Electrostatic Forces as Dominant Interactions Between Proteins and Polyanions: an ESI MS Study of Fibroblast Growth Factor Binding to Heparin Oligomers

Abstract

The interactions between fibroblast growth factors (FGFs) and their receptors (FGFRs) are facilitated by heparan sulfate (HS) and heparin (Hp), highly sulfated biological polyelectrolytes. The molecular basis of FGF interactions with these polyelectrolytes is highly complex due to the structural heterogeneity of HS/Hp, and many details still remain elusive, especially the significance of charge density and minimal chain length of HS/Hp in growth factor recognition and multimerization. In this work, we use electrospray ionization mass spectrometry (ESI MS) to investigate the association of relatively homogeneous oligoheparins (octamer, dp8, and decamer, dp10) with acidic fibroblast growth factor (FGF-1). This growth factor forms 1:1, 2:1, and 3:1 protein/heparinoid complexes with both dp8 and dp10, and the fraction of bound protein is highly dependent on protein/heparinoid molar ratio. Multimeric complexes are preferentially formed on the highly sulfated Hp oligomers. Although a variety of oligomers appear to be binding-competent, there is a strong correlation between the affinity and the overall level of sulfation (the highest charge density polyanions binding FGF most strongly via multivalent interactions). These results show that the interactions between FGF-1 and Hp oligomers are primarily directed by electrostatics, and also demonstrate the power of ESI MS as a tool to study multiple binding equilibria between proteins and structurally heterogeneous polyanions. Graphical Abstract ᅟ.

Keywords: Glycosaminoglycans; Heparin; Native electrospray ionization mass spectrometry; Noncovalent interactions; Polyelectrolytes.

Figure 1

ESI mass spectrum of a 5 μM solution of FGF-1 in 100 mM NH₄CH₃CO₂ at pH 6.8.

Figure 2

ESI mass spectra of FGF-1 incubated with dp8 (left panel) and dp10 (right panel) it 100 mM NH₄CH₃CO₂ at pH 6.8. The numbers in boxes indicate charge states of free FGF-1 (black), and its complexes with heparinoids: 1:1 (red), 2:1 (blue) and 3:1 (green). The r values shown in each row indicate the protein/heparinoid molar ratio.

Figure 3

ESI mass spectrum of dp10 incubated in a 5 μM solution of FGF-1/100 mM NH₄CH₃CO₂ at pH 6.8 (protein/heparinoid molar ratio r = 4).

Figure 4

Delphi-generated electrostatic potential contours (−0.5 kT/e, red and +0.5 kT/e, blue) generated around FGF-1 (PDB id: 1K5U) at pH 7.0 and ionic strength I = 100 mM (top). A representative dp10 molecule (a deca-saccharide segment of 3IRJ) is shown for comparison to provide the physical dimensions of the polyanionic chain relative to the protein dimensions. Diagrams at the bottom show possible arrangements of dp10 (shown schematically as a ruler) and FGF-1 in multi-valent complexes.

Figure 5

Mass distributions of the FGF-bound dp8 molecules at two different protein/heparinoid molar ratios. Mass distributions of the dp8 chains involved in formation of 1:1 complexes (red traces) are calculated based on the ionic signal of FGF·dp8⁺⁷ species, and those for 2:1 complexes (blue traces) are calculated based on the ionic signal of FGF₂·dp8⁺¹² species. The colored boxes indicate the mass ranges corresponding to the dp8 species with varying levels of sulfation.

Figure 6

Mass distributions of the FGF-bound dp10 molecules at several different protein/heparinoid molar ratios. Mass distributions of the dp10 chains involved in formation of 1:1 complexes (red traces) are calculated based on the ionic signal of FGF·dp10⁺⁷ species, those for 2:1 (blue traces) and 3:1 (green trace) complexes are calculated based on the ionic signals of FGF₂·dp10⁺¹² and FGF₃·dp10⁺¹⁴ species, respectively. The colored boxes indicate the mass ranges corresponding to the dp10 species with varying levels of sulfation.

Figure 7

Mass distributions of free dp10 molecules in solution in the absence (green trace) and in the presence of FGF-1 (r-values are shown on each panel) calculated based on the ionic signals of FGF⁺² species in ESI mass spectra. The colored boxes indicate the mass ranges corresponding to the dp10 species with varying levels of sulfation.