparum displays somewhat different
disease pathology compared with the other Plasmodium
species; perhaps the differences in MSP-119 proteins are
related to the differences in disease severity. For example, P. falciparum invades a broader spectrum of red blood cells and reaches higher levels of parasitemia compared with other malaria species, potentially due to loss of receptor specificity in P. falciparum. Some of this specificity may be conferred by the MSP-1 protein during red blood cell invasion. Although P. falciparum lacks the tryptophan defining the floor of the binding
pocket, all the other species of Plasmodium have this
tryptophan, which generates a site for binding histidine. We propose
the use of this histidine binding site as a target for the design of
compounds that bind, for example, P. vivax MSP-1. From such
a scaffold compound, new inhibitors that protect against merozoite
invasion of a red blood cell might be developed.
Our structure reveals a new feature of the MSP-119
molecule, a histidine-binding pocket in domain 1. Because antibodies
that protect against malarial infections often bind specifically to domain 1, this new property of the molecule might be used to engineer tighter binding and thus more protective antibodies. Alternatively, this histidine-binding pocket might be used to interfere with the
binding of blocking antibodies. For example, a small molecule inhibitor
containing an imidazole scaffold may be designed to overlap with the
footprint of blocking antibodies and thus disrupt the binding of
blocking antibodies. The specific disruption of MSP-119
blocking antibodies without perturbation of protective antibodies would
greatly enhance the immune response against the malaria parasite.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
V
3 (27). In MSP-1, disulfide bonds are
integral to the correct folding of the molecule (20). Although the
full-length MSP-1 is highly polymorphic, the C-terminal EGF-like
domains in MSP-119 are much less so, and they represent a
conserved segment of a variable molecule. MSP-119 shows
high sequence identity across Plasmodium species: P. knowlesi MSP-119 shares 82% sequence identity with P. cynomolgi and 51% sequence identity with P. falciparum.
Crystallographic statistics
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-factor pre-pro secretory signal and a C-terminal
hexahistidine tag. The plasmid is episomally maintained by supplying a
functional HIS4 gene for the host VK1 cell line (his4). Protein
expression is under control of the ADH2 promoter and induced by
introducing ethanol as a carbon source during fermentation. The
secreted protein product included 105 amino acids: 5 amino acids
(E-A-E-A-S) from the cleaved mating factor, the 92 amino acids from
MSP-1, and 8 amino acids (G-P-H6) from the affinity tag.
The protein was purified using nickel affinity, ion exchange, and gel
filtration chromatography.
mating factor secretory
signal. Refinement protocols in CNS included conjugate gradient
minimization, simulated annealing, and temperature factor refinement.
Models were built into simulated annealing composite omit maps
calculated in CNS. Tight 4-fold noncrystallographic symmetry restraints
(300 kcal/mol-Å2) were imposed on all atoms in the early
stages of refinement and later relaxed for atoms that differ among the
four copies in the asymmetric unit. Refinement steps were accepted only
if they reduced the R-free (of a test set comprised of 860 reflections, 5% of the total, selected using resolution shells). The
R-work and R-free are 23.4% and 26.4%,
respectively, using all reflections to 2.4 Å.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The structure of MSP-119 from
P. knowlesi. a, the two proteolytic
processing stages of MSP-1. In the first processing step, the
200-kDa precursor is cleaved into four pieces. In the second step, the
42-kDa C-terminal segment is cleaved into two fragments,
MSP-133 and MSP-119. Segments are not to scale.
b, the MSP-119 ribbon representation including
disulfide bonds. In each domain, strands 1-4 are shown as ribbons
labeled
1
4. Disulfide bonds connect random coil to
1, coil
to
2, and
2 to
4. This front view, with domain 1 on the
left and domain 2 on the right, is also shown in
c and in Figs. 3 and 4. The non-native residues from the
C-terminal purification epitope are colored gray.
c, the
carbons of the four molecules in the
crystallographic asymmetric unit are shown superimposed. Chain A is
yellow, chain B is green, chain C is
blue, and chain D is red. The structures differ
significantly only at the termini and loop 69-74.
strands (Fig. 1b). Typically, EGF-like domains contain six cysteines in
three disulfide bonds with a connectivity of 1-3, 2-4, and 5-6. The MSP-1 EGF-like domains follow this paradigm, except that in domain 1, valine and tryptophan replace the middle (2-4) disulfide. The valine
and tryptophan pack into the domain 1 hydrophobic core, in the same
volume as the disulfide bond in canonical EGF-like domains.
atoms, with
differences primarily at the two termini and residues 69-74 on the
loop between
strands 1 and 2 in domain 2 (Fig. 1c). This
loop shows differences across the four molecules in the
crystallographic asymmetric unit and also within one molecule (as
reflected by high B factors). The flexibility in this loop and in the
termini corresponds to the mobility seen in the NMR structure of the
P. falciparum MSP-119 (28).
Comparison of MSP-119 structures
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Fig. 2.
