Abstract
Vascular endothelial growth factor (VEGF) is a covalently linked homodimeric protein that functions as an endothelial cell-specific mitogen, and is an important mediator of pathological angiogenesis. Phage display has been used to select three different classes of novel disulfide-constrained peptides that bind to VEGF and disrupt receptor binding with IC50values between 0.2–10 µM. Mapping of peptide induced nuclear magnetic resonance (NMR) chemical shift changes shows that they target a region of the VEGF receptor-binding domain that overlaps with the contact surfaces of the receptors, Flt-1 and KDR. The structure of one of these 28-kDa VEGF/peptide complexes was determined by NMR spectroscopy. The structure is based on a total of 4416 internuclear distance and dihedral angle restraints derived from data obtained using samples of the complex containing either13C/15N-labeled peptide or protein. Incorporation of residual dipolar coupling restraints improved both the precision and accuracy of the structure (as judged by comparison with crystal structures of VEGF). Comparison with the structure of a different VEGF/peptide complex reveals different peptide binding modes that each resemble those of natural protein ligands (an anti-VEGF antibody and the VEGF-receptor Flt-1). Prospects for the development of small-molecule antagonists of VEGF, based on the VEGF-bound peptide structures, are discussed.
Highlights
Vascular endothelial growth factor (VEGF) is a covalently linked homodimeric member of the cystineknot family of growth factors [1]
VEGF signaling is mediated via dimerization of its tyrosine kinase receptors, Flt-1 and KDR, using a pair of identical receptor-binding sites localized at the poles of the dimeric VEGF receptor-binding domain [1,11,12,13,14]
nuclear magnetic resonance (NMR) chemical shift mapping reveals that the peptide-binding sites on VEGF overlap with the previously identified receptor-binding sites
Summary
Vascular endothelial growth factor (VEGF) is a covalently linked homodimeric member of the cystineknot family of growth factors [1]. KDR, the lower affinity receptor (Kd ∼ 75–125 pM) [15], is the primary signaling receptor of VEGF and its activation is sufficient to stimulate vascular endothelial cell mitogenesis [16], while the higher affinity receptor Flt-1 (Kd ∼ 10–20 pM) [17] is thought to function,. A high-resolution crystal structure of the complex between the VEGF receptor-binding domain and the second Ig-like domain of Flt-1 (Flt-1D2) shows in detail the mode of ligand–receptor interaction [12]. The binding sites for several neutralizing antibodies have been shown to overlap with the KDR- and Flt-1-binding sites on the VEGF receptor-binding domain (Fig. 1B) [1,20,21]. Prospects for the development of novel, small-molecule antagonists of VEGF, based on the interactions observed between the phage-derived peptide antagonists and the VEGF receptor-binding domain, are discussed
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