From the Department of Biology and Biochemistry,
University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom,
** Geymonat S.p.A., via S. Anna 2, 03012 Anagni (FR), Italy, and the
International Institute of Genetics and
Biophysics, CNR, Via G. Marconi, 12-80125, Naples, Italy
Received for publication, September 4, 2000, and in revised form, November 6, 2000
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ABSTRACT |
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The angiogenic molecule placenta growth factor
(PlGF) is a member of the cysteine-knot family of growth
factors. In this study, a mature isoform of the human PlGF protein,
PlGF-1, was crystallized as a homodimer in the crystallographic
asymmetric unit, and its crystal structure was elucidated at 2.0 Å resolution. The overall structure of PlGF-1 is similar to that of
vascular endothelial growth factor (VEGF) with which it shares 42%
amino acid sequence identity. Based on structural and biochemical data,
we have mapped several important residues on the PlGF-1 molecule that
are involved in recognition of the fms-like tyrosine kinase receptor
(Flt-1, also known as VEGFR-1). We propose a model for the
association of PlGF-1 and Flt-1 domain 2 with precise shape
complementarity, consider the relevance of this assembly for PlGF-1
signal transduction, and provide a structural basis for altered
specificity of this molecule.
Angiogenesis, the process of new blood vessel formation, is
essential for development, reproduction, wound healing, tissue regeneration, and remodeling (1). It also plays a major role in tumor
progression, diabetic retinopathy, psoriasis, and rheumatoid arthritis
(2). Angiogenesis involves proliferation of endothelial cells
(ECs)1 in an organized
fashion and is most likely regulated by polypeptide growth factors (3,
4) such as acidic and basic fibroblast growth factors (aFGF and bFGF,
Ref. 5), vascular endothelial growth factor (VEGF, Refs. 6-10), and
placenta growth factor (PlGF, Refs. 11-14). PlGF, VEGF (VEGF-A),
VEGF-B (15), VEGF-C (16), VEGF-D (17), VEGF-E (18), and
Fos-induced growth factor (FIGF, Ref. 19) are members of a
family of structurally related growth factors. Intra- and interchain
disulfide bonds among eight characteristically spaced cysteine residues
are involved in the formation of these active dimeric proteins and
hence termed as cysteine-knot proteins. They also share a number of
biochemical and functional features (for a review, see Ref. 20) such
that PlGF and VEGF can form heterodimeric molecules in cells in which
both genes are expressed (21, 22).
Alternative splicing of the PlGF primary transcript leads to three
forms of the mature human PlGF protein (22-24). The two predominant
forms, PlGF-1 and PlGF-2 (also known as PlGF-131 and PlGF-152,
respectively), differ only by the insertion of a highly basic 21-amino
acid stretch at the carboxyl end of the protein. This additional basic
region confers upon PlGF-2 the ability to bind to heparin (13, 23).
The exact role of PlGF in vascular development is yet to be
established. However, purification of PlGF-1 from overexpressing eukaryotic cells and measurement of angiogenic activity of the purified
PlGF-1 in vivo in the rabbit cornea and chick
chorioallantoic membrane (CAM) assays showed induction of a strong
neovascularization process that was blocked by affinity-purified
anti-PlGF-1 antibody. In the avascular cornea, PlGF-1 induced
angiogenesis in a dose-dependent manner and seemed to be at
least as effective (if not more effective) as VEGF and bFGF under the
same conditions and at the same concentration. PlGF-1 was shown to
induce cell growth and migration of endothelial cells from bovine
coronary postcapillary venules and from human umbilical veins (HUVECs).
In these two in vitro assays, PlGF-1 seemed to have a
comparable effect on the cultured microvascular endothelium
(e.g. capillary venule endothelial cells, CVECs) to that of
VEGF and bFGF. These results clearly demonstrate that PlGF-1 can induce
angiogenesis in vivo and stimulate the migration and
proliferation of endothelial cells in vitro (25). In the case of PlGF-2 it has been established that the recombinant, purified protein is able to stimulate bovine aortic endothelial cells (BAEC, Ref. 13) and HUVECs but not the ECs from hepatic sinusoids (26).
