Structural determinants of growth factor binding
and specificity by VEGF receptor 2
Veli-Matti Leppänena,1, Andrea E. Protab,1, Michael Jeltscha, Andrey Anisimova, Nisse Kalkkinenc,
Tomas Strandind, Hilkka Lankinend, Adrian Goldmanc, Kurt Ballmer-Hoferb,2, and Kari Alitaloa,2
aMolecular Cancer Biology Program, Biomedicum Helsinki, Department of Pathology, Haartman Institute and Helsinki University Central Hospital,
P.O. Box 63, University of Helsinki, Haartmaninkatu 8, FI-00014, Helsinki, Finland; bPaul Scherrer Institut, Biomolecular Research, CH-5232 Villigen PSI,
Switzerland; cInstitute of Biotechnology, University of Helsinki, Viikinkaari 1, FI-00014 , Helsinki, Finland; and dHaartman Institute, University of Helsinki,
Haartmaninkatu 3, FI-00014, Helsinki, Finland.
Communicated by Erkki Ruoslahti, Burnham Institute for Medical Research at University of California, Santa Barbara, CA, December 16, 2009 (received for
review September 27, 2009)
Vascular endothelial growth factors (VEGFs) regulate blood and
lymph vessel formation through activation of three receptor
tyrosine kinases, VEGFR-1, -2, and -3. The extracellular domain
of VEGF receptors consists of seven immunoglobulin homology do-
mains, which, upon ligand binding, promote receptor dimerization.
Dimerization initiates transmembrane signaling, which activates
the intracellular tyrosine kinase domain of the receptor. VEGF-C
stimulates lymphangiogenesis and contributes to pathological an-
giogenesis via VEGFR-3. However, proteolytically processed VEGF-C
also stimulates VEGFR-2, the predominant transducer of signals re-
quired for physiological and pathological angiogenesis. Here we
present the crystal structure of VEGF-C bound to the VEGFR-2
high-affinity-binding site, which consists of immunoglobulin
homology domains D2 and D3. This structure reveals a symmetrical
2∶2 complex, in which left-handed twisted receptor domains wrap
around the 2-fold axis of VEGF-C. In the VEGFs, receptor specificity
is determined by an N-terminal alpha helix and three peptide loops.
Our structure shows that two of these loops in VEGF-C bind to
VEGFR-2 subdomains D2 and D3, while one interacts primarily with
D3. Additionally, the N-terminal helix of VEGF-C interacts with D2,
and the groove separating the two VEGF-C monomers binds to the
D2/D3 linker. VEGF-C, unlike VEGF-A, does not bind VEGFR-1. We
therefore created VEGFR-1/VEGFR-2 chimeric proteins to further
study receptor specificity. This biochemical analysis, together with
our structural data, defined VEGFR-2 residues critical for the bind-
ing of VEGF-A and VEGF-C. Our results provide significant insights
into the structural features that determine the high affinity and
specificity of VEGF/VEGFR interactions.
angiogenesis ∣ lymphangiogenesis ∣ vascular endothelial growth factor C ∣
vascular endothelial growth factor receptor-2
Angiogenesis and lymphangiogenesis, the growth of new bloodand lymphatic vessels from preexisting ones, are important
biological processes during embryonic development, tissue
growth, wound healing, and in the pathogenesis of various dis-
eases. The mammalian vascular endothelial growth factors
(VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placenta growth
factor, PlGF) and their tyrosine kinase receptors (VEGFR-1,
VEGFR-2, and VEGFR-3) are the major mediators of angiogen-
esis. In addition, these receptors regulate vascular permeability
and vessel dilation [reviewed in Lohela et al. (1)]. VEGF-A
signaling through VEGFR-2 is the major pathway regulating en-
dothelial cell sprouting, migration, proliferation, and survival (2),
whereas VEGF-C signaling through VEGFR-3 is indispensable
for the development of lymphatic vessels (3, 4). VEGFR-2 acti-
vation is responsible for the angiogenic properties of VEGF-C in
many experimental conditions (5–7), but angiogenic signaling
also involves VEGFR-3 (8). In addition, VEGF-C promotes
the formation of VEGFR-2/VEGFR-3 heterodimers whose sig-
naling potential is not yet clear (9). VEGF signaling is modulated
through interactions with distinct heparan sulfate proteoglycans
and neuropilins, which act as coreceptors (10–14). VEGFs exist in
multiple isoforms that are generated by alternative splicing and
posttranslational processing and display distinct receptor specifi-
cities (15).
All VEGFs are antiparallel, cystine-knot polypeptide dimers
that are covalently linked by two intermolecular disulfide bonds
(16–19). In VEGF-C and VEGF-D, this VEGF homology
domain is flanked by C- and N-terminal propeptides that are se-
quentially cleaved, giving rise to VEGF homologs with distinct
functions. Interestingly, mature VEGF-C has been described as
a mixture of covalently and noncovalently bound dimers (20, 21).
