Identification and dynamics of a heparin-binding site in hepatocyte growth factor.
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. (15)N 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.
Zhou
H
H
Macromolecular NMR Section, ABL-Basic Research Program, SAIC Frederick, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201, USA.
Casas-Finet
J R
JR
Heath Coats
R
R
Kaufman
J D
JD
Stahl
S J
SJ
Wingfield
P T
PT
Rubin
J S
JS
Bottaro
D P
DP
Byrd
R A
RA
D000818:Animals; D001665:Binding Sites; D019281:Dimerization; D006493:Heparin / Q000378:metabolism*; D017228:Hepatocyte Growth Factor / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D013395:Sucrose / Q000031:analogs & derivatives / Q000378:metabolism; D013552:Swine
D010446:Peptide Fragments; D013395:Sucrose; D017228:Hepatocyte Growth Factor; D006493:Heparin; C053081:sucrose octasulfate