Influence of Heparin Mimetics on Assembly of the FGF·FGFR4 Signaling Complex

Fibroblast growth factor (FGF) signaling regulates mammalian development and metabolism, and its dysregulation is implicated in many inherited and acquired diseases, including cancer. Heparan sulfate glycosaminoglycans (HSGAGs) are essential for FGF signaling as they promote FGF·FGF receptor (FGFR) binding and dimerization. Using novel organic synthesis protocols to prepare homogeneously sulfated heparin mimetics (HM), including hexasaccharide (HM6), octasaccharide (HM8), and decasaccharide (HM10), we tested the ability of these HM to support FGF1 and FGF2 signaling through FGFR4. Biological assays show that both HM8 and HM10 are significantly more potent than HM6 in promoting FGF2-mediated FGFR4 signaling. In contrast, all three HM have comparable activity in promoting FGF1·FGFR4 signaling. To understand the molecular basis for these differential activities in FGF1/2·FGFR4 signaling, we used NMR spectroscopy, isothermal titration calorimetry, and size-exclusion chromatography to characterize binding interactions of FGF1/2 with the isolated Ig-domain 2 (D2) of FGFR4 in the presence of HM, and binary interactions of FGFs and D2 with HM. Our data confirm the existence of both a secondary FGF1·FGFR4 interaction site and a direct FGFR4·FGFR4 interaction site thus supporting the formation of the symmetric mode of FGF·FGFR dimerization in solution. Moreover, our results show that the observed higher activity of HM8 relative to HM6 in stimulating FGF2·FGFR4 signaling correlates with the higher affinity of HM8 to bind and dimerize FGF2. Notably FGF2·HM8 exhibits pronounced positive binding cooperativity. Based on our findings we propose a refined symmetric FGF·FGFR dimerization model, which incorporates the differential ability of HM to dimerize FGFs.

Fibroblast growth factor (FGF) signaling regulates mammalian development and metabolism, and its dysregulation is implicated in many inherited and acquired diseases, including cancer. Heparan sulfate glycosaminoglycans (HSGAGs) are essential for FGF signaling as they promote FGF⅐FGF receptor (FGFR) binding and dimerization. Using novel organic synthesis protocols to prepare homogeneously sulfated heparin mimetics (HM), including hexasaccharide (HM 6 ), octasaccharide (HM 8 ), and decasaccharide (HM 10 ), we tested the ability of these HM to support FGF1 and FGF2 signaling through FGFR4. Biological assays show that both HM 8 and HM 10 are significantly more potent than HM 6 in promoting FGF2-mediated FGFR4 signaling. In contrast, all three HM have comparable activity in promoting FGF1⅐FGFR4 signaling. To understand the molecular basis for these differential activities in FGF1/2⅐FGFR4 signaling, we used NMR spectroscopy, isothermal titration calorimetry, and size-exclusion chromatography to characterize binding interactions of FGF1/2 with the isolated Ig-domain 2 (D2) of FGFR4 in the presence of HM, and binary interactions of FGFs and D2 with HM. Our data confirm the existence of both a secondary FGF1⅐FGFR4 interaction site and a direct FGFR4⅐FGFR4 interaction site thus supporting the formation of the symmetric mode of FGF⅐FGFR dimerization in solution. Moreover, our results show that the observed higher activity of HM 8 relative to HM 6 in stimulating FGF2⅐FGFR4 signaling correlates with the higher affinity of HM 8 to bind and dimerize FGF2. Notably FGF2⅐HM 8 exhibits pronounced positive binding cooperativity. Based on our findings we propose a refined symmetric FGF⅐FGFR dimerization model, which incorporates the differential ability of HM to dimerize FGFs.
Four genes in mammalian organisms (FGFR1 (flg), FGFR2 (bek), FGFR3, and FGFR4) code for FGFRs (9). The ectodomain of a prototype FGFR comprises three Ig domains (D1-D3). A unique property of FGFR is the presence of a stretch of acidic residues in the linker connecting D1 to D2, referred to as the "acid box." A wealth of structural and biochemical studies has demonstrated that D2, D3, and D2-D3 linker are necessary and sufficient for FGF binding (8,29). Like FGFs, FGFRs are also HSGAG-binding proteins (30). HSGAG-binding site of FGFR resides in D2 and is composed of residues from g-helix A, ␤ strands B and D, and gA-␤AЈ and ␤AЈ-␤B loops (23,24). D1 and the D1-D2 linker are dispensable for FGF binding and in fact negatively regulate FGFR signaling (31)(32)(33).
Two major alternative splicing events take place in the ectodomains of FGFR1-FGFR3: one involving D1 and the D1-D2 linker, and the other D3 (9,34). Alternative splicing of D1 and the D1-D2 linker serves as a mechanism to modulate receptor autoinhibition, whereas alternative splicing in D3 determines ligand binding specificity (35,36). In FGFR1-FGFR3, the second half of D3 is encoded by two mutually exclusive exons ("b" and "c") that are used in a tissue-specific fashion (37)(38)(39)(40)(41). The "b" exon is used in epithelial tissues, whereas the "c" exon is preferentially used in mesenchymal tissues. As a result of this splicing event, the number of principal FGFRs is increased to seven isoforms namely FGFR1b, FGFR1c, FGFR2b, FGFR2c, FGFR3b, FGFR3c, and FGFR4. Importantly, alternative splicing of D3 sets a specificity barrier such that epithelially expressed FGFs activate FGFRc isoforms, whereas mesenchymally expressed FGFs activate FGFRb isoforms (42). Most FGFs bind and activate more than one of the seven principal FGFRs, although they do not cross the specificity barrier set by the D3 alternative splicing (43). For example, FGF2 exhibits high affinity to both FGFR1c and FGFR2c but does not bind to FGFR1b and FGFR2b. The affinity of FGF2 to FGFR3c and FGFR4 is also negligible (44). FGF1 is an exception, because it is capable of indiscriminately binding both "b" and "c" isoforms of FGFR1-3 and FGFR4 (42). Crystallographic studies of several FGF⅐FGFR complexes have shown that the D3 alternative splicing regulates FGF⅐FGFR binding specificity and promiscuity by modifying the primary sequences of the key ligand binding sites in D3 (14,16).
Genetic analysis of mice and flies deficient in enzymes involved in heparan sulfate (HS) biosynthesis, and cell-based assays using cell lines devoid of HSGAG have established that FGF signaling requires HSGAGs (45)(46)(47). HSGAGs impinges on FGF signaling through multiple mechanism, including promotion of FGF⅐FGFR binding and dimerization (8), control of FGF diffusion, and gradient formation in the extracellular matrix (18,48,49), providing thermal stability and protection from proteolytic degradation (50,51).
