Cooperative heparin-mediated oligomerization of fibroblast growth factor-1 (FGF1) precedes recruitment of FGFR2 to ternary complexes

Abstract

Fibroblast growth factors (FGFs) utilize cell surface heparan sulfate as a coreceptor in the assembly of signaling complexes with FGF-receptors on the plasma membrane. Here we undertake a complete thermodynamic characterization of the assembly of the FGF signaling complex using isothermal titration calorimetry. Heparin fragments of defined length are used as chemical analogs of the sulfated domains of heparan sulfate and examined for their ability to oligomerize FGF1. Binding is modeled using the McGhee-von Hippel formalism for the cooperative binding of ligands to a monodimensional lattice. Oligomerization of FGFs on heparin is shown to be mediated by positive cooperativity (α = 6). Heparin octasaccharide is the shortest length capable of dimerizing FGF1 and on longer heparin chains FGF1 binds with a minimal footprint of 4.2 saccharide units. The thermodynamics and stoichiometry of the ternary complex suggest that in solution FGF1 binds to heparin in a trans-dimeric manner before FGFR recruitment.

Figure 1

The interaction between FGF1, FGFR, and heparin is structurally well characterized with structures of (a) FGF1 bound to heparin hexasaccharide (PDB: 2ERM); (b) FGF1 dimerized on heparin decasaccharide (PDB: 1AXM); (c) the asymmetric FGF1:FGFR2c:heparin complex with 2:2:1 stoichiometry (PDB: 1E0O); and (d) a symmetric 2:2:2 FGF2:FGFR1c:heparin complex (PDB: 1FQ9). In images a–c, one FGF1 molecule and the heparin are in the same orientation. FGF is shown in cartoon representation and heparin in stick representation.

Figure 2

Analysis of FGF1-heparin hexasaccharide interaction by direct (a) and reverse (b) titrations. In the direct titration, 100 μM FGF1 was titrated into 7 μM hexasaccharide and in the reverse titration, 100 μM heparin was titrated into 12.5 μM FGF1. (Upper panel) Calorimetric titration trace with the integrated isotherms (shown in lower panel). (Solid lines) Best fit to the noncooperative McGhee-von Hippel model. (c) Thermodynamic dissection of the interaction between FGF1 and heparin hexasaccharide. (Shading) Free energy of binding (ΔG); (crosshatch) enthalpy of binding (ΔH); (diagonal shading) entropy of binding (−TΔS).

Figure 3

Analysis of FGF1-heparin octasaccharide interaction by direct (a) and reverse (b) titrations. In the direct titration, 100 μM FGF1 was titrated into 7 μM octasaccharide and in the reverse titration, 100 μM octasaccharide was titrated into 12.5 μM FGF1. (Upper panel) Calorimetric titration trace with the integrated isotherms (shown in lower panel). (Solid lines) Best fit to the cooperative McGhee-von Hippel model.

Figure 4

ITC analysis of FGF1-heparin 16-mer by direct (a) and reverse (b) titrations. A trace of calorimetric titration (upper panel) and integrated isotherms (lower panel). Solid lines represent the best fit to the cooperative McGhee-von Hippel model. (c) The evolution of the number of ligand molecules bound, ν, are shown as the summation of those bound in an isolated state (ν_iso); those bound in a singly contiguous state (ν_sc) and those in a doubly-contiguous state (ν_dc) are plotted as a function of the molar ratio for the direct titration of FGF1 to heparin 16-mer. (Inset) A theoretical model of four FGF molecules bound to heparin 16-mer showing two linear arrays in which doubly-contiguous molecules are in cis and trans conformations.

Figure 5

ITC analysis of FGF1 binding to FGFR2c in the absence (a) and presence of heparin hexa- (b) and octasaccharide (c). To determine the interaction between FGF1 and FGFR2c, 100 μM FGF1 was titrated into 10 μM FGFR2c. For panel b, 125 μM complex of FGF1–6-mer preincubated in a 1:1 ratio was titrated into 10 μM FGFR2c and for panel c, 100 μM FGF1–8-mer complex was titrated into 10 μM receptor. Trace of calorimetric titration (upper panel) and integrated isotherms (lower panel). (Solid lines) Best fit to a one-site model. For analysis of the FGF1-FGFR2c titration, n was fixed as 1.0.

Figure 6

The products of the ITC experiments were applied to a Superdex 200 10/300 column equilibrated with 20 mM Tris pH 8.0, 150 mM NaCl. Absorbance of the eluate was monitored at 280 nm. Peak labeling: (i) 1:1 FGF1:heparin hexasaccharide; (ii) 1:1:1 FGF1:FGFR2c:heparin hexasaccharide; 1), 2:1 FGF1:heparin octasaccharide; and 2), 2:2:1 FGF1:FGFR2c:heparin octasaccharide.