The VEGF homodimer binds to and induces autophosphorylation of two
distinct kinase receptors: the fms-like tyrosine kinase, Flt-1 (also
known as VEGFR-1) and the kinase insert domain-containing receptor/fetal liver kinase, KDR/Flk-1 (also known as VEGFR-2). Conversely, the PlGF-1 and -2 homodimer bind only to the Flt-1 receptor
(22, 26-28). Likewise, VEGF-B also binds selectively to Flt-1 and
hence appears to be a closer homolog of PlGF in its receptor-binding
profile (29). Purified heterodimeric VEGF/PlGF has been shown also to
bind KDR/Flk-1 (22). The extracellular portion of both receptors
consists of seven immunoglobulin (IgG)-like domains, and the receptors
share 44% amino acid sequence homology. The IgG-like domain 2 of the
Flt-1 receptor is responsible for the binding specificity of PlGF-1 and
-2 (30-32). Furthermore, it has been reported that only PlGF-2 can
recognize neuropilins-1 and -2, receptor molecules found at the
endothelial surface, in a heparin-dependent fashion (33,
34).
Since PlGF has been shown to bind and induce autophosphorylation of
Flt-1 but not KDR/Flk-1, it appears that PlGF should exert its
mitogenic and chemotactic effects on ECs through the activation of the
Flt-1 intracellular signaling pathway. PlGF induces DNA synthesis but
not migration of porcine aortic ECs (PAE) overexpressing Flt-1 (28).
However, recent findings that PlGF is mitogenic and chemotactic for
CVECs and HUVECs in vitro (25) (discussed above), raise the
question of whether PlGF induces Flt-1 directly to transduce mitogenic
and chemotactic signals inside the cell or whether PlGF acts indirectly
through a mechanism of decoy, as previously proposed by Park et
al. (14).
The recent observation that Flt-1 is able to mediate signaling in
HUVECs in response to both PlGF and VEGF, leading to distinct biological responses, suggests that Flt-1 does not act as a decoy receptor but is indeed able to signal intracellularly (35). Inhibition
of PlGF translation by antisense mRNA in the human dermal
microvascular endothelial cells in culture results in the inhibition of
cell proliferation under hypoxic conditions (36). These new findings
assign a role to PlGF in the direct control of endothelial cell
proliferation, probably competing with VEGF for binding to Flt-1 and
thereby forcing the binding of VEGF to the KDR/Flk-1 and activating
cell proliferation. In addition, both PlGF and VEGF are able to induce
migration of 39% and 51% of monocytes, respectively, through
activation of Flt-1 (35, 39). This suggests that PlGF may induce EC
migration and proliferation through activation of Flt-1, although the
existence of a yet unknown PlGF receptor cannot be ruled out.
A considerable amount of structural information is now available for
VEGF (VEGF-A). Muller et al. (38, 39) reported the crystal
structures of the receptor binding domain of VEGF in different crystal
forms and have identified the KDR binding site using mutational analysis. Also, Wiesmann et al. (40) have reported the
crystal structure of VEGF in complex with domain 2 of Flt-1
(Flt-1D2). To understand the specific molecular details of
the receptor binding site and critical components of the homodimer,
which will consequently help in understanding the differences in
specificity and cross-reactivity among the VEGF homologs, we have
embarked on a three-dimensional structural study of PlGF. Here we
report the crystal structure of PlGF-1 at 2.0 Å resolution. As
anticipated, the structure is similar to that of VEGF. However, it
shows subtle differences in molecular interactions at the receptor
recognition site that appear to be relevant to signaling.