Crystallographic analyses of FGF⅐FGFR⅐heparin ternary complexes have provided two conceptually different models by which HSGAG promote FGF⅐FGFR dimerization. According to a symmetric model (Mohammadi model, FGFR1⅐FGF2⅐heparin (decasaccharide), PDB entry 1FQ9) (23) HS promotes formation of a symmetric 2:2:2 FGF⅐FGFR signaling complex. The dimer interface is mediated by protein-protein contacts between the two adjacent FGF⅐FGFR halves and is strengthened by interaction of heparin with FGF and FGFR. The dimer interface comprises direct receptor-receptor contacts mediated by D2, and interactions of FGF from one FGF⅐FGFR half with D2 of FGFR in the second FGF⅐FGFR half (referred to as secondary FGF⅐FGFR interface). At the membrane distal end of the dimer, the individual HS-binding sites of two FGFs and FGFRs unite into one HS-binding canyon into which two HS chains bind. By engaging the HS-binding sites of FGF and FGFRs, HS augments FGF⅐FGFR affinity within each FGF⅐FGFR half as well as promotes the dimer interface (8,52).
In this structure, only the first six sugar units at the nonreducing end of the decasaccharide are in contact with the protein (FGFR or FGF). The remaining four sugar units of one of two decasaccharides are disordered. In the other decasaccharide, two additional sugar units are visible due to the favorable crystal contacts they make with an adjacent FGF molecule. Thus, based on the symmetric model, a hexasaccharide should be sufficient for dimerization and hence biological activity (8,52).
A different model has been proposed by Blundell and coworkers (Blundell model, FGFR2c⅐FGF1⅐HS, PDB entry 1E0O) (24). This model displays a 2:2:1 FGF⅐FGFR⅐HS stoichiometry in which a single HS chain bridges two FGFs in trans and each FGF binds to one FGFR only. In contrast to the symmetric model, there are no direct protein-protein contacts between the two FGF⅐FGFR halves in this model. In other words, the asymmetric dimer is held together solely by the ability of HS to dimerize FGFs, and consequently this mode of dimerization is strictly HS-dependent.
The entire co-crystallized decasaccharide is visible in this structure. Two sugar units at the non-reducing end of oligosaccharide engage the D2 domain of one of the two FGFR chains while the HSGAG-binding site of the other FGFR chain remains unoccupied. Seven sugar rings bridge the two FGFs in trans. Based on this model, the shortest biologically active HS would be an octasaccharide, although maximal activity would require a dodecasaccharide, because it will be long enough such that it could engage D2 of both FGFRs (24). It should be noted, however, that an alternative interpretation of this crystal structure also leads to a model similar to the symmetric one (52,53).
Numerous in vitro and cell-based studies have been attempted to test the salient features of each model. Most data support the physiological relevance of the symmetric dimerization model. For example, mutations that ablate interactions of FGF from one FGF⅐FGFR half to D2 of FGFR in the second FGF⅐FGFR half diminish the ability of the mutated FGF to signal while these mutated FGFs retain the ability to form a 1:1 FGF⅐FGFR complex (54). A further unbiased piece of evidence in support of a symmetric model comes from analysis of the naturally occurring A172F mutation in FGFR2. This gain-offunction mutation, responsible for the Pfeiffer syndrome, maps to the direct FGFR⅐FGFR interface and confers gain-of-function by promoting direct D2-D2 contacts and hence receptor dimerization (54).
Biological studies with size-fractioned heparin oligosaccharides have also been used to test the validity of each model. However, no consensus has been reached with regard to the minimal oligosaccharide length needed for FGF signaling. Some studies show that a hexasaccharide and even smaller sugars are capable of promoting FGF signaling (55)(56)(57)78). In contrast, other data show that a hexasaccharide has either poor or no activity at all and that an octasaccharide is the shortest biologically active heparin (53). In all these prior studies, the heparin oligosaccharides were prepared by either enzymatic hydrolysis or chemical cleavage of heparin isolated from natural sources. Therefore, a potential reason for the disparity between these data could be differences in the homogeneity of oligosaccharide preparations used. Our ability to de novo synthesize homogeneously sulfated heparin oligosaccharides of various degrees of polymerization, including hexasaccharide (HM 6 ), octasaccharide (HM 8 ), and decasaccharide (HM 10 ) (Fig.  1A), provided us with the unique opportunity to revisit the minimum oligosaccharide length requirement for FGF signaling. Using a BaF3 cell line overexpressing a chimeric FGFR4, we show differences in the abilities of these HM to promote FGF1 and FGF2 signaling through FGFR4. All three HM have comparable capacity to stimulate FGF1-FGFR4 signaling. In contrast, in the case of FGF2, a major activity difference in signaling is seen for the transition from HM 6 to HM 8/10 .
To understand the molecular basis for the observed prominent activity differences between HM in promoting FGF1-and FGF2⅐FGFR4 signaling, we used NMR spectroscopy, ITC, and SEC to study the interactions of FGF1 and FGF2 with the isolated D2 of FGFR4 (the HSGAG-binding domain of FGFR4) in the presence and absence of HM 6 and HM 8 . Because HM 8 and HM 10 displayed similar receptor activation level in biological assays, our biophysical studies were restricted to HM 8 . NMR chemical shift mapping, signal attenuation, and T 2 relaxation studies show the existence of a secondary FGF⅐FGFR D2 interaction site as well as direct D2-D2 interaction site corresponding to those seen in 1FQ9 thus providing direct evidence that the symmetric mode of dimerization occurs in solution. Our data show that the two HM make identical sets of contacts with FGF and FGFR D2 in the 2:2 FGF⅐D2 dimers. Therefore, the higher activity of HM 8 relative to HM 6 is not due to the additional contacts of the two extra sugar rings of HM 8 with FGF or FGFR D2. Rather, our data show that differences in the binding affinity of HM for FGFs along with differences in the ability of HM to dimerize FGFs correlate with the differences in the biologic activities of the FGFs. Based on our findings we propose a refined symmetric model that takes into account the differential abilities of HM to dimerize FGF2 in a cooperative fashion.

EXPERIMENTAL PROCEDURES
Cloning, Expression, and Purification of FGF and FGFR-DNA encoding the human FGF1 (Gly 21 -Asp 155 ), FGF2 (Pro 10 -Ser 155 ), and FGFR4 D2 (Asn 138 -Leu 242 ) was amplified by PCR and cloned into Escherichia coli vector pETTEV (N-terminal His tag followed by a tobacco etch virus protease cleavage site) or pET21b (C-terminal His tag) (Novagen). The identity of the clones was verified by sequencing, and the E. coli expression plasmids were transformed into BL21(DE3) CodonPlus-RIPL (Stratagene) cells. Expression and purification of FGF1 (58) and FGF2 (59) was performed as previously described. The successful expression, purification, and refolding of the D2 domain is similar for FGFR1, FGFR2, and FGFR4 with minor modifications (60,61).
