VEGF and the Fab fragment of a humanized neutralizing antibody: crystal structure of the complex at 2.4 å resolution and mutational analysis of the interface

AbstractBackground: Vascular endothelial growth factor (VEGF) is a highly specific angiogenic growth factor; anti-angiogenic treatment through inhibition of receptor activation by VEGF might have important therapeutic applications in diseases such as diabetic retinopathy and cancer. A neutralizing anti-VEGF antibody shown to suppress tumor growth in an in vivo murine model has been used as the basis for production of a humanized version.Results: We present the crystal structure of the complex between VEGF and the Fab fragment of this humanized antibody, as well as a comprehensive alanine-scanning analysis of the contact residues on both sides of the interface. Although the VEGF residues critical for antibody binding are distinct from those important for high-affinity receptor binding, they occupy a common region on VEGF, demonstrating that the neutralizing effect of antibody binding results from steric blocking of VEGF–receptor interactions. Of the residues buried in the VEGF–Fab interface, only a small number are critical for high-affinity binding; the essential VEGF residues interact with those of the Fab fragment, generating a remarkable functional complementarity at the interface.Conclusions: Our findings suggest that the character of antigen–antibody interfaces is similar to that of other protein–protein interfaces, such as ligand–receptor interactions; in the case of VEGF, the principal difference is that the residues essential for binding to the Fab fragment are concentrated in one continuous segment of polypeptide chain, whereas those essential for binding to the receptor are distributed over four different segments and span across the dimer interface

Vascular endothelial growth factor: Crystal structure and functional mapping of the kinase domain receptor binding site

Vascular endothelial growth factor (VEGF) is a homodimeric member of the cystine knot family of growth factors, with limited sequence homology to platelet-derived growth factor (PDGF) and transforming growth factor β2 (TGF-β). We have determined its crystal structure at a resolution of 2.5 Å, and identified its kinase domain receptor (KDR) binding site using mutational analysis. Overall, the VEGF monomer resembles that of PDGF, but its N-terminal segment is helical rather than extended. The dimerization mode of VEGF is similar to that of PDGF and very different from that of TGF-β. Mutational analysis of VEGF reveals that symmetrical binding sites for KDR are located at each pole of the VEGF homodimer. Each site contains two functional “hot spots” composed of binding determinants presented across the subunit interface. The two most important determinants are located within the largest hot spot on a short, three-stranded sheet that is conserved in PDGF and TGF-β. Functional analysis of the binding epitopes for two receptor-blocking antibodies reveal different binding determinants near each of the KDR binding hot spots