Utilizing the activation mechanism of serine proteases to engineer hepatocyte growth factor into a Met antagonist

• Published in: Proceedings of the National Academy of Sciences - PNAS 2007-03, Vol.104 (13), p.5306-5311
• Main Authors: Kirchhofer, Daniel, Lipari, Michael T, Santell, Lydia, Billeci, Karen L, Maun, Henry R, Sandoval, Wendy N, Moran, Paul, Ridgway, John, Eigenbrot, Charles, Lazarus, Robert A
• Format: Article
• Language: English
• Subjects: Active sites; Allosteric Site; Amino acids; Animals; Binding sites; Biochemistry; Biological Sciences; Cell adhesion & migration; Cell growth; Cell Line, Tumor; Cell lines; CHO Cells; Cricetinae; Cricetulus; Crystal structure; Crystallography, X-Ray; Electrostatics; Hepatocyte Growth Factor - metabolism; Hepatocytes; Humans; Kinases; Mutation; Neoplasms - metabolism; Phosphorylation; Proteases; Protein Engineering - methods; Protein Structure, Tertiary; Proteins; Proto-Oncogene Proteins c-met - antagonists & inhibitors; Proto-Oncogene Proteins c-met - metabolism; Receptors; Salts; Serine Endopeptidases - chemistry; Serine Endopeptidases - metabolism; Signal Transduction
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0700184104

Hepatocyte growth factor (HGF), the ligand for the receptor tyrosine kinase Met, is secreted as single chain pro-HGF that lacks signaling activity. Pro-HGF acquires functional competence upon cleavage between R494 and V495, generating a disulfide-linked α/β-heterodimer, where the β-chain of HGF (HGF β) has a serine protease fold that lacks enzymatic activity. We show that, like serine proteases, insertion of the newly formed N terminus in the β-chain is critical for activity, here by allosterically stabilizing interactions with Met. The HGF β crystal structure shows that V495 inserts into the "activation pocket" near the Met binding site where the positively charged N terminus forms a salt bridge with the negatively charged D672, and the V495 side chain has hydrophobic interactions with main- and side-chain residues. Full-length two-chain HGF mutants designed to interrupt these interactions (D672N, V495G, V495A, G498I, and G498V) displayed 50-fold decreases in Met binding of the low-affinity HGF β domain alone bearing the same mutations and further correlated with impaired N-terminal insertion. Because high-affinity binding resides in the HGF α-chain, full-length mutants maintained normal Met binding and efficiently inhibited HGF-mediated Met activation. Conversion of HGF from agonist to antagonist was achieved by as little as removal of two methyl groups (V495A) or a single charge (D672N). Thus, although serine proteases and HGF have quite distinct functions in proteolysis and Met signal transduction, respectively, they share a similar activation mechanism.