Structural and Functional Basis of the Serine Protease-like Hepatocyte

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 279, No. 38, Issue of September 17, pp. 39915–39924, 2004 Printed in U.S.A.

Structural and Functional Basis of the Serine Protease-like Hepatocyte Growth Factor ␤-Chain in Met Binding and Signaling* Received for publication, April 29, 2004, and in revised form, June 22, 2004 Published, JBC Papers in Press, June 24, 2004, DOI 10.1074/jbc.M404795200

Daniel Kirchhofer‡§, Xiaoyi Yao¶储, Mark Peek‡, Charles Eigenbrot¶, Michael T. Lipari‡, Karen L. Billeci**, Henry R. Maun¶‡‡, Paul Moran‡, Lydia Santell¶, Christian Wiesmann¶, and Robert A. Lazarus¶§§ From the Departments of ‡Physiology, ¶Protein Engineering, and **Assay and Automation Technology, Genentech, Inc., South San Francisco, California 94080 and ‡‡Institute for Biology III, University of Freiburg, Schaenzlestrasse 1, D-79104 Freiburg, Germany

Hepatocyte growth factor (HGF), a plasminogen-related growth factor, is the ligand for Met, a receptor tyrosine kinase implicated in development, tissue regeneration, and invasive tumor growth. HGF acquires signaling activity only upon proteolytic cleavage of single-chain HGF into its ␣/␤ heterodimer, similar to zymogen activation of structurally related serine proteases. Although both chains are required for activation, only the ␣-chain binds Met with high affinity. Recently, we reported that the protease-like HGF ␤-chain binds to Met with low affinity (Stamos, J., Lazarus, R. A., Yao, X., Kirchhofer, D., and Wiesmann, C. (2004) EMBO J. 23, 2325–2335). Here we demonstrate that the zymogen-like form of HGF ␤ also binds Met, albeit with 14-fold lower affinity than the protease-like form, suggesting optimal interactions result from conformational changes upon cleavage of the single-chain form. Extensive mutagenesis of the HGF ␤ region corresponding to the active site and activation domain of serine proteases showed that 17 of the 38 purified two-chain HGF mutants resulted in impaired cell migration or Met phosphorylation but no loss in Met binding. However, reduced biological activities were well correlated with reduced Met binding of corresponding mutants of HGF ␤ itself in assays eliminating dominant ␣ -chain binding contributions. Moreover, the crystal structure of HGF ␤ determined at 2.53 Å resolution provides a structural context for the mutagenesis data. The functional Met binding site is centered on the “active site region” including “triad” residues Gln534 [c57], Asp578 [c102], and Tyr673 [c195] and neighboring “activation domain” residues Val692, Pro693, Gly694, Arg695, and Gly696 [c214-c219]. Together they define a region that bears remarkable resemblance to substrate processing regions of serine proteases. Models of HGF-dependent Met receptor activation are discussed. * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The atomic coordinates and structure factors (code 1SI5) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). § To whom correspondence may be addressed: Dept. of Physiology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080. Tel.: 650-225-2134; Fax: 650-225-6327; E-mail: [email protected]. 储 Present address: Dept. of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409. §§ To whom correspondence may be addressed: Dept. of Protein Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080. Tel.: 650-225-1166; Fax: 650-225-3734; E-mail: [email protected]. This paper is available on line at http://www.jbc.org

Hepatocyte growth factor (HGF),1 also known as scatter factor, is the ligand for Met (1, 2), a receptor tyrosine kinase encoded by the c-met protooncogene (3). HGF binding to Met induces phosphorylation of the intracellular kinase domain resulting in activation of a complex set of intracellular pathways that lead to cell growth, differentiation, and migration in a variety of cell types; several recently published reviews (4 – 6) provide a comprehensive overview. In addition to its fundamental importance in embryonic development and tissue regeneration, the HGF/Met signaling pathway has also been implicated in invasive tumor growth and metastasis and as such represents an interesting therapeutic target (4, 5, 7, 8). HGF belongs to the plasminogen-related growth factor family and comprises a 69-kDa ␣-chain, containing the N-terminal finger domain (N) and four kringle (K1–K4) domains, and a 34-kDa ␤-chain, which has strong similarity to protease domains of chymotrypsin-like serine proteases from Clan PA(S)/ Family S1 (1, 9, 10). Like plasminogen and other serine protease zymogens, HGF is secreted as a single-chain precursor form (pro-HGF). A noncleavable single-chain form of HGF containing an R494E mutation binds Met with high affinity; however, it cannot activate the receptor (11–13). Acquisition of HGF signaling activity is contingent upon proteolytic cleavage (activation) of pro-HGF between Arg494 and Val495 resulting in the formation of mature HGF, a disulfide-linked ␣/␤ heterodimer (11–14). The protease-like domain of HGF (HGF ␤-chain) lacks the Asp [c102]-His [c57]-Ser [c195] (standard chymotrypsinogen numbering in brackets throughout) catalytic triad found in all serine proteases (15, 16), having a Gln534 [c57] and Tyr673 [c195] instead, and thus is devoid of any enzymatic activity. We investigated the importance of the protease-like domain of HGF for molecular interactions with Met based upon a significant similarity to serine proteases and their activation process (9). In serine proteases, cleavage of the zymogen effects a conformational rearrangement of the so-called “activation domain” giving rise to a properly formed active site and the substrate/inhibitor interaction region. The activation domain constitutes three surface-exposed loops designated the [c140],

1 The abbreviations used are: HGF, hepatocyte growth factor; N domain, N-terminal finger domain; K1–K4, Kringle domains 1– 4; proHGF, single-chain precursor form of HGF; scHGF ␤, a noncleavable single-chain form of HGF (Asn479–Ser728; R494E); scHGF, a noncleavable single-chain form of full-length HGF (R424A:R494E); MSP, macrophage-stimulating protein; wild type HGF ␤, refers to the native sequence Val495 [c16]-Ser728 [c250]; HGF ␤, refers to the Val495 [c16]Ser728 [c250] sequence containing the C604S [c128] mutation; FBS, fetal bovine serum; ELISA, enzyme-linked immunosorbent assay; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; KIRA, kinase receptor activation assay.