C-terminally cleaved VEGF-C and VEGF-D are high-affinity
ligands for VEGFR-3 and, upon removal of both propeptides,
they acquire binding affinity for VEGFR-2 (20, 22). All
VEGF-A isoforms bind to VEGFR-1 and VEGFR-2, whereas
PlGF and VEGF-B are specific for VEGFR-1. Furthermore,
pox viruses encode VEGF variants collectively called VEGF-E
that specifically bind to VEGFR-2 (23–25).
Crystal structures have been published for VEGF-A (26),
PlGF (27), VEGF-B (28), and VEGF-E (29). In addition, struc-
tures for VEGF-A (30) and PlGF (31) in complex with domain 2
of VEGFR-1 (VEGFR-1D2) are available. Analysis of VEGFR-1
and VEGFR-2 mutants showed that the second and third immu-
noglobulin homology domains are essential for high-affinity
VEGF-A binding (32, 33), in agreement with the recently pub-
lished EM structure of the VEGF-A/VEGFR-2 complex (34).
According to our EM data, receptor dimers are held together
by ligand interacting with immunoglobulin homology domains
2 and 3 and by homotypic receptor contacts mediated by the
membrane-proximal domains (34). This rigid conformation of
the extracellular domain may then instigate transmembrane sig-
naling resulting in the activation and autophosphorylation of the
intracellular kinase domain (12).
To obtain high-resolution structural information of VEGF/
VEGFR interactions and to understand VEGF receptor specifi-
city in molecular terms, we determined the crystal structure of
VEGF-C in complex with immunoglobulin homology domains
2 and 3 of VEGFR-2. This structure, in combination with our
mutational analysis, provides insights into the high affinity inter-
actions of VEGFs with their receptors.
Author contributions: V.-M.L., A.E.P., M.J., K.B.-H., and K.A. designed research; V.-M.L.,
A.E.P., M.J., A.A., N.K., T.S., H.L., A.G., K.B.-H., and K.A. performed research and analyzed
data; and V.-M.L., A.E.P., K.B.-H., and K.A. wrote the paper.
The authors declare no conflict of interest.
Data deposition: The coordinates and structure factors have been deposited in the Protein
Data Bank, www.pdb.org (PDB ID codes 2x1x and 2x1w).
1V.-M.L. and A.E.P. contributed equally to this work.
2To whom correspondence may be addressed: E-mail: Kurt.Ballmer@psi.ch or Kari.Alitalo@
Helsinki.Fi.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0914318107/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.0914318107 PNAS ∣ February 9, 2010 ∣ vol. 107 ∣ no. 6 ∣ 2425–2430
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Results
Biochemical Analysis. We expressed the human VEGFR-2 immu-
noglobulin homology domains 2 and 3 (VEGFR-2D23) with a
C-terminal Fc-tag and human VEGF-C (22) in insect cells.
VEGF-C was used with a Cys137Ala transversion for increased
protein stability (7) and is hereafter referred to as VEGF-C. The
complex was purified by protein A affinity chromatography
followed by size exclusion chromatography, resolving as a
major peak consisting of the VEGF-C/VEGFR-2D23 complex
(Fig. S1A). The molecular weight of the VEGF-C/VEGFR-
2D23 complex was 78.0 kDa (Fig. S1B) as determined by multi-
angle laser light scattering (MALS), suggesting that the complex
consists of two receptor molecules and one VEGF-C homodimer.
To further characterize the complexes, we measured the binding
affinity of VEGF-C for VEGFR-2D23 (D23; Fig. S1C) and
VEGFR-2D2 alone (D2; Fig. S1D) using isothermal titration
calorimetry (ITC). The data confirmed the 2∶2 ligand∶
receptor stoichiometry and showed that binding is enthalpically
and entropically favorable. VEGFR-2D2 (21) alone was suffi-
cient for VEGF-C binding, but the presence of VEGFR-2D3
was essential for high-affinity binding.
VEGF-C/VEGFR-2D23 Complex Structure. The crystal structure of
VEGF-C in complex with VEGFR-2D23 (Fig. 1) in an ortho-
rhombic crystal form was determined to 2.7 Å resolution by single
isomorphous replacement with anomalous scattering phases, and
it was refined to a crystallographic R value of 22.5% and an Rfree
of 27.7% (Table S1). The asymmetric unit contains two crystal-
lographically independent copies of 2∶2 VEGF-C/VEGFR-
2D23 complexes. The VEGF-C/VEGFR-2D23 complex structure
was solved also in a tetragonal spacegroup at 3.1 Å resolution
with an R value of 25.7% and an Rfree of 34.6% (Table S1). Here,
the asymmetric unit contains only one chain each of VEGF-C and
VEGFR-2D23. The two structures are highly similar, but some
loops and the VEGF-C extended N-terminal helix are differen-
tially resolved (SI Materials and Methods). The two VEGF-C and
the four VEGFR-2D23 N-linked glycans are not equally ordered
in all chains and were only partially modeled.
