Volume 91, Issue 5, Pages 695-704 (November 1997) Crystal Structure at 1.7 Å Resolution of VEGF in Complex with Domain 2 of the Flt-1 Receptor  Christian Wiesmann, Germaine Fuh, Hans W. Christinger, Charles Eigenbrot, James A. Wells, Abraham M. de Vos  Cell  Volume 91, Issue 5, Pages 695-704 (November 1997) DOI: 10.1016/S0092-8674(00)80456-0

Figure 1 Inhibition of VEGF165-Induced Growth of Human Umbilical Vein Endothelial Cells by Flt-1 Constructs Cells were incubated for 24 hr with 0.2 nM VEGF165 plus increasing concentrations of Flt-1D2 (closed circles) or the dimeric IgG fusion of domains 1–7 (open squares) and pulsed with 0.5 μCi of [3H]thymidine for 18 hr before harvesting. Cell 1997 91, 695-704DOI: (10.1016/S0092-8674(00)80456-0)

Figure 2 Ribbon Rendering of Flt-1D2, in Two Views Related by a Rotation of Approximately 90° about the Vertical Axis The termini and the secondary structure elements as defined by the program Procheck (Laskowski et al. 1993) are labeled; β strands are rendered as green arrows, the helical turn as a green ribbon, and the loop regions as gray tubes. The disulfide bond is shown in ball-and-stick rendering, with sulfur atoms colored yellow. The two potential N-linked glycosylation sites at Asn-164 and Asn-196 are colored blue. The VEGF binding site is located on the “bottom” end of the five-stranded sheet; residues in contact with VEGF in the complex are colored red. A segment near the N terminus, which forms strand βa in members of the I set of the immunoglobulin superfamily, bulges away from the core of the domain. This figure was created using the program MOLMOL (Koradi et al. 1996). Cell 1997 91, 695-704DOI: (10.1016/S0092-8674(00)80456-0)

Figure 3 Stereo Views in Ball-and-Stick Rendering of Structural Details Hydrogen bonds are shown as dotted lines; oxygen atoms are colored red, nitrogens dark blue, and carbons gray. This figure was generated using the programs MOLSCRIPT (Kraulis 1991) and RASTER3D (Merrit and Murphy 1994). (A) The environment of Phe-135 of Flt-1. (B) The region in Flt-1 corresponding to the “Y corner” found in most Greek key barrel proteins. (C) A region of the interface between VEGF (in dark gray) and Flt-1 (in light gray) around the interaction between Asp-63 and Arg-224, showing a chain of water molecules in the interface. Cell 1997 91, 695-704DOI: (10.1016/S0092-8674(00)80456-0)

Figure 4 Ribbon Representation of the Complex Between VEGF8–109 and Flt-1D2 The two monomers of VEGF8–109 are shown in red and blue, respectively; the two copies of Flt-1D2 in green. The secondary structure elements of VEGF8–109 as well as all terminal residues are labeled. (A) “Bottom” view, looking up from the membrane; (B) side view, with the membrane below. Ribbon representation generated using the program MOLSCRIPT (Kraulis 1991). Cell 1997 91, 695-704DOI: (10.1016/S0092-8674(00)80456-0)

Figure 5 Space-Filling Rendering of VEGF8–109 and Flt-1D2, in “Open Book” View (Left) Functional map of the KDR binding site on the receptor binding domain of VEGF (Muller et al. 1997a). Binding determinants are color-coded based on the magnitude of the decrease in binding upon alanine substitution (dark red, >100-fold; orange, 50- to 100-fold; yellow, 10- to 50-fold; light yellow, 3- to 10-fold). (Center and Right) Buried surface between VEGF8–109 (center) and Flt-1D2 (right). Contact residues are color-coded in different shades of red, reflecting the percentage of accessible surface buried in the interface (dark red, 75%–100%; orange, 50%–75%; yellow, 25%–50%; light yellow, 0%–25%). The two VEGF images have been adjusted to create a wall-eyed stereo view of the surface; note the groove between residues 64 and 43. Model generated using the program CONIC (Huang et al. 1991). Cell 1997 91, 695-704DOI: (10.1016/S0092-8674(00)80456-0)

Figure 6 Location of Other Domains of Flt-1 (A) Surface rendering of VEGF8–109 with the contact regions of Flt-1D2 in tube representation, generated using the program GRASP (Nicholls et al. 1993) and color-coded for electrostatic potential. Blue and red indicate positively and negatively charged patches on the surface of VEGF8–109, respectively; selected VEGF residues are labeled. The C terminus of Flt-1D2 reaches toward a groove between Glu-64 and Ile-43. Glu-64 is an important binding determinant of VEGF for binding to Flt-1 (Keyt et al. 1996b), and both of these residues are important for high-affinity binding of VEGF to KDR (Muller et al. 1997a). (B) Model of a complex between VEGF8–109 and domains 1–4 of Flt-1. The two VEGF subunits are colored red and blue, respectively, domain 2 of Flt-1 is in green, and the other domains of Flt-1 are in different shades of gray. Cell 1997 91, 695-704DOI: (10.1016/S0092-8674(00)80456-0)

Figure 7 Sequence Alignment of Domain 2 of the Members of the PDGF Receptor Family Secondary structure elements observed for Flt-1D2 are shown and labeled as in Figure 2, and key I-set positions are marked by closed circles. Homologous residues are shaded, Flt-1 residues in contact with VEGF are shown in red, and the two potential N-linked glycosylation sites of Flt-1D2 are in blue. Human sequences were taken from the SwissProt Data Bank (accession numbers P17948, P35968, P35916, P09619, P16234, P07333, and P10721). Cell 1997 91, 695-704DOI: (10.1016/S0092-8674(00)80456-0)