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Role of the HGF ␤-Chain in Met Binding and Signaling

[c180], and [c220] loops and the newly formed N terminus, which inserts into a hydrophobic pocket (17). We thus reasoned that rearrangements in the corresponding “activation domain” of the HGF ␤-chain might form a Met interaction site. In the homologous ligand/receptor pair macrophage-stimulating protein (MSP)/Ron, the serine protease-like MSP ␤-chain provides the main energy for receptor binding (18, 19). This is reversed from the HGF/Met system where the high affinity receptor binding site for Met resides in the HGF ␣-chain (20, 21). To better understand the binding requirements and mechanism for Met activation and signaling, we expressed and purified various forms of HGF ␤. Consistent with our recently published data (22), we find that the wild type protease-like HGF ␤ binds to Met. A zymogen-like single-chain form of HGF ␤ (scHGF ␤) also binds to Met, but with lower affinity, suggesting that this Met binding site is influenced by a conformational rearrangement upon cleavage. Met binding residues were identified by mutagenesis studies on both full-length HGF and the HGF ␤-chain and were mapped onto the crystal structure of HGF ␤, which was determined to a resolution of 2.53 Å. The mutagenesis results define a distinct Met binding site, which consists of residues of the “active site region” and “activation domain” of the serine protease-like HGF ␤-chain. Models of Met dimerization and activation by two-chain HGF are discussed. EXPERIMENTAL PROCEDURES

Expression and Purification of HGF ␤ Proteins—All HGF ␤ proteins (Val495 [c16] to Ser728 [c250]), which contained a His6 C-terminal tag, were expressed and purified to homogeneity (⬎95% purity) as described previously (22). Wild type HGF ␤ refers to the native sequence, and HGF ␤ refers to the C604S [c128] mutant. HGF ␤ mutants Q534A [c57], D578A [c102], Y619A [c143], R695A [c217], G696A [c219], and I699A [c221a] were made using QuikChangeTM site-directed mutagenesis (Stratagene, La Jolla, CA) in the C604S [c128] construct. scHGF ␤ encodes HGF from residues Asn479 to Ser728, a His6 C-terminal tag, and has an R494E mutation made using the oligonucleotide 5⬘-CAAAACGAAACAATTGGAAGTTGTAAATGGGATTC-3⬘. The cysteine was not altered in this construct to allow putative disulfide formation between Cys487 and Cys604 [c128]. Tranfection of Sf9 cells on plates in ESF 921 media (Expression Systems LLC, Woodland, CA) was carried out using the BaculoGoldTM Expression System according to the manufacturer’s instructions (Pharmingen). Virus amplification, cell culture, and purification was carried out essentially as described previously except that the elution buffer for the nickel-nitrilotriacetic-agarose column contained 500 mM imidazole instead of 250 mM imidazole (22). Proteins were analyzed by 12% SDS-PAGE stained with Coomassie Blue. Mutations were verified by DNA sequencing and mass spectrometry. Protein concentration was determined by quantitative amino acid analysis. N-terminal sequencing revealed a single correct N terminus present for scHGF ␤ and HGF ␤. Purified proteins showed the correct molecular mass on SDS-PAGE; multiple bands observed were likely due to heterogeneous glycosylation, consistent with the mass spectrometry data having molecular masses ⬃2 kDa higher than predicted from the sequence. Construction, Expression, and Purification of Full-length HGF Proteins—Recombinant proteins were produced in 1-liter cultures of Chinese hamster ovary (CHO) cells by transient transfection (23). Amino acid changes were introduced by site-directed mutagenesis (24) and verified by DNA sequencing. The expression medium (F-12/Dulbecco’s modified Eagle’s medium) contained 1% (v/v) ultra low IgG fetal bovine serum (FBS) (Invitrogen). After 8 days the medium was harvested and supplemented with FBS to give a final content of 5–10% (v/v). Additional incubation for 2–3 days at 37 °C resulted in complete single-chain HGF conversion. This step was omitted for expression of scHGF, a noncleavable single-chain form, which has amino acid changes at the activation cleavage site (R494E) and at a protease-susceptible site in the ␣-chain (R424A) (23). Mutant proteins were purified from the medium by HiTrap-Sepharose SP cation exchange chromatography (Amersham Biosciences) as described (23). Examination by SDS-PAGE (4 –20% gradient gel) under reducing conditions and staining with Simply Blue Safestain (Invitrogen) showed that all HGF mutants were ⬎95% pure and were fully converted into ␣/␤ heterodimers except for scHGF, which remained as a single-chain form. Protein concentration

