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  • Title: Energetic characterization of the basic fibroblast growth factor-heparin interaction: identification of the heparin binding domain.
    Author: Thompson LD, Pantoliano MW, Springer BA.
    Journal: Biochemistry; 1994 Apr 05; 33(13):3831-40. PubMed ID: 8142385.
    Abstract:
    Fibroblast growth factors (FGF's) interact on cell surfaces with "low-affinity" heparan sulfate proteoglycans (HSPG) and "high-affinity" FGF receptors (FGFR) to initiate cell proliferation. Previous reports have implicated the binding of heparin, or heparan sulfate, to FGF as essential for FGF-mediated signal transduction and mitogenicity. However, the molecular recognition events which dictate the specificity of this interaction have remained elusive. Amino acid residues on the surface of basic FGF (bFGF) were targeted as potential heparin contacts on the basis of the position of sulfate anions in the X-ray crystal structure of bFGF and of a modeled pentasaccharide heparin-bFGF complex. Each identified amino acid was replaced individually with alanine by site-directed mutagenesis, and the resulting mutant proteins were characterized for differences in binding to a low molecular weight heparin (approximately 3000) by isothermal titrating calorimetry and also for differences in [NaCl] elution from a heparin-Sepharose affinity resin. The combination of site-directed mutagenesis and titrating calorimetry permitted an analysis of the energetic contributions of individual bFGF residues in the binding of heparin to bFGF. The key amino acids which comprise the heparin binding domain on bFGF constitute a discontinuous binding epitope and include K26, N27, R81, K119, R120, T121, Q123, K125, K129, Q134, and K135. Addition of the observed delta delta G degrees of binding for each single site mutant accounts for 8.56 kcal/mol (> 95%) of the free energy of binding. The delta delta G degrees values for N27A, R120A, K125A, and Q134A are all greater than 1 kcal/mol each, and these four amino acids together contribute 4.8 kcal/mol (56%) to the total binding free energy. Amino acid residues K119 through K135 reside in the C-terminal domain of bFGF and collectively contribute 6.6 kcal/mol (76%) of the binding free energy. Although 7 out of the 11 identified amino acids in the heparin binding domain are positively charged, a 7-fold increase in [NaCl] decreases the affinity of wild-type bFGF binding to heparin only 37-fold (Kd at 0.1 M NaCl = 470 nM vs Kd at 0.7 M NaCl = 17.2 microM). This indicates that pure electrostatic interactions contribute only 30% of the binding free energy as analyzed by polyelectrolyte theory and that more specific nonionic interactions, such as hydrogen bonding and van der Waals packing, contribute the majority of the free energy for this binding reaction.
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