The histidine binding site.
a, a stereo diagram of a
2Fo Fc composite omit map,
contoured at 1.3
, in the vicinity of the histidine binding site of
P. knowlesi MSP-119 molecule C. The electron
density in red derives from a symmetry-related molecule.
b, a ribbon representation of a portion of domain 1 with
side chains shown for residues around the histidine binding site.
Histidines from four symmetry-related molecules are shown, colored as
described in Fig. 1b. Residues Glu33,
Trp34, Arg35, and Glu42 define the
walls of the pocket. c, a surface representation of the same
region, colored by electrostatic potential. The surface potential
ranges from
10 kT (red) to +10 kT
(blue). Bonds from the superimposed histidines are shown,
colored as described in b. In molecule D, only the imidazole
ring of the histidine is modeled.
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Fig. 3.
MSP-119 in crystal contacts and
across species. a, the P. knowlesi
MSP-119 molecular surface is shown, colored by the number
of crystal contacts each residue makes, using the four molecules in
the P. knowlesi asymmetric unit and the one molecule in
the P. cynomolgi asymmetric unit. The surface has a color
gradient from 0 (white; no contacts in any of the five
molecules) to 5 (blue; contacts in each of the five
molecules) crystal contacts/residue. The histidine binding site is
circled in red. b, carbon traces of
MSP-119 from P. knowlesi, P. cynomolgi, and P. falciparum are superimposed. The four
copies of P. knowlesi MSP-119 are colored
yellow, green, blue, and red; the
P. cynomolgi MSP-119 is colored
magenta; and the P. falciparum
MSP-119 NMR structure is colored white.
Non-native residues from the purification epitope of P. knowlesi are included at the bottom of domain 2.
atoms, whereas the P. falciparum structure
superimposes on the P. knowlesi structure with an
r.m.s. deviation of 1.7 Å for 65 C
atoms (Table II). A
superposition of the four P. knowlesi MSP-119
molecules with the P. cynomolgi and P. falciparum
homologues reveals that the differences occur primarily at the termini
and in the 69-74 loop, the same loop that shows variability among the
four molecules in the P. knowlesi crystal.
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Fig. 4.
Electrostatic potential and
hydrophobicity. a, the amino acid sequences from the
crystals of MSP-119 from P. knowlesi, P. cynomolgi, and P. falciparum. Cysteines are highlighted
in yellow. b d, surface representations of
MSP-119 from three Plasmodium species are shown,
colored by electrostatic potential (left) and hydrophobicity
(right). Molecules are shown in two views 180° apart.
Electrostatic potentials contoured from
10 kT (red) to +10
kT (blue) are plotted onto the molecular surface of
MSP-119 from P. knowlesi, P. cynomolgi, and P. falciparum, respectively.
Hydrophobicity values calculated from modified solvent transfer
experiments (45) are contoured from
2 kcal/mol (most hydrophobic, in
yellow) to 2 kcal/mol (hydrophilic, in green).
The hydrophobic patches found on the surface tend to be from exposed
portions of residues forming the interface between domains 1 and
2.
7 in the 92 residues of the two domains. There are no buried ion pairs in P. knowlesi MSP-119; rather, all charged residues are
surface-accessible. The overall charge is species-specific because the
equivalent segments of MSP-1 from P. cynomolgi and P. falciparum have overall charges of
4 and
2, respectively (Fig.
4, c and d). In P. knowlesi
MSP-119, the negative charge is concentrated in domain 2 (with an overall charge of
7), whereas domain 1 is neutrally charged
overall. The acidic regions in domain 1 cluster near the histidine
binding site.
d). Of the
26 residues contributing to the interface, 15 are hydrophobic, and 3 are cysteines in disulfide bonds. Because the molecule is only 15 Å thick, some hydrophobic residues in the interdomain interface
(including Tyr25, Tyr27, Leu37,
Leu38, and Phe88) remain partially
solvent-accessible. Surprisingly, the large patch of hydrophobic
residues on the back surface is among the least active surfaces in
protein binding, as judged by the crystal contact analysis (Fig.
3a).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank T. Allison and the other members of the Structural Biology Section for discussions.
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FOOTNOTES |
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* This work was supported by the Intramural Program of the NIAID.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1N1I) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ To whom correspondence may be addressed: Structural Biology Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Twinbrook II, 12441 Parklawn Dr., Rockville, MD 20852. E-mail: garman{at}alpha.niaid.nih.gov (for S. C. G.) or garboczi{at}nih.gov (for D. N. G.).
Present address: CSL Limited, 45 Poplar Rd., Parkville,
Victoria 3052, Australia.
Published, JBC Papers in Press, December 19, 2002, DOI 10.1074/jbc.M210716200
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ABBREVIATIONS |
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The abbreviations used are: MSP, merozoite surface protein; EGF, epidermal growth factor; r.m.s., root mean square.
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