for each mutant was determined by quantitative amino acid analysis. Binding of HGF ␤ to Met in a Competition Binding ELISA—To develop a competition ELISA, we first determined the direct specific binding of the HGF ␤-chain to Met in a plate ELISA. Microtiter plates were coated with Met-IgG fusion protein (25) as described (22) and incubated with wild type HGF ␤-chain. Bound HGF ␤ was detected using penta-His horseradish peroxidase conjugate (Qiagen, Valencia, CA) followed by addition of SureBlue TMB peroxidase substrate and TMB STOP (Kirkegaard & Perry Laboratories, Gaithersburg, MD). The determined effective concentration to give half-maximal binding (EC50) was 320 ⫾ 140 nM (n ⫽ 6). Based on these results, a competition binding ELISA was developed as described (22) using wild type HGF ␤ biotinylated with 20-fold molar excess of biotin-maleimide (Pierce). Briefly, plates were coated with Met-IgG fusion protein and incubated with a mixture of 250 nM biotinylated wild type HGF ␤ and various concentrations of unlabeled HGF ␤, HGF ␤ mutants, or scHGF ␤. After incubation for 1 h at room temperature, the amount of biotinylated wild type HGF ␤ bound on the plate was measured by using horseradish peroxidase-neutravidin (Pierce). IC50 values were determined by fitting the data to a fourparameter equation (Kaleidagraph, Synergy Software, Reading, PA). Binding of HGF Mutants to Met—Biotinylated HGF (two-chain full length) was prepared using the Sigma immunoprobe biotinylation kit (Sigma). Microtiter plates were coated with rabbit anti-human IgG Fc-specific antibody as described above. Plates were washed in PBS containing 0.05% (v/v) Tween 20 followed by a 1-h incubation with 0.5% (w/v) bovine serum albumin, 0.05% Tween 20 in PBS, pH 7.4, at room temperature. After washing, 1 nM biotinylated HGF and 0.2 nM MetIgG fusion protein (25) together with various concentrations of HGF mutants were added to the wells and incubated for 2 h. After washing, bound biotinylated HGF was detected by addition of diluted (1:3000) streptavidin horseradish peroxidase conjugate (Zymed Laboratories Inc., South San Francisco, CA) followed by SureBlue TMB peroxidase substrate and stop solution TMB STOP (Kirkegaard & Perry Laboratories). The A450 was measured, and IC50 values were determined as described above. Relative binding affinities are expressed as the IC50(mutant)/IC50(wild type HGF). HGF-dependent Phosphorylation of Met—The kinase receptor activation assay (KIRA) was carried out as follows. Confluent cultures of lung carcinoma A549 cells (CCL-185, ATCC, Manassas, VA), maintained previously in growth medium (Ham’s F-12/Dulbecco’s modified Eagle’s medium 50:50 (Invitrogen) containing 10% FBS (Sigma)), were detached using Accutase (ICN, Aurora, OH) and seeded in 96-well plates at a density of 50,000 cells per well. After overnight incubation at 37 °C, growth medium was removed, and cells were serum-starved for 30 – 60 min in medium containing 0.1% FBS. Met phosphorylation activity by HGF and HGF mutants was determined from addition of serial dilutions from 500 to 0.2 ng/ml in medium containing 0.1% FBS followed by a 10-min incubation at 37 °C, removal of media, and cell lysis with cell lysis buffer (Cell Signaling Technologies, Beverly, MA) supplemented with protease inhibitor mixture set I (Calbiochem). HGF ␤-chain studies were carried out similarly starting at 5 ␮g/ml. Cell lysates were analyzed for phosphorylated Met via an electrochemiluminescence assay using a BioVeris M-Series instrument (BioVeris Corp., Gaithersburg, MD). Anti-phosphotyrosine antibody 4G10 (Upstate Biotechnology, Inc., Lake Placid, NY) was labeled with ORI-TAG via Nhydroxysuccinimide ester chemistry according to the manufacturer’s directions (BioVeris). Anti-Met extracellular domain antibody 1928 (Genentech, Inc.) was biotinylated using biotin-X-N-hydroxysuccinimide (Research Organics, Cleveland, OH). The BV-TAG-labeled 4G10 and biotinylated anti-Met antibody were diluted in assay buffer (PBS, 0.5% Tween 10, 0.5% bovine serum albumin), and the mixture was added to the cell lysates. After incubation at room temperature with vigorous shaking for 1.5–2 h, streptavidin magnetic beads (Dynabeads, BioVeris) were added and incubated for 45 min. The beads with bound material (anti-Met antibody/Met/anti-phosphotyrosine antibody) were captured by an externally applied magnet. After a wash step, the chemiluminescent signal generated by the light source was measured as relative luminescent units on a BioVeris instrument. For each experiment, the Met phosphorylation induced by HGF mutants was expressed as a percentage of the maximal signal obtained with two-chain HGF. Cell Migration Assay—Breast cancer cells MDA-MB435 (HTB-129, ATCC, Manassas, VA) were cultured in recommended serum-supplemented medium. Confluent cells were detached in PBS containing 10 mM EDTA and diluted with serum-free medium to a final concentration of 0.6 – 0.8 ⫻ 106 cells/ml. This cell suspension (0.2 ml) was added in triplicate to the upper chambers of 24-well Transwell plates (8 ␮m pore size) (HTS MultiwellTM Insert System, Falcon, Franklin Lakes, NJ)


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FIG. 1. Binding and activity of HGF and HGF ␤ with Met. A, HGF ␤/Met competition ELISA. Competition binding of immobilized Met-IgG with 250 nM maleimide-coupled biotinylated wild type HGF ␤ and unlabeled HGF ␤ (●) and scHGF ␤ (f) was carried out as described under “Experimental Procedures.” Data from at least three independent determinations each were normalized, averaged, and fitted by a four-parameter fit using Kaleidagraph, from which IC50 values were determined; error bars represent S.D. B, HGF-dependent phosphorylation of Met in A549 cells was carried out as described under “Experimental Procedures” using HGF (Œ) and HGF ␤ (●). pre-coated with 10 ␮g/ml of rat tail collagen type I (Upstate Biotechnology, Inc.). Wild type HGF or HGF mutants were added to the lower chamber at 1 nM in serum-free medium, unless specified otherwise. HGF ␤-chain was also tested at 0.95 ␮M. After incubation for 13–14 h, cells on the apical side of the membrane were removed, and those that migrated to the basal side were fixed in 4% paraformaldehyde followed by staining with a 0.5% crystal violet solution. After washing and air drying, cells were solubilized in 10% acetic acid, and the A560 was measured on a Molecular Devices microplate reader. Pro-migratory activities of HGF mutants were expressed as percent of HGF controls after subtracting basal migration in the absence of HGF. Photographs of stained cells were taken with a Spot digital camera (Diagnostics Instruments, Inc., Sterling Heights, MI) connected to a Leitz microscope (Leica Mikroskope & Systeme GmbH, Wetzlar, Germany). Pictures were acquired by Adobe Photoshop 4.0.1 (Adobe Systems Inc., San Jose, CA). HGF ␤ X-ray Structure—Purified HGF ␤ was concentrated to 10 mg/ml using a Centriprep® YM-10 in 10 mM HEPES, pH 7.2, 150 mM NaCl, 5 mM CaCl2. Hanging drops (1 ␮l of protein and 1 ␮l of 30% PEG-1500) over a reservoir containing 500 ␮l of 30% PEG-1500 (Hampton Research, Laguna Niguel, CA) yielded crystalline rods (⬃25 ⫻ 25 ⫻ 500 ␮m) during incubation at 19 °C overnight. A crystal fragment was preserved directly from the mother liquor by immersion in liquid nitrogen. Data extending to 2.53 Å resolution were collected on a Quantum 4 ccd detector (ADSC, Poway, CA) at ALS beam line 5.0.2 with 1.0 Å wavelength x-rays. Data processing and reduction were performed using HKL (26) (HKL Research, Charlottesville, VA) and CCP4 (27). The structure was solved by molecular replacement using AMoRe (28) in space group P3121, using parts of the protease domain of coagulation factor VIIa (29) as the search probe. Refinement was performed using X-PLOR98 (Accelrys, San Diego, CA) and REFMAC (30). Inspection of electron density maps and model manipulation was performed using XtalView (31) (Syrrx, San Diego, CA). RESULTS

