HEPARIN-BINDING BY FIBRONECTIN MODULE-III-13 INVOLVES 6 DISCONTINUOUSBASIC RESIDUES BROUGHT TOGETHER TO FORM A CATIONIC CRADLE

The thirteenth type III domain of fibronectin binds heparin almost as well as fibronectin itself and contains a so-called heparin-binding co nsensus sequence, Arg(6)-Arg(7)-Ala(8)-Arg(9) (residues 1697-1700 in p lasma fibronectin). Barkalow and Schwarzbauer (Barkalow, F. J., and Sc hwarzbauer, J. E. (1991) J. Biol. Chem. 266, 7812-7818) showed that mu tation of Arg(6)-Arg(7) in domain III-13 of recombinant truncated fibr onectins abolished their ability to bind heparin-Sepharose. However, s ynthetic peptides containing this sequence have negligible affinity fo r heparin (Ingham, K. C., Brew, S. A., Migliorini, M. M., and Busby, T . F. (1993) Biochemistry 32, 12548-12553). We generated a three dimens ional model of fibronectin type III-13 based on the structure of a hom ologous domain from tenascin. The model places Arg(23), Lys(25), and A rg(54) parallel to and in close proximity to the Arg(6)-Arg(7)-Ala(8)- Arg(9) motif, suggesting that these residues may also contribute to th e heparin-binding site. Domain III-13 and six single-site mutants cont aining Ser in place of each of the above-mentioned basic residues were expressed in Escherichia coli. All of the purified mutant domains mel ted reversibly with a Tm near that of the wild type indicating that th ey were correctly folded. When fluorescein-labeled heparin was titrate d at physiological ionic strength, the wild type domain increased the anisotropy in a hyperbolic fashion with a K-d of 5-7 mu M, close to th at of the natural domain obtained by proteolysis of fibronectin. The R 54S mutant bound 3-fold weaker and the remaining mutants bound at leas t 10-fold weaker than wild type. The results point out that the Arg(6) -Arg(7)-Ala(8)-Arg(9) consensus sequence by itself has little affinity for heparin under physiological conditions, even when presented in th e context of a folded domain. Thus, the heparin-binding site in fibron ectin is more complex than previously realized. It is formed by a clus ter of 6 positively charged residues that are remote in the sequence b ut brought together on one side of domain III-13 to form a ''cationic cradle'' into which the anionic heparin molecule could fit.