Solution structure of the VEGF-binding domain of Flt-1: comparison of its free and bound states.

The extracellular portion of the VEGF and PlGF receptor, Flt-1 (or VEGFR-1), consists of seven immunoglobulin-like domains. The second domain from the N terminus (Flt-1D2) is necessary and sufficient for high affinity VEGF binding. The 1.7 A resolution crystal structure of Flt-1D2 bound to VEGF revealed that this domain is a member of the I-set of the immunoglobulin superfamily, but has several unusual features including a region near the N terminus that bulges away from the domain rather than pairing with the neighboring beta-strand. Some of the residues in this region make contact with VEGF, raising the possibility that this bulge could be a consequence of VEGF binding and might not be present in the absence of ligand. Here we report the three-dimensional structure of Flt-1D2 in its uncomplexed form determined by NMR spectroscopy. A semi-automated method for NOE assignment that takes advantage of the previously solved crystal structure was used to facilitate rapid analysis of the 3D NOESY spectra. The solution structure is very similar to the previously reported VEGF-bound crystal structure; the N-terminal bulge is present, albeit in a different conformation. We also report the 2.7 A crystal structure of Flt-1D2 in complex with VEGF solved in a different crystal form that reveals yet another conformation for the N-terminal bulge region. (1)H-(15)N heteronuclear NOEs indicate this region is flexible in solution; the crystal structures show that this region is able to adopt more than one conformation even when bound to VEGF. Thus, VEGF-binding is not accompanied by significant structural change in Flt-1D2, and the unusual structural features of Flt-1D2 are an intrinsic property of this domain.

Solution structure of the heparin-binding domain of vascular endothelial growth factor.

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 beta sheet; the carboxy-terminal subdomain also contains a short alpha 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.

1H, 13C, and 15N backbone assignment and secondary structure of the receptor-binding domain of vascular endothelial growth factor.

Nearly complete sequence-specific 1H, 13C, and 15N resonance assignments are reported for the backbone atoms of the receptor-binding domain of vascular endothelial growth factor (VEGF), a 23-kDa homodimeric protein that is a major regulator of both normal and pathological angiogenesis. The assignment strategy relied on the use of seven 3D triple-resonance experiments [HN(CO)CA, HNCA, HNCO, (HCA)CONH, HN(COCA)HA, HN(CA)HA, and CBCA-(CO)NH] and a 3D 15N-TOCSY-HSQC experiment recorded on a 0.5 mM (12 mg/mL) sample at 500 MHz, pH 7.0, 45 degrees C. Under these conditions, 15N relaxation data show that the protein has a rotational correlation time of 15.0 ns. Despite this unusually long correlation time, assignments were obtained for 94 of the 99 residues; 8 residues lack amide 1H and 15N assignments, presumably due to rapid exchange of the amide 1H with solvent under the experimental conditions used. The secondary structure of the protein was deduced from the chemical shift indices of the 1H alpha, 13C alpha, 13C beta, and 13CO nuclei, and from analysis of backbone NOEs observed in a 3D 15N-NOESY-HSQC spectrum. Two helices and a significant amount of beta-sheet structure were identified, in general agreement with the secondary structure found in a recently determined crystal structure of a similar VEGF construct [Muller YA et al., 1997, Proc Natl Acad Sci USA 94:7192-7197].

Protection of chimpanzees from infection by HIV-1 after vaccination with recombinant glycoprotein gp120 but not gp160.

The development of a vaccine to provide protective immunity to human immunodeficiency virus type 1 (HIV-1), the virus causing AIDS, would be the most practical method to control its spread. Subunit vaccines consisting of virus envelope glycoproteins, produced by recombinant DNA technology, are effective in preventing viral infections. We have now used this approach in the development of a candidate AIDS vaccine. Chimpanzees were immunized with recombinant forms of the HIV-1 glycoproteins gp120 and gp160 produced in Chinese hamster ovary cells, and then challenged with HIV-1. The control and the two animals immunized with the gp160 variant became infected within 7 weeks of challenge. The two animals immunized with the gp120 variant have shown no signs of infection after more than 6 months. These studies demonstrate that recombinant gp120, formulated in an adjuvant approved for human use, can elicit protective immunity against a homologous strain of HIV-1.