Properly Oriented Heparin−Decasaccharide-Induced Dimers Are the Biologically Active Form of Basic Fibroblast Growth Factor^,

Abstract

Interaction of basic fibroblast growth factor (FGF-2) with heparin or heparan sulfate proteoglycans (HSPGs) is required for receptor activation and initiation of biological responses. To gain insight into the mechanism of activation of the FGF receptor by FGF-2 and heparin, we have used NMR, dynamic light scattering, and HSPG-deficient cells and cell-free systems. The first 28 N-terminal residues in FGF-2 were found to be highly mobile and flexible, consistent with the disorder found in both the NMR and X-ray structures. The structure of an FGF-2-heparin-decasaccharide complex that binds to and activates the FGF receptor was compared to a heparin-tetrasaccharide-induced complex that does not promote an interaction with the receptor. The major change observed upon the addition of the tetrasaccharide to FGF-2 was an increase in the correlation time consistent with the formation of an FGF-2 dimer. The NMR line widths of FGF-2 in the presence of the decasaccharide are severely broadened relative to the tetrasaccharide, consistent with dynamic light scattering results which indicate FGF-2 is a tetramer. The interaction of these heparin species with FGF-2 does not induce a significant conformational change in the overall structure of FGF-2, but small chemical shift changes are observed in both heparin and receptor binding sites. A trans-oriented symmetric dimer of FGF-2 is formed in the presence of the tetrasaccharide whereas two cis-oriented dimers in a symmetric tetramer are formed in the presence of the decasaccharide. This suggests that the cis-oriented FGF-2 dimer is the minimal biologically active structural unit of FGF-2. These data allow us to propose a novel mechanism to explain the functional interaction of FGF-2 with heparin and its transmembrane receptor.

Probing Fibroblast Growth Factor Dimerization and Role of Heparin-like Glycosaminoglycans in Modulating Dimerization and Signaling

“…Taking advantage of the surface-exposed cysteine residues in the wild-type structure of FGF2, we performed oxidative cross-linking studies to test the proposed symmetrical mode of FGF2 dimerization in Fig. 2C, as this model predicts facile cross-linking between two FGF2 molecules (27). Under mild oxidative conditions, wild-type FGF2 showed very little oligomer formation in the presence and absence of heparin (Fig.…”

“…B, two FGF molecules are oriented in trans to the axis of HLGAG in a "head-to-head" fashion (28). C, four FGF molecules interact both in cis and in trans with HLGAG (27). Note that, for the cis-interaction, the two FGF molecules are symmetrically related, as opposed to the dimer in A.…”

“…However, NMR studies predict a different mode of FGF oligomerization, viz. a symmetrical FGF2 dimer with possible disulfide bond formation between two surface cysteines (27). Furthermore, the recently solved FGF1⅐decasaccharide co-crystal points to an FGF trans-dimer involving no FGF-FGF contacts (28), a mechanism for dimerization that may or may not extend to other members of the FGF family, viz.…”

Structure of fibroblast growth factor 9 shows a symmetric dimer with unique receptor- and heparin-binding interfaces

“…For FGF2, the crystal structure with the highest resolution (PDB code 1bgf) showed disorder for the ®rst 19±20 N-terminal residues (Ago et al, 1991), con®rmed by NMR studies of complete FGF2 (PDB code 1rml), which showed disorder for the N-terminal 28 residues (Moy et al, 1996). For FGF1 the crystal structure with the highest resolution (PDB code 2afg) showed disorder for the N-terminal 9±10 residues (Blaber et al, 1996); for the NMR structure (PDB code 1rml) a molecule N-terminally truncated at residue 25 was used (Lozano et al, 1998).…”

“…Several models have been proposed for the interaction between FGF2±heparin and its receptor Ruoslahti & Yamaguchi, 1991;Spivak-Kroizman et al, 1994;Kan et al, 1993;Guimond et al, 1993;. Previous work utilizing NMR demonstrated that FGF dimers in a symmetric tetramer are formed in the presence of an active heparin decasaccharide (Moy et al, 1997), suggesting that a cis-oriented dimer is the minimal biologically active structural unit of FGF2. Using de®ned heparin fragments and soluble FGF receptors further demonstrated that ligand dimerization can signi®cantly enhance the binding of FGF2 to FGFR1, the dimerization of the receptor and the induction of downstream signal transduction pathways (Safran et al, 2000).…”