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

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.

Mapping the heparin‐binding domain of boar spermadhesins

“…It is noteworthy that similar to our results concerning AWN 4RRSR8, the RRAR sequence, when present in a synthetic peptide, has been shown to have weak affinity for heparin [28,29]. Furthermore, this same RRAR sequence exists in the carboxylterminal lobe of lactoferrin where it is not functional as a GAG-binding site [30]. These results together with recent mutagenesis studies on fibronectin module III-13 have point out that the BBXB consensus sequence by itself has little affinity for heparin even when presented in the context of a folded domain [31].…”

Basic Residues in Azurocidin/HBP Contribute to Both Heparin Binding and Antimicrobial Activity

“…6A). In the rAzu three-dimensional structure, these regions are found in close proximity in 2 loops and appear to adopt a conformation similar to the "cationic cradle" structure used for heparin binding in proteins such as lactoferrin and fibronectin (25,26) (Fig. 6B).…”

Matrix Metalloproteinase Processing of CXCL11/I-TAC Results in Loss of Chemoattractant Activity and Altered Glycosaminoglycan Binding

“…Notably, the heparinbinding cationic cradle is rarely a simple consensus sequence, but rather a more complex combination of remote positively charged residues that are clustered in the folded conformation, as illustrated with fibronectin module III-13 (68). We hypothesized that the glycosaminoglycan-binding site of CXCL11 is located in the COOH terminus due to an abundance of basic amino acids and indeed the heparin binding interaction was strongly reduced in CXCL11-(5-58).…”