Protein Expression and Purification--
By polymerase chain
reaction, the region of the human PlGF-1 gene coding for the
mature protein was cloned into a prokaryotic expression vector as
described previously (11). The recombinant vector was used to transform
a DE3 Escherichia coli strain, and the synthesis of PlGF-1
was induced by 1 mM
isopropyl-2-D-thiogalactopyranoside. After preparation and
refolding of the inclusion bodies, the PlGF-1 protein was purified
first by anion exchange chromatography followed by reverse phase
chromatography. Final recovery of the active protein was about 140 mg
per liter of initial bacterial culture. The identity of the protein was
checked by various assays such as immunoblotting, SDS-polyacrylamide
gel electrophoresis under reducing and nonreducing conditions,
two-dimensional electrophoresis, reverse phase chromatography, and
amino-terminal sequencing. The angiogenic activity was tested using a
CAM assay (41); the purified bacterial-derived PlGF-1 was able to
induce a strong and dose-dependent angiogenic response
(42).
Crystallization--
Crystals of recombinant PlGF-1 were grown
using the hanging drop vapor diffusion method from drops containing 8 mg/ml protein at pH 6.0 in 0.05 M MES buffer, 10 mM CaCl2 and 7.5% (v/v)
2-methyl-2,4-pentanediol (MPD) equilibrated against reservoirs
containing 0.1 M MES buffer (pH 6.0), 20 mM
CaCl2 and 15% (v/v) MPD. Single crystals appeared after
5-6 days at 16 °C. These crystals could be flash-frozen at 100 K
using a cryoprotectant solution containing 0.1 MES buffer (pH 6.0), and
30% (v/v) MPD. The systematic absences and symmetry were consistent
with the tetragonal space group P41 or P43,
with unit cell dimensions a = b = 62.6 Å, and c = 84.1 Å. There was one PlGF-1 homodimer per crystallographic asymmetric unit
and ~50% of the crystal volume was occupied by solvent.
Data Processing and Reduction--
X-ray diffraction data to 2.0 Å were collected at 100 K from a single crystal using the Synchrotron
Radiation Source (station PX 9.5) at Daresbury (United Kingdom).
Seventy images were collected ( Phasing--
The structure of PlGF-1 was determined by molecular
replacement with the program AMoRe (45) using a polyalanine (homodimer) model based on the structure of VEGF at 1.93 Å resolution (PDB code
2VPF, Ref. 39). Data in the range 15.0-3.0 Å were used for both the
rotation and the translation function searches. No solution was found
in space group P41. In space group P43, the best solution after FITING had a correlation coefficient of 56% and an
R-factor of 51%. Rigid-body refinement with CNS version 0.9 (46) of this model corresponding to the highest peak using data in the
range 40.0-2.0 Å, resulted in an Rfree and
Rcryst of 44.6 and 40.6%, respectively.
Refinement--
All crystallographic refinement was carried out
using the program CNS version 0.9 (46). Procedures carried out with CNS included simulated annealing using a maximum likelihood target function, restrained individual B-factor refinement, conjugate gradient
minimization, and bulk solvent correction. The behavior of the
Rfree value (811 reflections) was monitored
throughout refinement. Several rounds of refinement (using all
reflections) and model building were performed until the
Rfree for the model could not be improved any
further. During the final stages of refinement, water molecules were
inserted into the model at positions where peaks in the
|F0| Accession Number--
Final atomic coordinates of human PlGF-1
have been deposited with the RCSB Protein Data Bank under the accession
code 1FZV.
