Minimum FGF2 Binding Structural Requirements of Heparin and Heparan Sulfate Oligosaccharides As Determined by NMR Spectroscopy

Heparin and heparan sulfate (HS) glycosaminoglycans (HSGAGs) are sulfated polysaccharides that play important roles in fundamental biological processes by binding to proteins. The prototypic example of HSGAG-protein interactions is that with the fibroblast growth factors (FGFs), specifically FGF1 and FGF2. Structural and biochemical studies have shown that the chain length, sulfation pattern, and conformation of HSGAGs play a critical role in FGF binding and activity. Previously, we showed that a tetrasaccharide of the form A(NS,6X)-I-2S-A(NS.6X)-I-2S-OPr (where X is OH or O-sulfate and Pr is propyl) with at least one of the A(NS,6X) residues having a 6-O sulfate group was the minimum binding motif for FGF1 [Guerrini, M., Agulles, T., Bisio, A., Hricovini, M., Lay, L., Naggi, A., Poletti, L., Sturiale, L., Torri, G., and Casu, B. (2002) Biochem. Biophys. Res. Commun. 292, 222-230]. We report NMR structural analysis using two-dimensional NOE spectroscopy (2D-NOESY) and transferred NOESY (trNOESY) on a non-6-O-sulfated synthetic tetrasaccharide TETRA (A(NS)-I-2S-A(NS)-I-2S-OPr) both in its free state and bound to FGF2. This tetrasaccharide comprises both the structural trisaccharide motif A(NS)-I-2S-A(NS) that forms "kinks" in longer heparin chains induced by FGF binding [Raman, R., Venkataraman, G., Emst, S., Sasisekharan, V., and Sasisekharan, R. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 2357-23621 and the common binding Motif I-2S-A(NS)-I-2S present in octasaccharides that exhibited strong FGF2 binding [Kreuger, J., Salmivirta, M., Sturiale, L., Gimenez-Gallego, G., and Lindahl, U. (2001) J. Biol. Chem. 276, 30744-30752]. These data suggest that TETRA could be the shortest HSGAG oligosaccharide that binds to FGF2. Furthermore, our study confirms that both the IdoA residues in TETRA adopt the chair (1)C4 conformation upon FGF2 binding to provide the best molecular fit in contrast to an analogous 6-O-sulfated tetrasaccharide motif observed in the FGF2-HSGAG cocrystal structure where one of the IdoAs adopts skew-boat S-2(O) conformation. Thus, our study highlights the fact that the conformational plurality of IdoA is able to accommodate the changes in the sulfation pattern to provide the necessary specificity for protein binding.