Identification and Dynamics of a Heparin-Binding Site in Hepatocyte Growth Factor

• Published in: Biochemistry (Easton) 1999-11, Vol.38 (45), p.14793-14802
• Main Authors: Zhou, Hongjun, Casas-Finet, José R, Coats, R. Heath, Kaufman, Joshua D, Stahl, Stephen J, Wingfield, Paul T, Rubin, Jeffrey S, Bottaro, Donald P, Byrd, R. Andrew
• Format: Article
• Language: English
• Subjects: Animals; Binding Sites; Dimerization; Heparin - metabolism; Hepatocyte Growth Factor - metabolism; Kinetics; Models, Molecular; Molecular Weight; Peptide Fragments - metabolism; Sucrose - analogs & derivatives; Sucrose - metabolism; Swine
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9908641

Hepatocyte growth factor (HGF) is a heparin-binding, multipotent growth factor that transduces a wide range of biological signals, including mitogenesis, motogenesis, and morphogenesis. Heparin or closely related heparan sulfate has profound effects on HGF signaling. A heparin-binding site in the N-terminal (N) domain of HGF was proposed on the basis of the clustering of surface positive charges [Zhou, H., Mazzulla, M. J., Kaufman, J. D., Stahl, S. J., Wingfield, P. T., Rubin, J. S., Bottaro, D. P., and Byrd, R. A. (1998) Structure 6, 109−116]. In the present study, we confirmed this binding site in a heparin titration experiment monitored by nuclear magnetic resonance spectroscopy, and we estimated the apparent dissociation constant (K d) of the heparin−protein complex by NMR and fluorescence techniques. The primary heparin-binding site is composed of Lys60, Lys62, and Arg73, with additional contributions from the adjacent Arg76, Lys78, and N-terminal basic residues. The K d of binding is in the micromolar range. A heparin disaccharide analogue, sucrose octasulfate, binds with similar affinity to the N domain and to a naturally occurring HGF isoform, NK1, at nearly the same region as in heparin binding. 15N relaxation data indicate structural flexibility on a microsecond-to-millisecond time scale around the primary binding site in the N domain. This flexibility appears to be dramatically reduced by ligand binding. On the basis of the NK1 crystal structure, we propose a model in which heparin binds to the two primary binding sites and the N-terminal regions of the N domains and stabilizes an NK1 dimer.