Identification and dynamics of a heparin-binding site in hepatocyte growthfactor

Hepatocyte growth factor (HGF) is a heparin-binding. multipotent growth fac tor that transduces a wide range of biological signals, including mitogenes is, motogenesis, and morphogenesis. Heparin or closely related heparan sulf ate 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. (19 98) Structure 6, 109-116]. In the present study, we confirmed this binding site in a heparin titration experiment monitored by nuclear magnetic resona nce spectroscopy, and we estimated the apparent dissociation constant (Kd) Of the heparin-protein complex by NMR and fluorescence techniques. The prim ary heparin-binding site is composed of Lys60, Lys62, and Arg73, with addit ional contributions from the adjacent Arg76, Lys78, and N-terminal basic re sidues. The Kd Of binding is in the micromolar range. A heparin disaccharid e analogue, sucrose octasulfate, binds with similar affinity to the N domai n and to a naturally occurring HGF isoform, NK1, at nearly the same region as in heparin binding. N-15 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 li gand 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.