NMR Spectroscopy-NMR spectra for all proteins and compounds were recorded at 298 K and were referenced to internal 3-trimethylsilyl-2,2,3,3-tetradeuteropropionate sodium salt (TSP). The experiments were carried out on a Bruker threechannel DRX600 and on a Bruker four-channel DRX800 spectrometer using standard pulse programs. Specific parameters, including buffer conditions and concentrations, are summarized in the figure legends.
ITC-ITC measurements were carried out on a VP-ITC ultrasensitive titration calorimeter (MicroCal LLC) and data analyses were performed as described previously (62).

Synthesis of Heparin
Mimetics-For the synthesis of hexameric HM 6 , we have developed a strategy based on chain elongation from a common disaccharide building block (63). The elongation may then be repeated multiple times until the fully protected hexasaccharide is obtained. As in previous heparin mimetics synthesis, acyl groups were employed to protect the hydroxyl groups to be sulfated, benzyl ethers were used for the free hydroxyl groups, and azido groups masked the amino groups to be sulfated. We have used a similar strategy for the synthesis of oligosaccharides HM 8 and HM 10 , and their preparation will be published elsewhere.
HM 8 and HM 10 Are More Effective Than HM 6 in Promoting FGF2-dependent FGFR4 Dimerization and Signaling-The murine pre-B cell line BaF3 requires interleukin-3 (IL-3) to proliferate. This cell line does not naturally express FGFRs and HSGAG. However, when transfected with FGFRs, BaF3 cells acquire the ability to proliferate in response to exogenous FGF and soluble heparin. Thus, the FGFR-overexpressing BaF3 cell lines are ideal for comparing the biological activity of heparin mimetics to promote FGF⅐FGFR signaling. To compare the efficacy of HM 6 , HM 8 , and HM 10 ( Fig. 1A) in promoting FGF-dependent dimerization of FGFR we established a stable cell line expressing chimeric FGFR4 (Fig. 1, B and C), termed FGFR4-hMpl, in which the ectodomain of FGFR4 was fused to transmembrane and intracellular domain of thrombopoietin receptor (hMpl). Addition of FGF and heparin causes the ectodomain of the FGFR4-hMpl to dimerize, which in turn juxtaposes the intracellular hMp1 regions, leading to subsequent Mpl activation and ultimately evoking a proliferation response. Therefore, cellular proliferation can be used as a readout to compare the capacity of FGFs and/or HM to dimerize the ectodomain of FGFR4.
FGFR4-hMpl-expressing BaF3 cells were deprived of IL-3 and treated with increasing concentrations of FGF1 or FGF2 in the presence or absence of 100 nM of HM 6 , HM 8 , or HM 10 . In the absence of HM, both FGF1 and FGF2 induced proliferation of the FGFR4-hMpl cells with a half-maximal effective concentration (EC 50 ) of 16.6 ng/ml and 11.8 ng/ml, respectively (Fig.  1C). HM 6/8/10 modestly decreased the EC 50 of FGF1. In contrast, HM 8/10 led to a 10-fold decrease in the EC 50 values for FGF2 (Fig. 1C), suggesting that HM 8/10 are more potent than HM 6 in assisting FGF2 to dimerize and activate FGFR4-hMpl.
The observation that FGF1 and FGF2 are able to dimerize FGFR4-hMpl in the absence of HM is intriguing and can only be reconciled by the symmetric FGF⅐FGFR⅐heparin dimerization assembly model (Mohammadi model). In contrast, because the asymmetric mode of dimerization (Blundell model) lacks protein-protein contacts between the two FGF⅐FGFR halves, the ability of FGF1 and FGF2 to dimerize and activate FGFR4-hMpl in the absence of HM cannot be reconciled with this model. On the other hand, the higher efficacy of HM 8/10 compared with HM 6 fits better with the asymmetric model, because according to the symmetric model a hexasaccharide should be sufficient to promote FGF⅐FGFR binding and dimerization.
NMR Studies Indicate a Symmetric FGF⅐FGFR Dimerization Model in Solution-We decided to use NMR spectroscopy techniques to investigate the molecular basis for the higher potency of HM 8 over HM 6 in promoting FGF2⅐FGFR4 dimerization and signaling. Crystal structures of several FGF⅐FGFR complexes have established that both D2 and D3 are needed for FGF binding (8). In contrast to the well resolved Ig domains D1 (64) and D2 (60,65), the D3 domain of all investigated FGFR (FGFR1/2/4) constructs (D1D2D3, D2D3, and D3) lead to line broadening beyond detection in solution. The structural integrity of the investigated D3 domain was confirmed by x-ray analysis (successful crystallization of FGFR2 D2D3⅐FGF1). Therefore, our NMR study focused on the Ig domain 2, the heparin binding domain of the receptor.
We used chemical shift perturbations (CSPs) and changes in the signal intensities of one 15 (58,59) and were used in this study. Consistent with published FGF⅐FGFR x-ray structures, no CSPs in two-dimensional 1 H, 15 Ncorrelation spectra of 15 N-labeled FGF1 or FGF2 were observed upon titration with unlabeled FGFR4 D2. We also prepared uniformly 15 N-labeled D2 and titrated it with unlabeled FGF1 and FGF2. No CSPs were observed in HSQC of 15 N-labeled D2 at protein concentrations of 50 -100 M. These data are consistent with published crystal structures indicating that both D2 and D3 are needed for FGF binding (8).
The crystal structures of FGF⅐ FGFR⅐heparin ternary complex show that D2 mediates binding of FGFR to HM. In both these structures heparin interacts simultaneously with HM binding on D2 of FGFR1 (23) and FGFR2 (24) and the HSGAG binding site of FGFs. This observation suggests that, although D2 alone is not sufficient for high affinity FGF binding by itself, the presence of heparin increases the binding affinity of FGF to D2. Indeed a pervious ITC study has shown that the presence of sucrose octasulfate, a heparin analog, enhances binding of FGFR2 D2 to FGFR1 (60).
To test if HM 6 and HM 8 can promote binding of FGF1 and FGF2 to D2 of FGFR4 we first confirmed that isolated D2 of FGFR4 is capable of binding heparin. Addition of HM 6 and HM 8 to 15 N-labeled FGFR4 D2 led to CSPs in two dimensional 1 H, 15 N-correlation spectra (supplemental Fig. 1). To identify the HSGAG binding residues of D2 we then assigned the NMR backbone resonances of FGFR4 D2 using standard triple resonance NMR experiments on a 13 C, 15 showing CSPs include Lys 158 , Leu 159 , Val 168 , and Lys 169 . These D2 residues are homologous to the heparin-binding residues of FGFR1 and FGFR2 D2 seen in the x-ray structures of ternary complexes (PDB entries 1FQ9 (23) and 1E0O (24)). Interestingly, in addition to CSPs close to the HM binding site, strong CSPs and severe line broadening were observed for D2 residues Ser 141 and Tyr 142 located at the N-terminal tip of D2 in the presence of either HM.