Binding of HGF ␤ to Met—Because single-chain HGF binds to Met with comparable affinity to that of two-chain HGF, but does not induce Met phosphorylation (11–13), we hypothesized that this may be due to the lack of a Met binding site in the uncleaved form of the ␤-chain. To test this hypothesis, we carried out competition binding ELISAs with expressed and purified HGF ␤ and scHGF ␤. scHGF ␤ is a zymogen-like form of HGF ␤ containing the C-terminal 16 residues from the HGF ␣-chain and a mutation at the cleavage site (R494E) to ensure that the single-chain form remained intact. Initial ELISA data showed that HGF ␤ directly bound to immobilized Met-IgG

fusion protein, albeit with relatively low affinity. This is consistent with our recent data from surface plasmon resonance experiments with immobilized Met extracellular domain, where a Kd of ⬃90 nM for the HGF ␤/Met interaction was found (22). In the HGF ␤/Met competition binding ELISA we determined IC50 values of 0.86 ⫾ 0.17 and 11.6 ⫾ 1.8 ␮M for HGF ␤ and the precursor form scHGF ␤, respectively, showing that scHGF ␤ bound Met with 14-fold lower affinity than HGF ␤ (Fig. 1A). Thus, although a Met binding site on the zymogenlike HGF ␤ does in fact exist, it is not optimal. The apparent affinity differences observed between Kd (22) and IC50 values are due to the different assays used; the higher IC50 values reflect the higher concentrations of HGF ␤ necessary to compete with 250 nM biotinylated HGF ␤ for binding to Met. We also measured the ability of HGF ␤ to stimulate Met phosphorylation of A549 lung cancer cells. Fig. 1B shows that HGF ␤ was completely inactive, even at concentrations that exceeded optimal phosphorylation activity by full-length HGF by ⬎1000-fold. Similarly, in MDA-MB435 cell migration assays, HGF ␤ at concentrations of up to 0.95 ␮M had no effect. These results are consistent with other studies demonstrating the inability of HGF ␤ to activate the Met receptor (11, 32). Effects on Cell Migration and Met Phosphorylation by HGF and HGF ␤-Chain Mutants—To identify the Met binding site in the ␤-chain, we systematically changed residues in regions corresponding to the activation domain and the active site of serine proteases, herein referred to as the “activation domain” and “active site region” of HGF. Initial expression of HGF mutants in CHO cells yielded a mixture of single- and twochain HGF forms, exemplified by mutant HGF I623A (Fig. 2A). Complete conversion of residual uncleaved HGF was accomplished by additional exposure of the harvested culture medium to 5–10% serum for several days (Fig. 2A). The purity of HGF I623A [c149] following purification by cation exchange chromatography is representative of all HGF mutants. The functional consequence of mutating ␤-chain residues in HGF was assessed by determining the ability of the HGF mutants to stimulate migration of MDA-MB435 cells. The results showed that 3 HGF mutants, R695A [c217], G696A [c219], and Y673A [c195], were severely impaired, having less than 20% of wild type activity, whereas 5 mutants Q534A [c57],

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FIG. 2. HGF-dependent cell migration with HGF mutants. A, representative purity of HGF mutants. The purity of all HGF mutants analyzed by SDS-PAGE under reducing conditions is illustrated for cation exchange purified HGF I623A [c149]. Incomplete conversion of the secreted single-chain form by CHO expression in 1% FBS (v/v) is shown in lane 1. Additional exposure to 5% FBS completed the activation process by yielding pure two-chain HGF I623A (lane 2). Molecular weight markers are shown as Mr ⫻ 10⫺3. B, migration of MDA-MB435 cells in a Transwell assay in the presence of 1 nM HGF mutants. Activities are expressed as percent migration of control cells exposed to 1 nM wild type HGF; full-length HGF sequence numbering [chymotrypsinogen numbering] is shown. Values represent the averages of 4 – 8 independent experiments ⫾ S.D. C, photographs of MDA-MB435 cell migration. a, absence of wild type HGF; b, 1 nM wild type HGF; c, 1 nM HGF R695A [c217]; and d, 1 nM HGF G696A [c219].

D578A [c102], V692A [c214], P693A [c215], and G694A [c216] had 20 – 60% of wild type activity (Fig. 2B). An additional set of 9 mutants (R514A [c36], P537A [c60a], Y619A [c143], T620A [c144], G621A [c145], K649A [c173], I699A [c221a], N701A [c223], and R702A [c224]) had 60 – 80% of wild type activity. The remaining 21 mutants had activities ⬎80% that of wild type and were considered essentially unchanged from HGF. As expected, scHGF did not stimulate cell migration (Fig. 2B). The complete inability of 1 nM R695A [c217] or G696A [c219] to promote cell migration is illustrated in Fig. 2C, showing that migration in the presence of either mutant is similar to basal migration in the absence of HGF. To examine whether reduced activities in cell migration correlated with reduced Met phosphorylation, a subset of HGF mutants was examined in a kinase receptor assay (KIRA). For wild type HGF and HGF mutants, maximal Met phosphorylation was observed at concentrations between 0.63 and 1.25 nM