Quality of the Structure--
The crystal structure of PlGF-1 was
determined at 2.0 Å resolution. Details of the data collection and
refinement statistics are shown in Table
I. The protein crystallizes as a
homodimer in the asymmetric unit. As in the VEGF structure (38), the
first 17 amino-terminal residues of both monomers are not visible in the electron density map and were excluded from crystallographic refinement. Both monomers A and B contain residues 18-117. Also, residues Ser18, Glu51,
Glu73, Asn74, and Ser94 in both
molecules and residues Glu53 and Arg117 in
molecule A have been modeled as alanines because of lack of sufficient
density beyond C Overall Structure--
The crystal structure of PlGF-1 consists of
a homodimer, organized in an antiparallel arrangement with the 2-fold
axis perpendicular to the plane of the
The structural core of the PlGF-1 monomer consists of a four-stranded,
highly irregular, solvent-accessible Comparison with VEGF Structure--
Overall, the structure of
PlGF-1 exhibits remarkable topological identity with that of VEGF (38,
39) (with which it has 42% amino acid sequence identity) despite
significant functional diversity (Fig. 1, B Receptor Recognition--
The extracellular domain of both KDR and
Flt-1 receptors consist of seven immunoglobulin domains. Mutational
analysis of VEGF has revealed that symmetrical binding sites for KDR
are located at each pole of the VEGF homodimer (38). Each site appears
to contain two functional regions composed of binding determinants presented across the intermolecular interface. This experimental evidence suggested that only a small number of VEGF residues are important for binding to KDR, and the binding epitope for KDR contains two hot-spots, each of which extends across the dimer interface (39, 54-56). Furthermore, analysis of the conformational variability of VEGF (based on the high resolution structure of VEGF,
Ref. 39) showed that the loop connecting strands
Recently, the crystal structure of VEGF in complex with
Flt-1D2 (at 1.7 Å) has revealed that domain 2 is
predominantly involved in hydrophobic interactions with the poles of
the VEGF dimer (40). Based on this structure and previous mutagenesis
data, Wiesmann et al. (40) have proposed a model of VEGF
bound to the first four domains of Flt-1. In the case of PlGF, it has
been shown that binding of PlGF to human ECs revealed a high affinity
site and a low affinity site (35, 37). The high affinity site is for
Flt-1 and PlGF can displace VEGF from both truncated and full-length Flt-1 receptors. However, at the present time it is yet be established whether both PlGF-1 and VEGF bind identically to Flt-1.
Flt-1 (VEGFR-1) Receptor Interactions--
The structure reported
here for PlGF-1 is an unliganded structure and hence it is not possible
to establish the precise nature of the interaction of PlGF-1 with
Flt-1. However, using the structural data on the
VEGF·Flt-1D2 complex, we have been able to construct a
model to visualize the binding mode between PlGF-1 and Flt-1. The
PlGF-1·Flt-1D2 complex was modeled by superimposing the
atomic coordinates of the VEGF·Flt-1D2 complex (Ref. 40,
PDB code 1FLT) onto the PlGF-1 model followed by energy minimization
using the program X-PLOR (57). The resultant model showed a reasonable fit between PlGF-1 and Flt-1 without any obvious stereochemical impediments between the two proteins (Fig.
2A). The interface of the
putative PlGF-1·Flt-1D2 complex appears to include some 22 amino acids from the PlGF-1 molecule: residues from the
Based on the PlGF-1·Flt-1D2 model, we speculate that both
PlGF-1 and Flt-1D2 form extensive contacts through
sidechain interactions (Table III, Fig. 2B). The contact
residues from the two individual components of the modeled complex are
shown in Fig. 2, C and D. Asp72 in
PlGF-1 appears to be the only residue to make direct H-bond interactions with Arg224 of Flt-1D2. (In the
VEGF·Flt-1D2 complex structure, the conserved VEGF
residue Asp63 makes similar interactions with Flt-1, Ref.
40, Table III.) PlGF-1 residues Gln27, Tyr34,
Ala74, Tyr100, and Glu112 in one
molecule (either A or B) are predicted to make both polar and van der
Waals interactions with Flt-1, whereas residues Phe26,
Trp30, Gly31, Glu73,
Leu75, Gln88, Leu90,
Ile92, and Pro115 seem to participate in van
der Waals interactions with Flt-1 residues (Table III). Additional
PlGF-1 residues Pro25, Cys70,
Gly71, Pro98, Cys111,
Cys113, and Arg114 also appear to be part of
the interface.