Next, we tested whether HM can support binding of FGF1 and FGF2 to D2. Two complementary titration experiments were performed: In the first experiment, uniformly 15 N-labeled FGFR4 D2 was titrated with a 1:1 mixture of unlabeled FGF with HM 6 or with HM 8 . In the second experiment, 1:1 mixtures of 15 N-labeled FGF and HM were titrated with unlabeled FGFR4 D2. In both these titration experiments, a major reduction in signal intensity of backbone resonances of the 15 N-labeled component (FGF1, FGF2, or D2) was observed suggesting that indeed, in the presence of HM, D2 is capable of binding FGF. Analysis of the line width of individual backbone amide resonances of D2 and FGF allowed us to identify residues in FGF and D2 that are involved in protein-protein and protein-HM interactions (supplemental Table I).
No structure of FGFR4 D2 has been reported so far. However, the sequence identity between the extracellular domains of FGFR1, FGFR2, and FGFR4 is very high (over 50%). Therefore, NMR results on FGFR4 D2 can be analyzed using the available ternary complex structures of FGFR1 and FGFR2. The regions in FGF1 and D2 that are affected in the FGF1⅐D2⅐HM 8 titration experiment are mapped onto both x-ray structures (the symmetric 2:2:2 complex 1FQ9 (Fig. 2) and the asymmetric 2:2:1 complex 1E0O (Fig. 3)). FGFR4 D2 residues that undergo signal attenuation upon titration with an unlabeled FGF1⅐HM 8 mixture include the predicted HSGAG-binding site of FGFR4 D2 and the predicted primary ligand binding site of D2. These two regions are identical between the two crystallographic models. Interestingly, other affected D2 residues include Gly 165 , Asn 166 , Thr 167 , and Glu 212 and correspond to the D2 regions that are expected to mediate the direct D2-D2 interface (Figs. 2 and 3, cyan circles) and the secondary FGF⅐FGFR D2 interface (Figs. 2 and 3, yellow circles) observed in the symmetric model. The affected residues are located at the interface between FGF, FGFR, and HM 8 . In addition to the FGF⅐FGFR interaction sites within the heteromeric half complexes (FGFR⅐FGF or FGFRЈ⅐FGFЈ), also interactions between the heteromeric half complexes (FGFR⅐FGFRЈ (cyan circles); FGFR⅐FGFЈ and FGFRЈ⅐FGF (yellow circles)) are detected. Orange circles indicate the receptor-receptor-contacts only seen in the asymmetric x-ray structure (Fig. 3). B, NMR mapping of 15 N FGF1 amide signals with strong line broadening observed upon addition of unlabeled FGFR4 D2 in the presence of HM 8 . The interaction sites between FGF and HM 8 as well as the binding surface to FGFR and FGFRЈ are affected. The unique FGF-heparin interaction of the asymmetric x-ray structure is depicted as a dark blue circle.  Fig. 2) but do not fit to the depicted complex are marked with circles: yellow and cyan circles show contacts in 1FQ9 between an FGF and a receptor molecule and between two receptor molecules, respectively. Dark blue circles mark contacts between heparin and FGF, and orange circles between the two receptor molecules present in 1E0O that could not be confirmed by CSP data.
Residues in FGF1 that undergo signal attenuation upon titration with unlabeled D2 correspond to the FGF1 HSGAGbinding site and primary receptor binding site (supplemental Table I). Interestingly, in addition CSPs and/or signal attenuation were observed for the residues mapping to the region on FGF1 that is predicted to mediate the secondary FGF⅐FGFR D2 interface (Figs. 2 and 3, yellow circles) based on the symmetric dimerization model. This secondary FGF⅐D2 interaction site was also described by Kochoyan and co-workers for a complex between FGFR1 D2 and FGF1 in the absence of heparin (65). Notably, the NMR data show that HM 6 and HM 8 generally induce perturbations and signal attenuation of the same residues in FGFR4 D2 and in FGF1 and FGF2 (FGFR4 D2⅐FGF2⅐HM 6/8 mapped on the 1FQ9 x-ray structure (supplemental Figs. 2 and 3)). We therefore conclude that NMR data show the symmetric dimerization model in solution.
Strong line broadening of D2 and FGF residues indicates that the FGF⅐D2⅐HM complex is highly dynamic with exchange rates between different oligomerization states on the microsecond to millisecond timescale. Thus, we carried out T 2 NMR relaxation measurements to gain more insights into the dynamics and stoichiometry of the FGF⅐D2⅐HM complexes. T 2 -derived apparent molecular weights of FGF1/2 and FGFR4 D2 in the absence and presence of either HM 6 or HM 8 are summarized in Table 1. Consistent with the results of NMR titration experiments described above in the absence of HM, the predicted molecular weights for monomeric FGF1, FGF2, and FGFR4 D2 were observed. In the presence of HM, however, higher molecular weight species were detected ( Table 2). The highest molecular weights were observed for FGF1 and FGFR4 D2 in the presence of HM 8 . The measured apparent molecular mass of FGF1 increased from 21 to 44 kDa for 15 N-FGF1 in complex with unlabeled FGFR4 D2 and HM 8 . At the same time, the apparent molecular mass of FGFR4 D2 increased from 21 to 37 kDa for 15 N-FGFR4 D2 in the complex. For a stable 2:2 dimeric FGF⅐D2 complex, values of ϳ60 kDa would have been expected for both complexes. Therefore, smaller apparent molecular masses together with different apparent molecular masses for FGF and FGFR4 D2 suggest that both proteins are in a dynamic equilibrium between the free form and higher molecular mass complexes with a stoichiometry greater than 1:1:1 FGF⅐D2⅐HM (theoretical molecular mass of 32 kDa) but smaller than 2:2:2 in solution at concentrations of 50 M. The increase in T 2 -derived apparent molecular weights was greater for FGF1 than for FGF2, and greater with HM 8 than with HM 6 indicating that the equilibrium between ternary complex and lower molecular weight species in solution is shifted prominently toward the ternary complex for FGF1 versus FGF2 and for HM 8 versus HM 6 .
