Interaction of heparin with a synthetic pentadecapeptide from the C-terminal heparin-binding domain of fibronectin

The synthetic pentadecapeptide FN-C/H II (KNNQKSEPLIGRKKT-NH2) has the sequ ence of the carboxy-terminal heparin-binding domain of module III14 of fibr onectin. Interaction of FN-C/H II with bovine lung heparin has been studied by H-1 and Na-23 NMR spectroscopy and by heparin affinity chromatography. FN-C/H II binds to heparin from pD <2 up to pD similar to 10; at higher pD, the binding decreases as the lysine side-chain ammonium groups are titrate d. Na+ counterions are displaced from the counterion condensation volume th at surrounds sodium heparinate by FN-C/H II, which provides direct evidence that the binding involves electrostatic interactions. The pK(A) values for each of the five ammonium groups of FN-C/H II increase upon binding to hep arin which, together with chemical shift data, indicates that the binding i nvolves both delocalized and direct electrostatic interactions between ammo nium groups of FN-C/H II and carboxylate and/or sulfate groups of heparin. NMR data also provide evidence for the direct interaction of the guanidiniu m group of the arginine side chain with anionic sites on heparin. The affin ity of heparin for FN-C/H II and for 13 analogue peptides in which lysine a nd arginine residues were systematically substituted with alanine increases as the number of basic residues increases. The relative contribution of ea ch lysine and arginine to the affinity of heparin for FN-C/H II is R-12 > K -13 > K-14 > K-1 > K-5. Nuclear Overhauser enhancement (NOE) data indicate that, while FN-C/H II is largely unstructured in aqueous solution, the boun d peptide interconverts among overlapping, turn-like conformations over the L-9 - T-15 Segment of the peptide. NOE data for the interaction of FN-C/H II with a heparin-derived hexasaccharide, together with the number of Naf i ons displaced from heparin by FN-C/H II was determined by Na-23 NMR, indica tes that the peptide binds to a hexasaccharide segment of heparin. Identica l NMR and heparin affinity chromatography results were obtained for the int eraction of FN-C/H II and its D-amino acid analogue peptide with heparin, w hich is of interest for the potential use of peptides as therapeutic agents for diseases in which cell adhesion plays a critical role.