Heparin binding by fibronectin module III-13 involves six discontinuous basic residues brought together to form a cationic cradle

J Biol Chem. 1995 Aug 4;270(31):18558-62. doi: 10.1074/jbc.270.31.18558.

Abstract

The thirteenth type III domain of fibronectin binds heparin almost as well as fibronectin itself and contains a so-called heparin-binding consensus sequence, Arg6-Arg7-Ala8-Arg9 (residues 1697-1700 in plasma fibronectin). Barkalow and Schwarzbauer (Barkalow, F.J., and Schwarzbauer, J.E. (1991) J. Biol. Chem. 266, 7812-7818) showed that mutation of Arg6-Arg7 in domain III-13 of recombinant truncated fibronectins abolished their ability to bind heparin-Sepharose. However, synthetic peptides containing this sequence have negligible affinity for heparin (Ingham, K.C., Brew, S.A., Migliorini, M. M., and Busby, T.F. (1993) Biochemistry 32, 12548-12553). We generated a three-dimensional model of fibronectin type III-13 based on the structure of a homologous domain from tenascin. The model places Arg23, Lys25, and Arg54 parallel to and in close proximity to the Arg6-Arg7-Ala8-Arg9 motif, suggesting that these residues may also contribute to the heparin-binding site. Domain III-13 and six single-site mutants containing Ser in place of each of the above-mentioned basic residues were expressed in Escherichia coli. All of the purified mutant domains melted reversibly with a Tm near that of the wild type indicating that they were correctly folded. When fluorescein-labeled heparin was titrated at physiological ionic strength, the wild type domain increased the anisotropy in a hyperbolic fashion with a Kd of 5-7 microM, close to that of the natural domain obtained by proteolysis of fibronectin. The R54S mutant bound 3-fold weaker and the remaining mutants bound at least 10-fold weaker than wild type. The results point out that the Arg6-Arg7-Ala8-Arg9 consensus sequence by itself has little affinity for heparin under physiological conditions, even when presented in the context of a folded domain. Thus, the heparin-binding site in fibronectin is more complex than previously realized. It is formed by a cluster of 6 positively charged residues that are remote in the sequence but brought together on one side of domain III-13 to form a "cationic cradle" into which the anionic heparin molecule could fit.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Amino Acids, Diamino / metabolism
  • Binding Sites
  • Cations
  • DNA Mutational Analysis
  • Fibronectins / genetics
  • Fibronectins / metabolism*
  • Fluorescein
  • Fluoresceins
  • Fluorescence Polarization
  • Heparin / metabolism*
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism*
  • Recombinant Proteins / metabolism
  • Structure-Activity Relationship

Substances

  • Amino Acids, Diamino
  • Cations
  • Fibronectins
  • Fluoresceins
  • Peptide Fragments
  • Recombinant Proteins
  • Heparin
  • Fluorescein