We also studied the interaction of FGFR4 D2 with FGF1 and FGF2 in the absence and presence of HM using ITC. In the absence of HM, titrations of FGFR4 D2 into FGF1 or FGF2 resembled control titrations of FGFR4 D2 into buffer (supplemental Fig. 4). Titration of D2 into a 1:1 mixture of FGF1⅐HM 6 and FGF2⅐HM 6 also generated no heat signal. In contrast, titration of FGFR4 D2 into a 1:1 mixture of FGF1⅐HM 8

TABLE 1 Thermodynamic parameters for the interactions measured by ITC
Binding parameters were obtained from a fit of the calorimetric data to a single site binding model. Where applicable, the standard error from 2-3 measurements is indicated. All titrations were performed in 50 mM NaH 2 PO 4 / Na 2 HPO 4 , 100 mM NaCl, 25 mM arginine, pH 6.8, T ϭ 25°C. Protein concentrations in the sample cell, 5-10 M; ligand concentrations in the syringe, 50 -100 M; final ratios of ligand over sample protein was 2-3.   15 N-correlation spectra. AUGUST 20, 2010 • VOLUME 285 • NUMBER 34

FGF⅐FGFR⅐Heparin Interaction
and FGF2⅐HM 8 showed a binding event with a K D of ϳ1 M and a stoichiometry near unity (Table 1 and supplemental Fig. 4). These data indicate that HM 8 forms a ternary complex with FGF and D2, whereas complexes mediated by HM 6 are not stable at these protein concentrations (10 M). The prominent dilution heat signal that was observed in the titrations of FGFR4 D2 into buffer possibly indicates dissociation of protein oligomers; a tendency for self-association of FGFR4 D2 was also observed by NMR spectroscopy (discussed below). Taken together, NMR and ITC experiments indicate differences between HM 6 and HM 8 in their ability to promote FGF⅐D2 complex formation and dimerization. However, NMR CSP and signal attenuation studies show HM 6 and HM 8 generally induce effects on the same residues in FGFR4 D2 and in FGF1 and FGF2 suggesting that the greater efficacy of HM 8 relative to HM 6 to promote dimerization cannot be explained by additional contacts of two extra sugar units of HM 8 with FGFR D2 or FGF. These findings imply that differences in the binary interactions of HM with FGF and/or FGFR D2 may underlie the observed differences in the biological potency of these two HM.
HM Display Major Differences in Their Ability to Dimerize FGFs-It has been reported previously that heparin can induce dimerization/higher order oligomerization of FGF1 or FGF2 (66,67). Therefore, we compared the binding affinities of HM for FGF1 and FGF2 and the abilities of HM to induce oligomerization of FGF1/2 using NMR spectroscopy. As expected, titration of HM 6 and HM 8 to both 15 N-labeled FGF1 and FGF2 (NMR backbone assignments (58, 59)) caused strong CSPs and line broadening of the amino acid residues comprising the HSGAG-binding site of the ligands (supplemental Fig. 5 and Table II). Compared with HM 6 , HM 8 induced stronger CSPs and signal attenuation of 15 N-labeled FGF1 and FGF2 than HM 6 in agreement with higher affinity of HM 8 over HM 6 to FGF1 and FGF2.
Next we used NMR 1 H T 2 relaxation measurements to study HM-induced FGF dimerization. The 1 H T 2 -derived apparent molecular weights of the FGF⅐HM complexes are given in supplemental Table III. The data show that both HM 6 and HM 8 are capable of dimerizing FGF. However, the smaller apparent molecular weight of the FGF dimer in the presence of HM 6 pointed to dynamic monomer-dimer equilibrium on the NMR timescale. The dynamic equilibrium in the case of HM 6 was also confirmed by size exclusion chromatography (SEC) (supplemental Fig. 6 and Table IV). In the presence of HM 6 , FGF eluted at a retention time midway between the expected retention times for monomeric and dimeric FGF. In contrast, upon addition of HM 8 , the FGF2 peak shifted to a retention volume, which correspond to the FGF2 dimer (supplemental Table IV).
Stepwise titration of HM to either FGF followed by NMR 1 H T 2 relaxation measurements allowed us to further elucidate the dimerization mechanism. The T 2 -derived apparent molecular weight as a function of the HM:FGF ratio was measured (Fig.  4E). Maximum dimerization was achieved at a HM:FGF2 ratio of 0.5. This finding indicates an HM:FGF2 stoichiometry of 1:2, suggesting a two-step binding model (Fig. 4F). At ratios higher than 0.5, the apparent molecular weight of FGF2 decreased again, in line with a partial dissociation of the HM⅐FGF dimer in the presence of an excess of HM. Although dimer association at low HM:FGF ratios was similar for both HM, dimer dissociation was more pronounced for HM 6 than for HM 8 .
Theoretical fitting of the HM 6 titration into FGF2 resulted in a two-step binding model with similar K D values for the two binding events (Fig. 4E, open triangles and black line; K D1 ϭ 160 nM, K D2 ϭ 120 nM). In contrast, the HM 8 ⅐FGF2 interaction showed a pronounced positive cooperativity (Fig. 4E, filled triangles and black line; K D1 ϭ 100 nM and K D2 ϭ 5.8 nM). Taken together, the NMR data suggest that for the HM 8 ⅐FGF2 interaction, the recruitment of the second FGF2 to form the dimeric complex HM 8 ⅐(FGF2) 2 occurs with higher affinity than the first step indicating positive cooperativity of binding, in agreement with previous reports (68).
The ability of HM to dimerize FGFs was also compared using ITC at 10-fold lower protein concentration than in the NMR 1 H T 2 relaxation measurements (Fig. 4, C and D). Both HM bound to FGF with sub-micromolar affinities (Table 1). Within experimental error, HM 6 exhibited similar affinity for FGF1 and FGF2. Compared with HM 6 , HM 8 bound 2-fold tighter to FGF1. In the case of FGF2 the difference in binding affinities between the HM was substantially larger: HM 8 bound with 15-fold higher affinity than HM 6 (54 versus 843 nM) to FGF2.
We also used ITC to compare the efficacy of HM to dimerize FGF1 and FGF2. ITC experiments showed that HM 6 bound to FGF with a 1:1 binding stoichiometry, whereas a stoichiometry of 0.5:1 was observed reproducibly for HM 8 ⅐FGF binding (Table  1). This finding indicates that one molecule of HM 8 bound two FGF molecules at half-saturation, whereas HM 6 bound only one FGF molecule. However, we cannot exclude the possibility that HM 6 also induces the formation of a transient dimer, which dissociates upon titration of excess ligand; this scenario would require two comparable K D values, i.e. no or only weak cooperative binding. HM 8 displayed a binding enthalpy ⌬H that was on average 2-fold higher than the binding enthalpy of HM 6 . This feature is indicative of positive cooperativity and is in agreement with NMR data (described above). Apparently, with HM 8 , the binding process of two FGF molecules titrated simultaneously, resulted in an additive binding enthalpy ⌬H1 ϩ ⌬H2. However, the symmetric sigmoid-shaped titration curves did not allow deconvolution of two separate binding events with a two-site binding model. Therefore, only apparent macroscopic K D values could be determined (Table 1).
