The intermolecular NOEs that allowed locating of the hexasaccharide at the protein surface are described in Table 2. These intermolecular NOE crosspeaks involved the side chain protons of positively charged amino acid residues, Lys127, Ser130, Arg133 and Lys142 (Fig. 5), suggesting a electrostatically driven interaction between the protein and the negatively charged sulfate groups of the hexasacharide. A close view of the electrostatic interactions in the binding site is given in Fig. 6. Consistently, similar NOEs (involving Lys127, Arg133 and Lys142) have been detected in previous NMR studies of FGF-1 with nonoligosacharidic molecules . The protein residues giving intermolecular NOEs are in the protein region that experiences the largest chemical shift perturbation upon complex formation. Moreover, 15N relaxation studies have shown that the order parameter substantially increases in this region from that observed in the free state upon complex formation .
A comparison of the heparin binding site of our NMR structure with the crystallographic ones of heparin–FGF-1 , and heparin–FGF-2–FGFR  was then performed. The overlays are shown in Fig. 7. The three complexes share a common FGF–carbohydrate interface, in which the hexasaccharide sulfate groups N-SO3(GlcN-6), 2-OSO3(IdoA-5) and 6-OSO3(GlcN-4) fit nicely with the negatively charged groups COO–(IdoA)-N-SO3(GlcN) and 2OSO3(IdoA), which lie juxtaposed on the same side of the heparin helix (Fig. 7). This sequence GlcN(N-SO3)-IdoA(2-OSO3)-GlcN(6-OSO3) has been described as the minimal binding sequence for FGF-1 . In the crystallographic models of FGF–heparin [27,28] and FGF–FGFR–heparin [30,31] complexes, the N-sulfate group of one glucosamine residue, and the 2-O-sulfate of the following iduronic acid pack closely against the protein backbone joining Q141 and K142 of FGF-1 (Q134 and K135 of FGF-2) and K126 and K127 of FGF-1 (K119 and R120 of FGF2), respectively, and are surrounded by the side chains of N32, Q141, K142, A143, K126, K127 and K132 of FGF-1 (N28, Q134, K135, A136, K119, R120, and K125 of FGF-2). A similar binding region is observed in our complex, where K142 is close to the N-sulfate group of GlcN-6 and K127 is close to the 2-O-sulfate group of IdoA-5 (Fig. 6). Residues N32, Q141, A143, and K132 are also directly involved in the interaction with this hexasaccharide region (GlcN6-GlcN4). Indeed, from the experimental data, they show important chemical shift changes upon complex formation (Δδ = 0.18, 0.14, 0.12 and 1.54 p.p.m., respectively). Moreover, R136, K126 and R133 are in close contact with the IdoA3-IdoA1 hexasaccharide region (Δδ = 0.22, 0.10 and 0.10 p.p.m., respectively) (Fig. 6). This sugar end is closer to the protein in our complex than in the complexes with natural heparin, because in our case, due to the differences in the sulfation pattern, there is an additional sulfate group in this sugar region that can interact with K126 and R133.
Figure 7. Comparison of the heparin binding sites. (A) Crystal structure of an FGF2–FGFR1–heparin complex, with 2 : 2 : 2 stoichiometry (PDB entry 1FQ9) . FGFR and FGF are in ribbon representation and heparin is coloured red. For the sake of simplicity, only one of the FGF2–heparin units (red) is overlaid with our complex (blue, PDB entry 2ERM). (B) Heparin–FGF-1 dimer (red, PDB entry 2AXM)  overlaid with our complex (blue). The three complexes share a common FGF–carbohydrate interface in which the hexasaccharide sulfate groups N-SO3(GlcN-6), 2-OSO3(IdoA-5) and 6OSO3(GlcN-4) fit very nicely with the negatively charged groups COO–(IdoA)-NSO3(GlcN) and 2OSO3(IdoA) that lie juxtaposed on the same side of the heparin helix.
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