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. 2007 Mar 27;104(13):5306-11.
doi: 10.1073/pnas.0700184104. Epub 2007 Mar 19.

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

Daniel Kirchhofer  1 , Michael T Lipari, Lydia Santell, Karen L Billeci, Henry R Maun, Wendy N Sandoval, Paul Moran, John Ridgway, Charles Eigenbrot, Robert A Lazarus
Affiliations

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

Daniel Kirchhofer et al. Proc Natl Acad Sci U S A. .

Abstract

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 alpha/beta-heterodimer, where the beta-chain of HGF (HGF beta) has a serine protease fold that lacks enzymatic activity. We show that, like serine proteases, insertion of the newly formed N terminus in the beta-chain is critical for activity, here by allosterically stabilizing interactions with Met. The HGF beta 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 <10% activity in Met receptor phosphorylation, cell migration, and proliferation assays. Impaired signaling of full-length mutants correlated with >50-fold decreases in Met binding of the low-affinity HGF beta domain alone bearing the same mutations and further correlated with impaired N-terminal insertion. Because high-affinity binding resides in the HGF alpha-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.

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Conflict of interest statement

Conflict of interest statement: The authors of this work are employed by Genentech, Inc.

Figures

Fig. 1.
Fig. 1.
N-terminal “activation pocket” of trypsin and HGF β. (A) Crystal structure of the trypsin/BPTI complex [Protein Data Bank (PDB) ID code 2FTL]. Trypsin D194 is shown with the carboxylate in light red. The N terminus for I16 of trypsin is shown as a blue sphere. The Cα of V17, G18, and G19 are shown as green spheres. The salt bridge between D194 and I16 is ≈3 Å and is shown as a dotted line. (B) Crystal structure of the HGF β/Met Sema PSI complex (PDB ID code 1SHY). The HGF β carboxylate at D672 [c194] is shown in light red, and the N terminus of V495 [c16] is shown as a blue sphere. The Cα of V496, N497, and G498 are shown as green spheres. The salt bridge between D672 [c194] and V495 [c16] is ≈3 Å and is shown as a dotted line.
Fig. 2.
Fig. 2.
Relative extent of structurally buried N terminus of HGF β-chain mutants. The percent of N terminus buried relative to WT is plotted at various quenching times. For comparative purposes, the peak height for each mutant was first normalized relative to itself at t0 (t0 for all proteins = 100%) and then normalized to HGF β (WT) at 30, 60, or 120 min to determine the relative abundance of buried (nonmodifiable) N terminus.
Fig. 3.
Fig. 3.
HGF-dependent Met phosphorylation and cell proliferation. (A and B) Phosphorylation of Met in A549 cells and quantification by KIRA assay was carried out as described in Materials and Methods. (C) Representative immunoblots of phospho-Met from BxPC3 and PC3 cells after stimulation with 100 ng/ml HGF (WT) or HGF mutant. (D) Quantification of immunoblots for phospo-Met normalized to HGF (% of WT). (E) BxPC3 cell proliferation by 0.1 μg/ml HGF (WT) or HGF mutants at 0.1 μg/ml (black bars) or 2.0 μg/ml (gray bars).
Fig. 4.
Fig. 4.
Inhibition of Met phosphorylation, cell proliferation, and migration by HGF mutants. (A) HGF mutants (0.003–25 μg/ml) together with HGF (50 ng/ml) were added to A549 cell layer for 10 min at 37°C. Met phosphorylation by HGF mutants V495G (▿), D672N (▵), and G498I (♦) was quantified by using a KIRA assay and normalized to WT HGF activity (control). (B) Proliferation of BxPC3 cells was measured with 0.025 μg/ml HGF together with 2 μg/ml (gray bars) and 20 μg/ml (black bars) of HGF mutants. (C) MDA-MB435 cell migration was carried out in transwell plates in the presence of 0.1 μg/ml HGF together with HGF mutants at 20 μg/ml. HGF mutants were compared with WT HGF by using Student's t test: *, P < 0.05; **, P < 0.005.
Fig. 5.
Fig. 5.
Inhibition of in vitro endothelial tube formation by HGF mutants. Human umbilical vein endothelial cells attached to Cytodex 3 beads (average bead diameter was 170 μM) were embedded in a fibrin gel that was covered by Detroit 551 fibroblasts. HGF mutants in PBS or PBS alone (control) were added to the medium at a concentration of 10 μg/ml and tube formation was quantified after 6 days of culture. (A) Representative photographs of control and HGF mutant D672N. (B) Quantification of the number of endothelial sprouts per bead. HGF mutants were compared with a control by using Student's t test: *, P < 0.02; **, P < 0.005.
Fig. 6.
Fig. 6.
Model of allosteric linkage of N-terminal insertion in the HGF β domain with its Met binding site. Labels refer to the state of the HGF β domain in each conformation. Single chain pro-HGF contains a disulfide bond (thin line) between the α-chain (orange) and β-chain (yellow). Pro-HGF first binds Met (light purple) at the NK1 region of the α-chain (Mα). The HGF β-chain is initially in the pro-HGF state (HGF βpro) where the β-chain activation pocket (AP*) and Met binding site (Mβ*) are in disordered conformational states and incompetent for Met signaling. Upon cleavage at R494–V495 (arrow), the positively charged HGF β-chain N terminus (blue triangle, +) is released, but not yet buried in its activation pocket (HGF β*). The N terminus can then insert into its activation pocket, which locks in the conformation (AP) observed in the HGF β crystal structure (HGF β) (19). A conformational change at its Met binding site (Mβ) results in Met binding at this site (HGF β:Met). In the HGF β and HGF β:Met states, the N-terminal V495 forms a salt bridge with the negatively charged D672 [c194] (red circle, −), and the V495 side chain engages in hydrophobic interactions (green). An equilibrium exists between the free N terminus (HGF β*) and the buried N terminus (HGF β), which favors the HGF β* state for the V495G, D672N, and G498I mutants. Pro-HGF activation, N-terminal insertion, and Mβ formation could also occur before any Met binding at Mα (model not shown).
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