Finally, the interaction of FGF and HM was also monitored using differential scanning fluorometry. Both HM 6 and HM 8 increased the melting temperature of FGF1 and FGF2, (supplemental Fig. 5 and Table V). HM 6 had a similar effect on both FGFs, leading to an increased melting temperature T m ϭ 54.29°C (FGF1, T m ϭ 42.55°C) and T m ϭ 53.03°C (FGF2, T m ϭ 46.48°C). HM 8 led to a small increase of the melting temperature in the case of FGF1 (T m ϭ 57.99°C). However, a much stronger shift to higher T m values was observed for HM 8 with FGF2 (T m ϭ 68.89°C). These observations support the finding that HM 8 has a higher affinity especially toward FGF2. Taken together, our NMR, ITC, and SEC data consistently show that both HM can dimerize FGF, but dimerization is more efficient for HM 8 compared with HM 6 .

FGF Interacts with All HM Units in a Degenerate and Dynamic
Fashion-Based on the published FGF-heparin crystal structures FGF binds to an internal trisaccharide binding motif (21,22). Thus we decided to study binding of HM to both FGF1 and FGF2 by monitoring the anomeric proton resonances of HM in 1 H one-dimensional NMR spectra (Fig. 4, A and B). Conveniently, the anomeric protons of the oligosaccharides fall in the region between 5.0 and 5.5 ppm where no protein signals occur. At an HM:FGF ratio of 1:1, the anomeric resonances of all sugar units of HM 6 and HM 8 become broadened beyond detection indicating the presence of multiple bound conformations, which interchange on the microsecond to millisecond timescale. No difference could be detected in the line broadening between the terminal and non-terminal sugar units. Based on the FGF-heparin crystal structures we would have expected to observe differences in line broadening between the internal trisaccharide binding motif and the terminal units. The lack of difference in the extent of line broadening between the terminal and internal sugar units indicate that all sugar units participate in ligand binding. Thus, based on our NMR data, FGF interacts in a degenerate fashion with all sugar units of HM with exchange of the units between the free and different protein-bound states on a time scale of microseconds to milliseconds.
Mathematical formulation of the degeneracy in the FGF2⅐HM interaction shows that an increased degeneracy of FGF binding sites for HM 8 relative to HM 6 alone is not sufficient to explain the differences in the affinity of HM 6 and HM 8 for FGF2. Based on a trisaccharide binding motif (see below), HM 8 offers three degenerate binding sites compared with only two binding sites on HM 6 . Supplemental Derivation I demonstrates that the increased number of FGF binding sites results for HM 8 in a higher affinity for both binding events (1.5-  HM⅐FGF2 interaction (A and B). Anomeric regions of the one-dimensional 1 H NMR spectra of HM 6 (A) and HM 8 (B) in the absence (bottom) and presence (top) of FGF2 are shown. Spectra were recorded with 50 M HM and 50 M FGF2 in 50 mM NaH 2 PO 4 /Na 2 HPO 4 buffer, pH 6.8, 100 mM NaCl. C and D, ITC titrations of FGF with heparin mimetics. C, sample titration of FGF2 with HM 8 . Top panel: raw heating power data; the first peak represents a small pre-injection (5 l) that is omitted in the integrated data. Bottom panel: data after peak integration and concentration normalization. Curve fit of the data to a single site binding model.  Table 2. E, HM-induced FGF2 dimerization determined by NMR 1 H T 2 relaxation. Apparent molecular weight of FGF2 upon titration with HM 6 (open triangles) and HM 8 (filled triangles) determined from 1 H T 2 measurements of the bulk imino protons at 30 M FGF2 concentration. Equilibrium concentrations and apparent molecular masses were fitted assuming the two-step mechanism depicted in F as a model. A least square fit to the experimental data was performed by variation of K D1 and K D2 . The fitted theoretical masses are depicted as solid lines. For HM 6 , K D1 ϭ 160 nM and K D2 ϭ 120 nM were found, for HM 8 , K D1 ϭ 100 nM and K D2 ϭ 5.8 nM. F, the assumed two-step interaction scheme of FGF2 (gray) with HM (black) with binding constants K D1 and K D2 . fold for binding of the first FGF; 1.3-fold for the second FGF). This theoretical formulation agrees well with the experimental data for the first HM⅐FGF interaction. However, for the second binding event, a 20-fold difference in affinity is observed experimentally for HM 8 compared with HM 6 . In addition, binding site degeneracy alone results in a lower affinity for the second FGF⅐HM interaction compared with the first one (HM 6 : 4-fold higher affinity; HM 8 : 4.5-fold) and therefore cannot explain the observed positive cooperativity.
Interaction of Heparin Mimetics with FGFR4 D2-We also studied the ability of HM to induce dimerization/oligomerization of FGFR4 D2. T 2 experiments indicate an increase in apparent molecular mass from 21 Ϯ 3 kDa in the absence of HM 8 to 28 Ϯ 7 kDa in the presence of HM 8 (expected monomeric molecular mass for FGFR4 D2: 15 kDa). These findings provide structural evidence for a FGF-independent, HM-induced dimerization of FGFR4, which was previously indicated by biological data (69).
Binding of HM to FGFR4 D2 was also measured by ITC (supplemental Fig. 8). For both HM, similar affinities in the low micromolar range (HM 6 : 2.4 M, HM 8 : 3.4 M) and stoichiometries near unity were obtained (Table 1). In a previous study, similar affinities of 0.3-0.4 M have been reported for the HM⅐FGFR4 interaction (70,71). Lower stoichiometries for HM 8 than for HM 6 (Table 1) possibly indicate a partial HM 8induced dimerization as observed by SEC (supplemental Table IV) and NMR. Binding thermodynamics differed strongly for the two HM. HM 8 had a much larger binding enthalpy compared with HM 6 (Ϫ9.3 versus Ϫ1.2 kcal/mol). In contrast, binding of D2 to HM 6 was primarily entropy-driven, indicating a different binding mode for HM 6 as compared with HM 8 . It has been reported previously that a heparin octamer constitutes the minimal chain length for stable HM binding to FGFR4 (70). The deviating binding thermodynamics observed with HM 6 could therefore point to an alternative, possibly nonspecific binding mode. Taken together, our data show that HM 6 and HM 8 differ greatly in their affinity for FGF1/2 and have different ability to dimerize FGFs, whereas both HM exhibit comparable affinities for D2 and have poor ability to dimerize FGFR4 D2.

DISCUSSION
Using organic chemistry methods we were able to de novo synthesize chemically pure sulfated heparin hexasaccharide (HM 6 ), octasaccharide (HM 8 ), and decasaccharide (HM 10 ). We showed that HM 8 is significantly more potent than HM 6 , while HM 10 displays similar potency as HM 8 in promoting FGF2mediated FGFR4 signaling. Interestingly, the prominent effect in biological activity of the octasaccharide relies on its interaction with FGF2, because FGF1⅐HM 6/8/10 activation only resulted in relatively small changes in FGFR4 signaling.