A general mechanism of Flt-1 recognition by PlGF-1 can be postulated
based on the proposed dimeric model of the receptor binding domain of
VEGF in complex with domains 1-4 of Flt-1 (40). A similar picture may
be visualized for PlGF-1·Flt-1 recognition with domain-1 pointing
away from PlGF-1, the domain 2-3 linker region occupying the groove
(6.8 Å wide) between the two monomers, and domain 3 making contact
with its bottom face, which would bring domain 4 into direct
inter-receptor contacts and hence involved in dimer formation. In the
PlGF-1 structure, the walls of the groove are formed by residues
Asp72, Glu73, Val52,
Met55, Val45, Asp43, and
Ser59. The corresponding groove in VEGF was formed by
residues Asp63, Glu64, Ile43,
Ile46, Phe36, Asp34, and
Ser50, which was implicated for recognition of domain 3 of
Flt-1 (40). This comparison illustrates a high degree of conservation
of residues in this region between the two molecules (Fig.
1C). However, at the structural level, one can visualize
significant changes in conformation (Fig. 2E), which could
be one of the contributing factor for the distinct receptor specificity
for PlGF-1 (see below).
Lack of KDR (VEGFR-2) Recognition--
A detailed mutagenesis
study of VEGF by Muller et al. (38) identified eight amino
acid residues forming part of the KDR binding site. These were grouped
as major and minor hot-spots for receptor recognition. The major
hot-spot consists of two important residues Ile46 and
Ile83 (VEGF numbering) and four additional residues
Ile43, Glu64, Lys84, and
Pro85 with slightly lesser importance. The minor hot-spot
contains residues Phe17 and Gln79 (38).
Furthermore, recently a variant of VEGF, which had amino acids 83-89
replaced with the analogous region of the related PlGF demonstrated
significantly reduced KDR binding compared with wild-type VEGF
emphasizing the point that this region is important for VEGF-KDR
interaction (58). Amino acid sequence alignment (Fig. 1C)
shows that of the six most important KDR binding determinants (the
major hot-spot) of VEGF, only two residues from VEGF (Glu64
and Ile83) are conserved in PlGF-1 (Glu73 and
Ile92) and both residues from the minor hot-spot are
conserved in the two structures. Observation of the PlGF-1 structure
indicates significant conformational rearrangement corresponding to
regions 43-45 and 83-85 (the major hot-spot residues for KDR
recognition in VEGF, Fig. 2E). This provides a possible
structural explanation for the inability of PlGF-1 to recognize KDR.
Based on the amino acid sequence alignment of VEGF-A, PlGF-1, and
VEGF-B, similar arguments can be put forward for VEGF-B, where
considerable changes have appeared in the KDR binding determinants and
hence may not recognize this receptor.
We have also performed modeling studies on the PlGF-1·VEGF
heterodimer in complex with Flt-1D2 (in a similar way to
that described above for the PlGF-1·Flt-1D2 complex).
From this model, it appears that the putative contact residues in the
heterodimer are similar to those listed in Table III for Flt-1 recognition.
Concluding Remarks--
Recent structural studies on polypeptide
growth factors in complex with their receptors have provided a wealth
of information in the area of protein-receptor signaling. In the case
of cysteine-knot proteins, the target molecule (e.g. VEGF,
PDGF, or NGF) seems to form complexes with one or two domains of the
receptor molecule. In this report, we have tried to address this
question with another member of this family, PlGF-1, referring to the
molecular details of a closely related molecule, VEGF. As in the case
of VEGF, PlGF-1 appears to use only a small number of residues in
receptor recognition. These details would be the starting point for the
design of small mimics of PlGF. Such agonists could be useful for the
design of PlGF antagonists, which prevent the interaction with the
receptor, and may serve to be important for the treatment of
pathological disorders involved in neovascularization during tumor growth.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
= 1.0 Å, oscillation range of
1.5°, 45 s exposure time) using a MAR-CCD detector system. Data
processing was performed with the HKL package (43). Data reduction was
carried out using the program TRUNCATE of the CCP4 suite (44). Details
of data processing statistics are presented in Table I.