(Fig. 3). The maximal Met phosphorylation achieved by mutants Y673A [c195], R695A [c217], and G696A [c219] was less than 30% of wild type, agreeing with their minimal or absent pro-migratory activities. Mutants Q534A [c57], D578A [c102], and V692A [c214] had intermediate activities (30 – 60%) in cell migration assays; they also had intermediate levels of Met phosphorylation, having 56 – 83% that of wild type HGF. In agreement with its lack of cell migration activity, scHGF had no Met phosphorylation activity (Fig. 3). Effect of ␤-Chain Mutations on Binding of HGF and HGF ␤-Chain to Met—The affinity of each mutant for Met-IgG fusion protein was analyzed by HGF competition binding. Except for K649A [c173] and Y673A [c195] (both ⬃4-fold weaker binding), all HGF mutants had essentially the same binding affinity as two-chain HGF (IC50 ⫽ 0.83 ⫾ 0.32 nM; n ⫽ 30), indicated by their IC50 ratios (IC50mut/IC50WT), which ranged from 0.36 to 2.25 (Table I). We also examined the cell migration activities of


FIG. 3. HGF-dependent phosphorylation of Met by HGF mutants. Phosphorylation of Met of A549 cells was carried out as described under “Experimental Procedures” using various concentrations of HGF (●), scHGF (⽧), HGF Q534A [c57] (E), HGF D578A [c102] (Œ), HGF Y673A [c195] (‚), HGF V692A [c214] (〫), HGF R695A [c217] (䡺), and HGF G696A [c219] ().

selected mutants at 10- and 50-fold higher concentrations; no increase in pro-migratory activity was observed (Table II). Therefore, the impaired function of HGF mutants is not due to reduced overall binding to Met, since an increase in concentration of up to 50-fold had no compensatory effect. The poor correlation between HGF mutant binding to Met and either HGF-dependent cell migration or Met phosphorylation is likely due to the high affinity between Met and the HGF ␣-chain. The HGF ␣-chain dominates the overall binding and thus masks any altered Met interactions stemming from the low affinity HGF ␤-chain. Therefore, we made mutations in HGF ␤ itself to eliminate any ␣-chain effects. The binding of selected HGF ␤ mutants (Q534A [c57], D578A [c102], Y619 [c143], R695A [c217], G696A [c219], and I699A [c221a]) to the Met receptor was tested in an HGF ␤/Met competition binding ELISA. Mutants were made in the HGF ␤ C604S [c128] background to avoid any potential dimerization during purification, although this mutation had no effect on binding to Met (Fig. 4). All mutants had reduced binding affinity to Met and R695A [c217] and G696A [c219] did not compete for binding at all (Fig. 4). The binding affinities of the mutants were then normalized to HGF ␤, which had an IC50 ⫽ 0.55 ⫾ 0.38 ␮M (n ⫽ 16). The results are summarized in Table III and include binding data of previously characterized HGF ␤ mutants Y673A [c195] and V692A [c214] (22). We found that mutants R695A [c217], G696A [c219], and Y673A [c195] had the greatest loss in migration activity (as two-chain full-length HGF mutants) and also had the greatest loss in Met binding (as HGF ␤ mutants). Conversely, mutants with a small reduction of migration activity (Y619A [c143] and I699A [c221a]) also had a small (less than 10-fold) reduction in Met binding (Table III). Thus, the elimination of HGF ␣-chain binding contribution in this Met binding assay revealed that the reduced migration activity of full-length HGF mutants was due to an impaired binding interaction of the HGF ␤-chain with the Met receptor. Location of the Met Binding Site in the Crystal Structure of HGF ␤—To better interpret Met binding and activity data from HGF mutants, we determined the HGF ␤ structure at 2.53 Å resolution. Data reduction, refinement statistics, and final model metrics appear in Table IV. HGF ␤ adopts the fold of chymotrypsin-like serine proteases, comprising two tandem distorted ␤-barrels. There are two poorly ordered and untraceable segments, His645–Thr651 [c170a-c175] and the C-terminal region beginning with Tyr723 [c245]. The active site region of

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HGF ␤ clearly differs from those of true proteases (Fig. 5A). Only Asp578 [c102] of the canonical “catalytic triad” is present, Ser and His being changed to Tyr673 [c195] and Gln534 [c57], respectively. As a result, the interaction between Ser and His, supported by an Asp-His hydrogen bond, is impossible, and Tyr673 [c195] significantly narrows the entrance to the “S1 pocket.” In addition, Pro693 [c215] is distinct from Trp [c215] found in all serine proteases. Indeed, normal substrate binding via main chain hydrogen bonds to segment [c214-c216] would be severely hampered by the main chain conformation and side chains of Val692 [c214] and Pro693 [c215] (Fig. 5B). Furthermore, there are structural differences in the nominal “S1 pocket,” where Gly667 [c189] at the bottom of the pocket and Pro668 [c190] are also distinct from residues found in serine proteases. Thus we have a structural basis to understand why mutations in HGF creating the Asp [c102]-His [c57]-Ser [c195] catalytic triad would be insufficient to impart catalytic activity. HGF ␤ residues important for interactions with Met are shown in Fig. 5, C and D, according to their relative activities in cell migration assays. The Met binding site is compact and centered on the “active site region.” This core region comprises “catalytic triad” residues (Gln534 [c57], Asp578 [c102], and Tyr673 [c195]) and residues of the [c220] loop (Val692 [c214], Pro693 [c215], Gly694 [c216], Arg695 [c217], and Gly696 [c219]). None of our Ala substitutions require large changes of main chain conformation except perhaps at Gly696 [c219]. Here, the ␾/␺ main chain torsion angles are in a restricted portion of the Ramachandran plot, suggesting that a non-Gly residue could cause conformational changes in the [c220] loop, which may explain the reduced activity of the G696A [c219] mutant. The electrostatic surface charge distribution in the binding site is diverse, being nonpolar at Tyr673 [c195] and Val692 [c214], polar at Gln534 [c57], negatively charged at Asp578 [c102], and positively charged at Arg695 [c217]. The outer limit of the functional Met binding site extends to distal portions of the [c220] loop (residues 699 and 701), the [c140] loop (residues 619 – 621), and residues 514 and 537 (Fig. 5, C and D). Recently, the HGF ␤/Met binding site in the crystal structure of HGF ␤ bound to the N-terminal portion of Met comprising the Sema and PSI domains was identified (22). Superimposition (33) of unbound HGF ␤ (present study) and HGF ␤ bound to Met gave a root mean square deviation value of 0.5 Å for the 203 C␣ pairs, which excludes residues Gly638–Asn653 (some are not seen in the present study and others are strongly shifted in the Met complex), Gly694 [c216] and Arg695 [c217] which are shifted by about 2 Å, and six others far from the Met binding site. While keeping these differences in mind, we can use our HGF ␤ structure to delineate the structural binding site from the HGF ␤䡠Met structure in order to compare the functional and structural binding sites (Fig. 5D). Fig. 5D shows an almost complete overlap of the Met binding sites identified by functional and structural approaches. The four residues that lie outside the structural binding site, i.e. Arg514 [c36], Thr620 [c144], Gly621 [c145], and Asn701 [c223], are all situated at the periphery, consistent with their small effects in cell migration assays. Intermolecular Environment of HGF ␤ Crystals—In HGF ␤ crystals each molecule has three intermolecular contacts (Fig. 6A). The smallest contact (⬃360 Å2 on each side) involves residues in the Ile550–Lys562 [c70-c80] loop on one molecule and residues near the ␣-chain connecting Cys604 [c128] (mutated to Ser in this construct) site on the other molecule. Two larger intermolecular contacts are derived from 2-fold crystallographic symmetry. Residues following the N terminus (Val496–Arg502 [c17-c23]) plus residues from the [c140] and [c180] loops lose ⬃640 Å2 of solvent-accessible area (each side), and residues centered on Gln534 [c57] share a contact area of ⬃930 Å2 (each side).