The crystal structure of human VEGF-C is an antiparallel
homodimer, covalently linked by two disulfide bridges between
Cys156 and Cys165 (Fig. S2A–C). The structure of the monomer
is similar to that of other cystine-knot proteins with an antipar-
allel four-stranded β-sheet, three connecting loops (L1–L3), and
an extended N-terminal α-helix (α1) that folds on top of the sec-
ond monomer, providing several van der Waals and ionic inter-
actions for the dimer interface.
VEGFR-2D2 (residues 120–218) and VEGFR-2D3 (residues
222–326) are immunoglobulin homology domains with two anti-
parallel β-sheets. They adopt the topology of an intermediate
I-set domain (35), with part of the β-strand A (A0; I-set β-strand
naming) moved to the opposite layer. D2 is a globular domain
with relatively short β-strands. The N-terminal bulge (30)
between strands A and A0 is disordered and was omitted from
the model. D3 is an elongated domain with long β-strands in both
sheets. Residues 265–269 between the C–D strands are partially
disordered and the C0 strand is absent from all D3 domains. Both
the D2 and the D3 domains have a disulfide bridge between the
β-sheets that is buried in the hydrophobic core. They have an
overall extended structure and are separated by a three-residue
(Val-Gly-Tyr) linker peptide such that there are only a few inter-
actions between the domains.
Consistent with the biochemical studies (Fig. S1), the indepen-
dent complexes in the two crystal forms follow the approximate
2-fold symmetry of the VEGF-C dimers with 2∶2 stoichiometry
(Fig. 1). VEGFR-2 immunoglobulin homology domains 2 and 3
are positioned perpendicular to the long axis of VEGF-C and D2
is approximately in the same plane as VEGF-C, wheras D3 is
located below this plane (Fig. 1). The bending angle between
D2 and D3 is 122–149° and results in a left-handed twisted do-
main arrangement about the VEGF-C 2-fold axis (Fig. S2D). The
superpositions of the VEGF-C molecules in the independent
complexes result in rmsd between 0.7 and 1.1 Å for 192
VEGF-C C
α
atoms, and the whole complexes superimpose with
an rmsd of about 3.5 Å for 567 C
α
atoms. The differences in the
superpositions result mainly from variation in D3 orientations re-
lative to the rest of the structure (Fig. S2E). The variation in D3
orientation and the VEGF-C loops 1 and 3 result in differences in
the surface area buried at the ligand–receptor interface, in
particular between VEGF-C and D3 (Table S2), whereas the
VEGF-C/D2 interfaces are essentially identical.
VEGF-C/VEGFR-2D23 Interface. VEGF-C binds to the D2-D3 junc-
tion so that the D2 strand G and the linker between the receptor
domains occupy the groove between the VEGF-C monomers.
Both VEGF-C monomers interact with the VEGFR-2 domains
D2 and D3, and the VEGF-C binding surface is continuous.
To better describe the numerous interactions, we assigned two
binding sites, 1 and 2, that mediate the VEGF-C monomer A
and B interactions with VEGFR-2 (Fig. 2A–C). The buried sur-
face area at the interface varies in the independent complexes but
can be divided into 1160–1410 Å2 (48–58% of the total
buried surface area) for site 1 and 1040–1250 Å2 (42–52% of
the total buried surface area) for site 2.
The VEGF-C site 1 interface (Fig. 2B) consists of the N-ter-
minal helix (residues 113–129) and loop L2 (residues 167–171).
The VEGFR-2D2 hairpin turn C-C0 (residues 164–166) packs
against the VEGF-C helix α1 (Fig. 2D) while the connecting loop
E–F and the beginning of strand F (residues 194–197) interact
with the VEGF-C loop L2 and the N-terminal helix. The
VEGFR-2D3 loop B–C (residues 250–257) and strand E also in-
teract with loop L2. The major hydrophobic contacts of site 1 con-
sist of VEGF-C Trp126 and Arg127 interacting with Gly196,
Met197, and Tyr165 of VEGFR-2 (Fig. 2 and Table S2). Most
of the site 1 interactions are hydrophilic and involve hydrogen
bonds between VEGF-C N167 N
δ
2 and the carbonyl oxygen of
VEGFR-2 Tyr194, and between VEGFR-2 Asn253 O
δ
1 and
the main chain amide of VEGF-C Glu169 (Fig. 2E). Moreover,
Glu169, which is highly conserved in the VEGF family (29), forms
a salt bridge with VEGFR-2 Lys286 and a hydrogen bond with the
main chain amide of VEGFR-2 Asn253, and VEGF-C Asp123 is
in contact with the D2 Arg164 and Tyr165.
Fig. 1. Structure of the VEGF-C/VEGFR-2D23 complex in a cartoon represen-
tation. The VEGF-C homodimer is shown in orange and green, and the two
VEGFR-2 receptor chains are colored in light blue. The sugar moieties and the
disulfide bonds are shown in purple and yellow sticks, respectively. VEGF-C
binds to the VEGFR-2 interface between domains 2 and 3.
2426 ∣ www.pnas.org/cgi/doi/10.1073/pnas.0914318107 Leppänen et al.