|Fc|
electron density maps had heights greater than 3
and were at
hydrogen bond forming distances from appropriate atoms. 2 ||F0|
|Fc||
calc maps were also used to verify the
consistency in peaks. Water molecules with a temperature factor greater
than 65 Å2 were excluded from the model and subsequent
refinement. One bound MPD molecule per monomer from the crystallization
medium was identified (interacting with the main-chain carbonyl oxygen
atom of Thr-104 at one end and a water molecule at the other end) and
was included in the final stages of the refinement. The details of
refinement are presented in Table I. Map calculations were performed
with CNS with the SigmaA protocol (47), using all the reflections in
the resolution range 40.0-2.0 Å. The program PROCHECK (48) was used
to assess the quality of the final structure. Analysis of the
Ramachandran (
-
) plot showed that all residues lie in the allowed
regions. The program "O" (49) was used for map visualization and
model building.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
atoms. The arrangement of the homodimer and the
nomenclature used throughout the text are shown in Fig. 1A. The final model
(homodimer) includes 1,546 non-hydrogen protein atoms, 132 water
molecules, and two MPD molecules with a crystallographic R-factor (Rcryst) of 21.6% in the
resolution range 40.0-2.0 Å. The Rfree value
is 24.7% with 4% of the reflections excluded from the refinement
(50). The mean coordinate error calculated from a plot of ln
A versus (sin
/
)2 is 0.3 Å.
The root mean square (r.m.s.) deviation in C
atoms between each
monomer of the pair is 0.43 Å (for 100 C
atoms). Regions that
deviate most include residues 18-19 from the amino-terminal tail, part
of the loop connecting strands
3 and
4 (residues 72-73), and the
carboxyl-terminal residue 117. Excluding these residues improves the
r.m.s deviation to 0.17 Å (for 94 C
atoms). Examination of the
Ramachandran plot shows 91.5% of the residues in most favorable
regions and no residues in disallowed regions.
Crystallographic statistics
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Fig. 1.
Structural comparison of PlGF-1 and
other members of the cysteine-knot super family. A,
representation of the PlGF-1 homodimer structure. Disulfide bonds are
shown in a ball-and-stick representation. The
inset presents the organization of three intra- (in
yellow) and one interdisulfide bridge (in green)
in the cysteine-knot motif. Each monomer in the homodimer is colored
differently to enhance clarity. Orange, monomer A;
cyan, monomer B. B, representatives of known
structures from the cysteine-knot protein family of dimeric
molecules. a, VEGF (PDB code 2VPF, Ref. 39);
b, PDGF-BB (PDB code 1PDG, Ref. 51); c, TGF- 2
(PDB code 1TFG, Ref. 52); and d, NGF (PDB code 1BTG, Ref.
53). With the exception of NGF, the homodimer 2-fold axis is
perpendicular to the plane of the
-sheet. The cysteine knots are
highlighted. C,
structure-based sequence alignment of PlGF-1 with VEGF (38, 39).
Amino acid residues that form part of the secondary structural elements
(
-strands and helices) as determined by DSSP (60) are shown in
blue and red, respectively. The cysteine residues
are shaded pink. VEGF residues involved in Flt-1 (VEGFR-1)
binding (40), and the equivalent residues in PlGF-1 (based on a
modeling study) are boxed and shaded in yellow.
The conserved glycine residue in both structures is
underlined. This figure was created with the program
ALSCRIPT (61). D, stereo view displaying the C
traces of PlGF-1 (orange) and VEGF (cyan) (39)
homodimers after alignment of the two structures with the program
"O" (49). A, B, and D were created
with the program MOLSCRIPT (59).