To delineate the underlying molecular basis for higher efficacy of HM 8 relative to HM 6 we used NMR, ITC, and SEC to characterize binding interactions of FGF1 and FGF2 with isolated Ig-domain 2 (D2) of FGFR4 in the presence of heparin mimetics, and binary interactions of FGFs and D2 with HM. Our NMR data in solution support the symmetric dimerization model proposed by Mohammadi and colleagues (23) for HM 6 and HM 8 in contrast to the conclusion of Goodger and coworkers (53) who proposed an asymmetric ternary complex formation (24) for octasaccharides and larger heparin fragments. Our observed symmetric complex in solution is transient, and HM 8 (compared with HM 6 ) shifts the equilibrium more strongly toward the ternary complex for both FGF1 and FGF2. Importantly, the NMR data show that the higher efficacy of HM 8 relative to HM 6 is not due to the ability of HM 8 to make additional contacts with FGF or D2.
Analysis of the binary interaction of HM with the D2 domain of FGFR4, which harbors the heparin binding site, shows that HM 6 and HM 8 bind D2 with ϳ1 M K D , in agreement with previous reports (70,71). Therefore, the differences in affinity appear too small to account for the observed differences in potency between HM 6 and HM 8 . A tendency of HM 8 to dimerize the receptor already in the absence of FGF could be observed, an interesting finding in light of the reported heparin sensitivity of FGFR4 (69).
In contrast to the findings of Goodger and coworkers (53) that an octasaccharide is the shortest heparin fragment to form a 2:1 FGF⅐heparin complex, in our study both HM 6 and HM 8 were able to dimerize FGF1 and FGF2, above a protein concentration of 50 M. NMR-based epitope mapping revealed closely overlapping binding sites of HM 6 and HM 8 on FGF1 and FGF2. The observed binding epitopes correspond to the heparin binding sites observed in the published crystallographic studies (21,22). Importantly, FGF2 dimerization upon HM 8 addition reveals strong positive cooperativity, in agreement with previous studies on both FGF1 (68) and FGF2 (53). Furthermore, HM 8 has a stronger propensity to dimerize FGF than HM 6 and binds with 15-fold higher affinity to FGF2. By contrast, for FGF1 the affinity for HM 8 is only 2-fold higher than for HM 6 .
There are two potential mechanisms that could account for the differences in affinities of HM 6 and HM 8 toward FGFs. Firstly, the affinity can be increased due to a degeneracy effect observed in multivalent interactions. Secondly, the interactions of HM with both FGF and FGFR are highly dynamic, as observed by NMR line broadening experiments. Our findings are in line with recent surface plasmon resonance data (72), where rapid association and dissociation kinetics for the FGF⅐HM complex and rapid dissociation for the receptor⅐HM complex were demonstrated. As the oligosaccharides offer multiple binding motifs for FGF, the fast dynamics result in multivalent binding. As a consequence, for both HM 6 and HM 8 all sugar units are involved in the interaction with at least one FGF molecule in agreement with a previous NMR study (73). The presence of multiple, overlapping binding sites in the longer oligosaccharide could result in apparent higher FGF binding affinities (57). However, a mathematical formulation of the influence of multivalency on the affinities of the hexameric versus octameric oligosaccharide (supplemental Derivation I) shows that the degeneracy effect can explain neither the observed cooperativity nor the difference between FGF1 and FGF2. Rather, we detect strong cooperativity for the second binding event only in the case of FGF2 with HM 8 .
To gain insights into the molecular basis of the observed cooperativity in FGF2⅐HM 8 binding on the basis of the reported structures, we analyzed the interactions of FGF1 and FGF2 with heparin oligosaccharides in the published crystal structures (PDB-entries 2AXM, 1E0O, 1FQ9, 1BFB, and 1BFC). Striking differences between the binding modes of FGF1 and FGF2 to heparin oligosaccharides were observed. Based on the structures of FGF1 in complex with a decasaccharide (22) (PDB entry 2AXM) or FGFR2 D2D3-decasaccharide (24) (PDB entry 1E0O), FGF1 interacts with the HM sulfate groups of the sequence GlcN-IdoA-GlcN (GIG) (supplemental Fig. 9, left). In contrast to FGF1, the available crystal structures of FGF2 in complex with heparin differ in their sugar interaction pattern: in the FGF2⅐FGFR1c-decasaccaride structure (1FQ9) FGF2 binds to a IdoA-GlcN-IdoA (IGI) motif, whereas, in the FGF2tetra/hexasaccharide structure (1BFB and 1BFC (21)), FGF2 interacts with a IdoA-GlcN (IG) motif. Based on our NMR CSP mapping data (Gly 28 , see supplemental Table I) we suggest that The sulfate group at the non-reducing end of HM, which is required for interaction with the receptor, is depicted in red. A, the first association of FGF2 to the HM 6 (I) is possible in two ways for HM 6 (IIa and IIb). The following dimerization step leads to one unique species (III). Direct binding of this complex to the receptor (IVa) is not possible for sterical reasons. The 1:1:1 FGF2⅐FGFR⅐HM complex (IVb) can either be formed by interaction of the FGF2⅐HM 6 complex IIb with FGFR or in a concerted step of binding of (FGF2) 2 ⅐HM 6 to the receptor and simultaneously dissociation of the second FGF. B, HM 8 (I) can form three different 1:1 FGF⅐heparin complexes (IIa, IIb, and IIc). The dimerization step can lead to two different FGF2 dimer forms (IIIa and IIIb). The favored (FGF2) 2 ⅐HM 8 complex (IIIb) can then form a 2:1:1 FGF2⅐FGFR⅐HM 8 complex (IVb) or dimerize to a 4:2:2 FGF2⅐FGFR⅐HM 8 complex. Alternatively, the ternary 1:1:1 complex (V) formed from the interaction of FGFR⅐FGF2⅐HM 8 complex (IIb) with FGFR can also result to a signaling competent assembly (2:2:2).
