Solution structure of the heparin-binding domain of vascular
endothelial growth factor
Wayne J Fairbrother1*, Mark A Champe1, Hans W Christinger1, Bruce A Keyt2
and Melissa A Starovasnik1
Background: Vascular endothelial growth factor (VEGF) is an endothelial
cell-specific mitogen and is a potent angiogenic and vascular permeabilizing
factor. VEGF is also an important mediator of pathological angiogenesis
associated with cancer, rheumatoid arthritis and proliferative retinopathy. The
binding of VEGF to its two known receptors, KDR and Flt-1, is modulated by
cell-surface-associated heparin-like glycosaminoglycans and exogenous heparin
or heparan sulfate. Heparin binding to VEGF165, the most abundantly expressed
isoform of VEGF, has been localized to the carboxy-terminal 55 residues;
plasmin cleavage of VEGF165 yields a homodimeric 110-residue amino-terminal
receptor-binding domain (VEGF110) and two 55-residue carboxy-terminal
heparin-binding fragments. The endothelial cell mitogenic potency of VEGF110
is decreased significantly relative to VEGF165, indicating that the
heparin-binding domains are critical for stimulating endothelial cell proliferation.
Results: The solution structure of the 55-residue heparin-binding domain of
VEGF165 has been solved using data from two-dimensional homonuclear and
three-dimensional heteronuclear NMR spectroscopy. The structure has two
subdomains, each containing two disulfide bridges and a short two-stranded
antiparallel β sheet; the carboxy-terminal subdomain also contains a short
α helix. Hydrophobic interactions are limited to sidechains packing against the
disulfide bridges.
Conclusions: The heparin-binding domain of VEGF has no significant
sequence or structural similarity to any known proteins and thus represents a
novel heparin-binding domain. Most of the positively charged amino acid
sidechains are localized on one side of the carboxy-terminal subdomain or on an
adjacent disordered loop in the amino-terminal subdomain. The observed
distribution of surface charges suggests that these residues constitute a
heparin interaction site.
Introduction
Vascular endothelial growth factor (VEGF) is an endo-
thelial cell-specific mitogen that is involved in the induc-
tion of angiogenesis [1,2]. The importance of VEGF as a
regulator of developmental angiogenesis is highlighted
by recent studies showing that heterozygous knock-out
mice lacking a single VEGF allele have severe, embryonic
lethal, deficiencies in vascularization [3,4]. VEGF is also
critical for the vascularization of solid tumors and blood
vessel growth associated with proliferative retinopathy and
rheumatoid arthritis [5]. The observation that tumor growth
can be suppressed by anti-VEGF antibodies [6] indicates
that VEGF is a viable target for therapeutic antagonists of
pathological angiogenesis.
VEGF comprises two identical polypeptide chains cova-
lently linked by a pair of disulfide bonds [7,8]. Four iso-
forms of VEGF, having 121, 165, 189, or 206 residues per
monomer (VEGF121, VEGF165, VEGF189, and VEGF206,
respectively), are produced by alternative splicing of
mRNA [7–10]. The different isoforms share a common
amino-terminal receptor-binding domain of 115 residues
(Figure 1). Isoforms VEGF165, VEGF189, and VEGF206
bind heparin with increasingly greater affinity, whereas
VEGF121 does not bind heparin [11,12]; the heparin-
binding forms share the same carboxy-terminal 50 residues
(Figure 1), which constitute a heparin-binding domain
([13]; see below).
Two receptors for VEGF have been identified, KDR/Flk-1
(kinase insert domain-containing receptor/fetal liver kinase-
1; [14,15]) and Flt-1 (fms-like tyrosine kinase-1; [16]); only
KDR appears to have a mitogenic response [17]. The
binding of VEGF to its receptors is modulated by both
endogenous cell-surface-bound heparin-like glycosamino-
glycans (GAGs) and exogenous heparin or heparan sulfate
Addresses: 1Department of Protein Engineering,
Genentech Inc., 1 DNA Way, South San Francisco,
CA 94080, USA and 2Department of
Cardiovascular Research, Genentech Inc., 1 DNA
Way, South San Francisco, CA 94080, USA.
*Corresponding author.
E-mail: fairbro@gene.com
Key words: heparin, NMR, vascular endothelial
growth factor
Received: 19 December 1997
Revisions requested: 12 February 1998
Revisions received: 2 March 1998
Accepted: 23 March 1998
Structure 15 May 1998, 6:637–648
http://biomednet.com/elecref/0969212600600637
© Current Biology Ltd ISSN 0969-2126
Research Article 637