Crystal structures of NK1-heparin complexes reveal the basis for NK1 activity and enable engineering of potent agonists of the MET receptor

NK1 is a splice variant of the polypeptide growth factor HGF/SF, which cons ists of the N-terminal (N) and first kringle (K) domain and requires hepara n sulfate or soluble heparin for activity. We describe two X-ray crystal st ructures of NK1-heparin complexes that define a heparin-binding site in the N domain, in which a major role is played by R73, with further contributio ns from main chain atoms of T61, K63 and G79 and the side chains of K60, T6 1, R76, K62 and K58. Mutagenesis experiments demonstrate that heparin bindi ng to this site is essential for dimerization in solution and biological ac tivity of NK1. Heparin also comes into contact with a patch of positively c harged residues (K132, R134, K170 and R181) in the K domain. Mutation of th ese residues yields NK1 variants with increased biological activity. Thus, we uncover a complex role for heparan sulfate in which binding to the prima ry site in the N domain is essential for biological activity whereas bindin g to the K domain reduces activity. We exploit the interaction between hepa rin and the K domain site in order to engineer NK1 as a potent receptor ago nist and suggest that dual (positive and negative) control may be a general mechanism of heparan sulfate-dependent regulation of growth factor activit y.