-sheet (Fig. 1A).
The homodimer is covalently linked by two interchain disulfide bonds
between Cys60 and Cys69. The most prominent
feature of the structure is the presence of a cysteine-knot motif,
positioned symmetrically opposite at one end of each monomer. This
motif is found in other closely related growth factors such as VEGF
(38, 39), platelet-derived growth factor-BB (PDGF-BB, Ref. 51),
transforming growth factor-
2 (TGF-
2, Ref. 52) and nerve growth
factor (NGF, Ref. 53) (Fig. 1B). The knot consists of an
eight residue ring formed by one interchain
(Cys60-Cys69) and three intrachain
(Cys35-Cys77,
Cys66-Cys111,
Cys70-Cys113) disulfide bonds (Fig.
1A). The ring structure is formed between two adjacent
-strands,
3 and
7, with the third intrachain disulfide bond
penetrating the covalent linkage and connecting strands
1 and
4.
The cysteine ring contains a conserved glycine residue at position 68, which seems to be important in optimizing the conformation of the
sidechains in the knot. As in the VEGF structure (38, 39), this residue
adopts positive dihedral
angles of 141 and 149° in monomers A and
B, respectively. Thus the cysteine-knot motif appears to be important
for the stabilization of the dimer as there are only a few contacts
between the
-strands (
1 and
1') at the dimer center. One
peptide bond in the PlGF-1 structure adopts a cis
conformation: that connecting Ser57 and Pro58
in both monomers.
-sheet (Fig. 1A).
The total buried surface area at the interface between the two monomers
is 2,627 Å2. A considerable proportion of this (1,830 Å2 or 69%) is accounted for by the extensive
intermolecular hydrophobic core interactions at the interface on the
opposite end of the cysteine-knot and provides additional stability to
the central portion of the structure. The hydrophobic core is formed by
residues from both monomers and is known to be part of the receptor
binding region of PlGF-1 (see under "Receptor Recognition").
Fourteen potential hydrogen bond interactions were observed between the two monomers (Table II). Two
water-mediated hydrogen bonds between Glu39 from each
monomer forms a bridge between two strands (
1 and
1') across the
center of the dimer interface.
Hydrogen bond interactions between the two monomers at the dimer
interface in the PlGF-1 structure
D, r.m.s
deviation of 1.47 Å using 95 C
atoms). The mode of dimerization for
PlGF-1 is similar to that of VEGF. Conformational differences between
PlGF-1 and VEGF are observed at the amino-terminal residues (18-25),
some residues from loop regions (loops connecting
3-
4,
5-
6, and
2-
2) and the carboxyl-terminal residues
(116). Interestingly, these loop regions appear to be part of the
receptor-binding face in both molecules (see below). Approximately 70 water molecules are conserved in PlGF-1 and VEGF and appear to be
important for the structural integrity of the homodimer in both molecules.
5 to
6 undergoes
a concerted movement. This loop is important for binding to both Flt-1
and KDR, suggesting that these receptor molecules have overlapping
binding sites on the target molecule. It has also been established that
minimally domains 2 and 3 of Flt-1 are necessary and sufficient for
binding VEGF with near native affinity, and domain 2 alone binds to
VEGF (60-fold less tightly than wild-type, Ref. 38). Similar results
have been found for deletions in the KDR (56).
1 helix,
3-
4 loop, and
7 strand of one monomer, and residues from
strands
5,
6, and the
5-
6 loop of the second monomer. In
the modeled complex, nineteen residues from the Flt-1D2
segments 141-147, 171-175, 199-204, and 219-226 form part of this
contact surface. Modeling studies based on the
VEGF·Flt-1D2 complex structure (40) predict that binding
between PlGF-1 and Flt-1D2 might also be mediated through
hydrophobic interactions involving planar surfaces from both the ligand
and the receptor (Table III). Such shape
complementarity is energetically favorable for maximizing the
contribution of van der Waals contacts.