39920

TABLE I Relative binding affinity of two-chain full-length HGF mutants to Met

a

HGF mutant

IC50mut/IC50WT ⫾ S.D.

HGF mutant

IC50mut/IC50WT ⫾ S.D.

I499A 关c20兴 R514A 关c36兴 N515A 关c38兴 Q534A 关c57兴 P537A 关c60a兴 R539A 关c60c兴 I550A 关c70兴 D552A 关c72兴 V553A 关c73兴 E559A 关c77兴 E575A 关c99兴 G576A 关c100兴 D578A 关c102兴 Y619A 关c143兴 T620A 关c144兴 G621A 关c145兴 L622A 关c146兴 I623A 关c149兴 N624A 关c150兴 Y625A 关c151兴

0.52 1.41 ⫾ 0.23 1.16 2.04 ⫾ 0.86 1.67 0.94 1.39 1.23 0.99 ⫾ 0.26 1.34 ⫾ 0.07 1.19 ⫾ 0.05 0.78 1.86 ⫾ 0.82 1.52 ⫾ 0.28 1.89 ⫾ 0.32 1.08 ⫾ 0.30 1.04 ⫾ 0.22 0.49 ⫾ 0.10 0.71 ⫾ 0.18 0.65 ⫾ 0.26

M637A 关c163兴 K641A 关c167兴 K649A 关c173兴 A661N 关c184a兴 K663A 关c186兴 G665A 关c188兴 E670A 关c192兴 Y673A 关c195兴 V692A 关c214兴 P693A 关c215兴 G694A 关c216兴 R695A 关c217兴 G696A 关c219兴 A698G 关c221兴 I699A 关c221a兴 P700A 关c222兴 N701A 关c223兴 R702A 关c224兴 scHGF Wild type HGFa

1.38 1.04 3.66 1.04 ⫾ 0.34 0.73 ⫾ 0.20 0.36 ⫾ 0.03 1.77 4.41 ⫾ 1.03 1.74 ⫾ 0.16 1.37 ⫾ 0.46 1.76 ⫾ 0.72 1.48 ⫾ 0.52 2.03 ⫾ 1.04 0.73 ⫾ 0.35 1.79 ⫾ 0.70 1.30 ⫾ 0.46 1.48 ⫾ 0.59 2.25 4.03 ⫾ 1.05 1

Wild type two-chain HGF had an IC50 ⫽ 0.83 ⫾ 0.32 nM (n ⫽ 30) in the HGF/Met competition binding ELISA.

TABLE II Pro-migratory activities of two-chain full-length HGF mutants at different concentrations Mutant

Y673A 关c195兴 V692A 关c214兴 G694A 关c216兴 R695A 关c217兴 G696A 关c219兴

Pro-migratory activity at 1 nM



% control

% control

% control

13.9 ⫾ 8.9 49.5 ⫾ 17.7 47.6 ⫾ 19.7 ⫺8.9 ⫾ 5.4 ⫺13.6 ⫾ 13.7

9.8 ⫾ 8.3 20.9 ⫾ 6.9 23.2 ⫾ 11.4 ⫺4.4 ⫾ 11.6 4.0 ⫾ 19.8

9.1 ⫾ 8.6 29.8 ⫾ 6.3 21.2 ⫾ 5.9 5.3 ⫾ 10.2 2.8 ⫾ 7.1

TABLE III Effects of mutations of the ␤-chain in HGF ␤ and two-chain full-length HGF Met competition binding of HGF ␤ mutants and cell migration activity of HGF mutants are shown.

Mutant

Q534A 关c57兴 D578A 关c102兴 Y619A 关c143兴 Y673A 关c195兴 V692A 关c214兴 R695A 关c217兴 G696A 关c219兴 I699A 关c221a兴

HGF ␤ mutant

Two-chain full-length HGF mutant

Relative binding affinitya (IC50 mutant/IC50 HGF ␤)

Percent reduction of pro-migratory activityb

12.5 ⫾ 3.6 16.6 ⫾ 8.2 5.1 ⫾ 0.2 ⬎100c ⬎50c ⬎100 ⬎100 7.3 ⫾ 0.9

45 ⫾ 16 65 ⫾ 9 27 ⫾ 13 86 ⫾ 9 51 ⫾ 18 109 ⫾ 14 114 ⫾ 14 20 ⫾ 17

a Values were determined by using the HGF ␤/Met competition binding ELISA. b Values are relative to wild type two-chain HGF activity ( ⫽ 100%) in the cell migration assay. c Data were taken from Stamos et al. (22).

