Heparin Binding by Fibronectin Module III-13 Involves Six Discontinuous Basic Residues Brought Together to Form a Cationic Cradle (∗)

• Published in: The Journal of biological chemistry 1995-08, Vol.270 (31), p.18558-18562
• Main Authors: Busby, Thomas F., Argraves, W. Scott, Brew, Shelesa A., Pechik, Igor, Gilliland, Gary L., Ingham, Kenneth C.
• Format: Article
• Language: English
• Subjects: 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
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.31.18558

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 T m 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 μM, 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.