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Fig. 2.
Proposed model for the
PlGF-1·Flt-1D2 complex based on the crystal structures of
PlGF-1 (present study) and the VEGF·Flt-1D2 complex (40),
PDB accession code 1FLT. A, PlGF-1 homodimer is shown
in orange (molecule A) and cyan (molecule B),
whereas the Flt-1D2 molecules are shown in
purple and green. B, stereo views of
contact residues (C atoms plus sidechain atoms) at the putative
PlGF-1·Flt-1D2 interface. Residues from PlGF-1 monomers A
and B are marked in orange and cyan,
respectively. Residues from Flt-1 (figure based on model shown in
A) are colored in green. The sidechains for
Glu73 and Asn74 in free PlGF-1 are disordered
and hence are treated as alanines. C, stereo views of
contact residues (C
atoms plus sidechain atoms) for PlGF-1. Residues
from monomer A and B are marked in orange and
cyan, respectively (figure based on model shown in
A). The sidechains for Glu73 and
Asn74 in free PlGF-1 are disordered and hence are treated
as alanines. D, stereo views of contact residues (C
atoms
plus sidechain atoms) for Flt-1D2 (figure based on model
shown in A). E, stereo views showing the location
of the groove in PlGF-1 (residues Asp72, Glu73,
Val52, Met55, Val45,
Asp43, and Ser59) and VEGF (Asp63,
Glu64, Ile43, Ile46,
Phe36, Asp34, and Ser50),
implicated for recognition of domain 3 of Flt-1 (40). The figure also
shows the difference in conformation for segment 90-95 in PlGF-1 and
81-86 in VEGF. Residues Ile83, Lys84, and
Pro85 are implicated in KDR recognition in VEGF (38), and
the corresponding residues in PlGF-1 are Ile92,
Ser94, and Arg93. The sidechains for PIGF-1 and
VEGF are shown in orange and cyan, respectively.
Ser94 and Glu73 in the PlGF-1 structure are
represented as alanines because of insufficient electron density beyond
the C
atom. A-E was generated using MOLSCRIPT
(59).
Putative intermolecular contacts
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ACKNOWLEDGEMENTS |
---|
We thank the staff at the Synchrotron radiation source, Daresbury (UK) for their help with X-ray data collection and members of the Acharya laboratory for constructive criticism of the manuscript.
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FOOTNOTES |
---|
* This work was supported by Medical Research Council (UK) Programme Grant 9540039 and Wellcome Trust (UK) Equipment Grant 055505/98/Z (to K. R. A.).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 1FZV) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ These authors contributed equally to this work.
¶ Postgraduate training bursary from the University of Bath, United Kingdom.
Present address: Inst. of Biological Research and
Biotechnology, The National Hellenic Research Foundation, 48 Vas
Constantinou Ave., Athens 11635, Greece.
§§ Recipient of the Associazione Italiana Ricerca sul Cancro (Italy) Research Grant.
¶¶ To whom correspondence should be addressed. Tel.: 44-1225-826238; Fax: 44-1225-826779; E-mail: K.R.Acharya@bath.ac.uk.
Published, JBC Papers in Press, November 7, 2000, DOI 10.1074/jbc.M008055200
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ABBREVIATIONS |
---|
The abbreviations used are:
ECs, endothelial
cells;
PlGF, placenta growth factor;
VEGF, vascular endothelial growth
factor;
FIGF, Fos-induced growth factor;
PDGF-BB, platelet-derived
growth factor-BB;
TGF-2, transforming growth factor-
2;
NGF, nerve
growth factor;
Flt-1, fms-like tyrosine kinase;
KDR/Flk-1, kinase
insert domain-containing receptor/fetal liver kinase;
VEGFR, VEGF
receptor;
HUVECs, human umbilical veins endothelial cells;
MPD, 2-methyl-2,4-pentanediol;
r.m.s., root mean square.
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