FIG. 4. Met competition binding of HGF ␤ mutants. The HGF ␤/Met-IgG competition ELISA described in Fig. 1A was used to assess Met binding of wild type HGF ␤ (‚), HGF ␤ (●), and HGF ␤ mutants Q534A [c57] (E), D578A [c102] (Œ), Y619A [c143] (〫), R695A [c217] (䡺), G696A [c219] (), and I699A [c221a] (⽧). Data were fit by a fourparameter fit by using Kaleidagraph; representative individual competition assays are shown for multiple independent determinations where n ⱖ 3. DISCUSSION

HGF acquires biological activity only upon proteolytic conversion of the single-chain precursor pro-HGF into two-chain HGF (11–14). Based on the structural similarity of HGF with chymotrypsin-like serine proteases and plasminogen in particular, we propose that this activation process is associated with structural changes occurring in the HGF ␤-chain. Here we provide evidence that the “activated” form of the HGF ␤-chain contains a distinct Met binding site located in a region that

corresponds to the substrate/inhibitor binding site of chymotrypsin-like serine proteases. HGF Binding Interactions with Met—Binding studies with purified HGF ␤-chains revealed that the “activated form” of HGF ␤ binds to Met with ⬃14-fold higher affinity than its precursor form, scHGF ␤, consistent with the view that optimization of the Met binding site is contingent upon processing of single-chain HGF. This suggested that the Met binding site includes the HGF region undergoing conformational rearrangements after pro-HGF cleavage, i.e. the “activation domain.” Indeed, functional analysis of HGF variants with amino acid substitutions in the “activation domain” led to the identification of the functional Met binding site. However, HGF mutants with the greatest reduction in pro-migratory activities (Q534A [c57], D578A [c102], Y673A [c195], V692A [c214], P693A [c215], G694A [c216], R695A [c217], G696A [c219], and R702A [c224]) displayed essentially unchanged binding affinities for Met, except for Y673A [c195] (4-fold reduction), because HGF affinity is dominated by the HGF ␣-chain (20, 21). Consistent with this, the reduced activities remained unchanged upon increasing the concentration of HGF mutants by more than 50-fold (Table II). Therefore, the reduced activities of HGF mutants were interpreted as resulting from perturbed molecular interactions of HGF ␤-chain with its specific, low affinity binding site on Met. In support of this, we found that the


39921

TABLE IV Structure statistics for HGF ␤ Data in space group P3121, a ⫽ 63.7 Å and c ⫽ 135.1 Å. Resolution

Nmeasa

Nref b

5835 5882 5896 5790 5724 5903 5875 5575 5005 3350 54,835

1219 1143 1134 1107 1097 1115 1117 1072 1077 886 10,967

I/␴

Completec

Rmerged

Rworke

Rfree f

0.032 0.035 0.043 0.060 0.086 0.126 0.190 0.269 0.367 0.368 0.064

0.274 0.211 0.216 0.237 0.265 0.295 0.287 0.278 0.294 0.323 0.246

0.309 0.277 0.260 0.291 0.330 0.352 0.356 0.327 0.253 0.385 0.303

Å

5.45–50.0 4.33–5.45 3.78–4.33 3.43–3.78 3.19–3.43 3.00–3.19 2.85–3.00 2.73–2.85 2.62–2.73 2.53–2.62 2.53–50.0

100 100 100 100 100 100 100 100 98 83 98

44 43 36 28 20 13 8.8 5.9 3.6 2.7 24

Final model Contents of model Residues

227

Atomsg

1798 (106)

Root mean square deviations Waters

Bonds

Angles

B-factor

33

0.012 Å

1.5°

5 Å2

a

Nmeas is the total number of observations measured. Nref is the number of unique reflections measured at least once. Complete is the percentage of possible reflections actually measured at least once. d Rmerge ⫽ ⌺储I兩 ⫺ 兩具I典储/⌺ 具I典兩, where I is the intensity of a single observation, and 具I典 is the average intensity for symmetry equivalent observations. e Rwork ⫽ ⌺兩Fo ⫺ Fc兩/⌺ 兩Fo兩, where Fo and Fc are observed and calculated structure factor amplitudes, respectively. f Rfree ⫽ Rwork for 531 reflections (5%) sequestered from refinement, selected at random from 99 resolution shells. R for all reflections is 0.249. g Number in parentheses is number of atoms assigned zero occupancy. b c

reduced biological activities of selected HGF mutants (twochain full-length) were well correlated with reduced Met binding of their corresponding HGF ␤ mutants in an assay that eliminated the binding contribution of the HGF ␣-chain (Table III). For instance, HGF ␤ mutants R695A [c217], G696A [c219], and Y673A [c195] had no measurable Met binding, correlating with greatly impaired biological functions as full-length mutants. As mapped onto the crystal structure of HGF ␤, the functional Met binding site is centered on “catalytic triad” residues Gln534 [c57], Asp578 [c102], and Tyr673 [c195] and five residues of the [c220] “activation domain” loop (Val692 [c214], Pro693 [c215], Gly694 [c216], Arg695 [c217], and Gly696 [c219]). Together, these residues define a region that bears a remarkable resemblance to the substrate-processing region of true serine proteases. The functional importance of the [c220] loop has precedent in the well described family of chymotrypsin-like serine proteases (15, 16). The extended canonical conformation of substrates and inhibitors includes residues that can form main chain interactions from [c214] to [c216]. This region is also recognized as an allosteric regulator of thrombin catalytic activity (34) and as an interaction site with its inhibitor hirudin (35). In addition, residues in factor VIIa and thrombin that correspond to HGF Arg695 [c217] are important for enzymecatalyzed substrate processing (36, 37). In MSP, the closest structural homolog of HGF, this residue (Arg683 [c217]) plays a pivotal role in the high affinity interaction of MSP ␤-chain with its receptor Ron (38). Arg683 [c217] is part of a cluster of five surface-exposed arginines proposed to be involved in Ron binding (19). Although only Arg695 [c217] and possibly Lys649 [c173] are conserved in HGF, these residues are all located within the Met binding region of the HGF ␤-chain, suggesting that the Ron binding site on the MSP ␤-chain is highly homologous. The functional binding site identified herein is in excellent agreement with the structural Met binding site revealed in the crystal structure of the complex of HGF ␤ bound to soluble Met Sema/PSI domain (22). The 17 identified functional binding residues are located within or proximal to the structural binding region (Fig. 5D), which mainly interacts with three sepa-

