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

The extracellular portion of the VEGF and P1GF receptor, Flt-1 (orVEGFR-1), consists of seven immunoglobulin-like domains. The second domain from the N terminus (Flt-1(D2)) is necessary and sufficient for high affinity VEGF b inding. The 1.7 Angstrom resolution crystal structure of Flt-1(D2) bound to VEGF revealed that this domain is a member of the I-set of the immunoglobu lin superfamily, but has several unusual features including a region near t he 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 o f VEGF binding and might not be present in the absence of ligand. Here we r eport the three-dimensional structure of Flt-1(D2) 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; th e N-terminal bulge is present, albeit in a different conformation, We also report the 2.7 Angstrom crystal structure of Flt-1(D2) in complex with VEGF solved in a different crystal form that reveals yet another conformation f or the N-terminal bulge region. H-1-N-15 heteronuclear NOEs indicate this r egion 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-1( D2), and the unusual structural features of Flt-1(D2) are an intrinsic prop erty of this domain. (C) 1999 Academic Press.