Different symmetries are observed for the interactions of FGF1 and FGF2 with HM in the ternary complexes. The conformations of the sugars bound to FGF1 and FGF2 are therefore also different (supplemental Fig. 9). The affinity for the first binding event of HM 8 is similar for FGF1 and FGF2. Because the structure of free HM (PDB entry 1HPN) differs from the conformation of bound HM, the first binding event of both FGF1 and FGF2 must change the conformation of the HM. In structural terms, positive cooperativity is consistent with the reported data that binding of the first FGF induces a kink (74) in the structure of HM, which then, together with entropic factors, facilitates binding of a second FGF. However, the affinity of the second binding event for FGF1 is in the same K D range as the first step. Therefore, there is no indication for a difference in the binding mode for the first and second step for FGF1. In stark contrast, for FGF2 the second step affinity is 17-fold stronger than the first association of FGF2 to HM 8 . Thus, according to our data, the strong ability of HM 8 to bind and dimerize FGF2 molecules in a cooperative fashion correlates with the higher efficacy of HM 8 relative to HM 6 to promote FGF2 signaling. Recent studies of heparin dendrimers (75) induced FGF-oligomers, synthetically polymerized FGFs (76), and covalently cross-linked FGF-dimers (77) suggest that oligomerization of FGF is required for an agonistic effect of HM on FGFR signaling. Therefore, the oligomerization of FGF in the presence of longer HM such as HM 6 and HM 8 needs to be considered in the formation of FGF⅐FGFR cell surface signaling assembly. Based on our findings we propose a refined symmetric FGF⅐FGFR dimerization model (Fig. 5), which incorporates the differential ability of HM to dimerize FGFs and the presence of a symmetric 2:2:2 ternary complex in solution (Fig. 5, A and B).
The (FGF2) 2 ⅐HM dimer is structurally not part of the symmetric ternary crystal complex, which therefore implies a dissociation of the (FGF2) 2 ⅐HM for the assembly of the ternary crystal complex. Due to the cooperativity of the FGF2⅐HM 8 dimerization, it is unlikely that a FGF2⅐HM 8 (1:1) monomer interacts with the receptor. This discrepancy is addressed in Assuming that FGF2 binds to an IGI motif (supplemental Fig. 9), the initial 1:1 FGF2⅐HM 6 complex can form through two alternative ways for HM 6 (Fig. 5A, IIa and IIb). Binding of a second FGF molecule leads to one unique dimeric species (Fig. 5A, III). This dimeric species cannot directly bind to the receptor because of potential steric clashes between FGF2 and the receptor (Fig. 5A, IVa, Fig. 6A). Therefore, the 1:1:1 FGF2⅐FGFR⅐HM 6 complex (Fig. 5A, IVb) can only be formed when the binding of (FGF2) 2 ⅐HM 6 to the receptor, and dissociation of the second FGF2 are synchronous or from the FGF2⅐HM 6 complex with a single FGF2 bound to HM 6 directly. In contrast to HM 6 , HM 8 can form three different 1:1 FGF2⅐HM 8 complexes (Fig. 5B, IIa, IIb, and IIc). Presuming the conformation of the oligosaccharide in the symmetric ternary complex, the dimerization step can either lead to an FGF dimer in analogy to the HM 6 complex (Fig. 5B, IIIa), or the FGF molecules bind one to the middle binding site and one to the reducing end site of the saccharide (Fig. 5B, IIIb). The cis-binding mode where both FGF2s are at the outer binding sites can be excluded, because it would lead to direct FGF2⅐FGF2 contacts that are not observed in two-dimensional NMR experiments. The dimerization step has a significantly higher affinity compared with the first FGF2 association step for HM 8 . Because HM 6 is chemically contained within HM 8 , it can be assumed that the 2:1 FGF2⅐HM 8 (Fig. 5B, IIIa) has a similar structure and is equally stable as the corresponding complex with HM 6 (Fig.  5A, III). Therefore, the high cooperativity upon HM 8 -induced dimerization is likely a result of complex formation involving the additional sugar units of HM 8 at the reducing end (Fig. 5B,  IIIb). We speculate that cooperativity is mediated by a different binding mode of the second FGF2 binding to HM 8 leading to a GIG binding motif shown in IIIb to be the favored dimer. The crystal structure of FGF2⅐FGFR1⅐decasaccharide (1FQ9) shows a GIG binding motif for the second FGF2 at the reducing end of decasaccharide (23). The fact that eight sugars are necessary for the biological effect further supports that the binding mode  A and B, FGF2 modeled to the 1:1:1 FGF2⅐FGFR1 D2D3⅐HM 8 heteromeric half-complex of the crystal structure. FGF2 is depicted as green schematics, HM in sticks. FGFR1 D2D3 is symbolized by a blue surface. A, modeled structure according to the HM 6 like (FGF2) 2 ⅐HM complex (Fig. 5, A and B, IVa). FGFR1 D2 and FGF2 overlap sterically. This complex cannot be formed. B, modeled structure with the second possible (FGF2) 2 ⅐HM 8 binding mode (Fig. 5B, IVb). No sterical clashes indicate that this complex is a possible intermediate of ternary complex formation. C, modeled ternary complex of a 4:2:2 FGF⅐FGFR⅐HM stoichiometry according to the heteromeric halfcomplexes depicted in B. This proposed complex is sterically possible as an intermediate assembly state for FGF-induced FGFR signaling.
of the formation of IIIb is according to a GIG motif. Additionally, the contacts of HM to the second FGF2 are formed by three sulfate groups (GIG) instead of two sulfates and a carboxyl group for the IGI motif, which could also explain higher affinity for the second FGF2 binding. The direct binding of the (FGF2) 2 ⅐HM 8 (Fig. 5B, IIIa) complex in analogy to the (FGF2) 2 ⅐HM 6 (Fig. 5A, III) complex to the receptor would also lead to the same steric clashes as for HM 6 (Fig. 5A, IVa, Fig. 6A). The favored (FGF2) 2 ⅐HM 8 complex, however, can form a 2:1:1 FGF2⅐FGFR⅐HM 8 complex (Fig. 5B, IVb) as an intermediate state. The ability of the (FGF2) 2 ⅐HM dimer to interact directly with FGFR constitutes the key difference between the octameric as compared with the hexameric HM. The formation of a symmetric ternary assembly of stoichiometry 2:2:2 in analogy to PDB entry 1FQ9 would require the dissociation of one FGF2 and subsequent dimerization of two 1:1:1 complexes. Alternatively, we propose that the resulting complex could dimerize directly to a 4:2:2 FGF2⅐FGFR⅐HM 8 complex. As depicted in Fig. 6B and 6C, the proposed complex is sterically possible. This mechanism could explain the increased potency of HM 8 versus HM 6 based on a symmetric complex, because HM-induced dimerization is one of its steps, and therefore this step can also be rate-limiting. On the basis of our model, it remains unclear if the 4:2:2 complex induces signaling directly or if it constitutes an intermediate state.
Our biophysical studies provide a detailed view on the interaction of FGF, FGF receptors, and heparin. The striking biological observation that oligosaccharide length and specificity toward FGF2 translates into differences in signaling is based on unique dimerization properties of the FGF2⅐HM 8 complex due to both multivalent, dynamic interaction of FGF with HM and the positive cooperativity only observed for FGF2⅐HM 8 . Given the fact that there is significant interest in pharmaceutical development of mimetics of HSGAG as potential drugs to modulate FGF signaling, our data should facilitate the rational design of heparin mimetics as agonists of FGF2⅐FGFR signaling.