rate loops from the Met Sema domain. Notably, residues of the functional “core” region, e.g. Tyr673 [c195], Val692 [c214], Pro693 [c215], Gly694 [c216], Arg695 [c217], and Gly696 [c219], also make the most important contacts to the Met receptor in the crystal structure. For example, Met residues Tyr125 and Tyr126 that are in the core of the binding interface pack against the HGF ␤-chain residue Arg695 [c217]. Thus, the results derived from three different experimental approaches, functional studies with HGF mutants, Met binding assays with HGF ␤ mutants, and the HGF ␤䡠Met crystal structure, are consistent and provide strong evidence for a distinct Met binding site located at the active site region of the HGF ␤-chain. Our findings with HGF Ala mutants agree with a previous study where Tyr673 [c195] and Val692 [c214] were each replaced by serine (12). The normal biological activity measured for HGF variant Q534H [c57] in two previous studies (12, 39) may reflect functional compensation of Gln by His, a relatively close isostere. However, our results contrast with previous studies demonstrating that HGF ␤-chain itself did not bind to Met (11, 32). In one instance, the HGF ␤-chain was different from ours, having extra ␣-chain residues derived from elastase cleavage of HGF, which could adversely affect Met binding. However, it is more likely that either the concentrations used, the sensitivity of the assays, or the extent of pro-HGF processing may have been insufficient to observe binding to this low affinity site. HGF ␤-chain has been reported to bind to Met although only in the presence of NK4 fragment from the ␣-chain (32). Signaling Mechanisms—In principle, the existence of two Met binding sites, one high affinity and one low affinity, in one HGF molecule supports a 2:1 model of a Met䡠HGF signaling complex, analogous to the proposed 2:1 model of the related Ron䡠MSP system (19). As found with HGF, the individual ␣and ␤-chains of MSP bind to their receptor but do not induce signaling (18, 38). High affinity binding is also dominated by one of the chains, although in the case of MSP it is the ␤-chain. Compared with full-length MSP, the MSP ␣-chain alone binds with ⬃100-fold reduced affinity. However, biochemical studies have not identified any 2:1 complexes of Met䡠HGF (40). This could be due to the low affinity interaction between Met and

39922


FIG. 5. HGF ␤ x-ray structure and Met binding site. A, structure and electron density of HGF ␤ “active site region.” The “active site catalytic triad residues” Asp578 [c102]-Gln534 [c57]-Tyr673 [c195] are depicted. Pro693 [c215] adopts a different conformation than Trp [c215] found in serine proteases and partially blocks the entrance to the “S1 pocket,” which has a Gly667 [c189] at the bottom. B, stereo view of active site regions of HGF ␤ (green) and plasmin (gray). The pseudo-substrate inhibitor Glu-Gly-Arg-chloromethyl ketone from the plasmin structure (yellow) fills the S1 pocket and interacts with its Asp [c189] side chain. The main chain amide nitrogen atoms that stabilize the oxyanion hole (blue spheres) are structurally conserved in HGF ␤. C, location of Met binding site on HGF ␤. Worm depiction of HGF ␤ showing mutated residues with ⬍20% (red), 20 – 60% (orange), 60 – 80% (yellow), and ⬎80% (blue) of wild type HGF pro-migratory activity data in Fig. 2B. The N terminus and three activation domain loops are in black. Residue Lys649 [c173] would be colored yellow but is disordered in the crystal structure and is not depicted. D, solvent-accessible surface of HGF ␤ showing residues colored as in C. The dotted line depicts the Met binding region from the crystal structure of the complex of HGF ␤ with the Sema/PSI domains of Met (22).

HGF ␤; perhaps more stable complexes are only found on cell surfaces with membrane-anchored Met or with additional contributions by heparin-like surface molecules. Further experiments may shed light on this possibility. Alternatively, the HGF ␤-chain may have critical functions in receptor activation beyond those involved in direct interactions with Met that would favor a 2:2 complex of HGF䡠Met.

Upon inspection of intermolecular contacts in the HGF ␤ crystal lattice, we observed that one of the dimer interfaces (green and blue molecules in Fig. 6A) borders the Met binding site and comprises parts of the N-terminal peptide (Val496–Arg520 [c17c23]) and adjacent residues from the [c140] and [c180] loops. This contact site must be very different in the single-chain form of HGF as it includes the activation cleavage site. If such an


39923

FIG. 6. HGF ␤ intermolecular contacts and comparison to other proteins. A, intermolecular contacts in HGF ␤ x-ray structure. The reference molecule (green) has three crystal contacts. The blue molecule arises from a 2-fold axis relating the N-terminal regions Val496–Arg502 [c17-c23] and adjacent residues. The N-terminal Val495 [c16] of the green and blue molecules are depicted as spheres. The HGF ␤-chain/␤-chain interface involving the N terminus and adjacent residues from the [c140] and [c180] loops is shown by the arrow. The salmon-colored molecule arises from a 2-fold axis relating “active site regions.” The side chains of Tyr673 [c195] are shown. Residue C604S [c128] (sphere) in the yellow molecule contacts the reference molecule in the [c70] loop. B, partial sequences for HGF and homologous proteins at the border between ␣- and ␤-chains. HGF and chymotrypsinogen numbering are above and below sequences, respectively. The boxed Cys in the ␣-chain forms a disulfide bond with a Cys in the ␤-chain. t-PA, tissue plasminogen activator.

HGF ␤-chain dimer interaction is important for Met signaling, it would explain why the single-chain form lacks any biological activity, despite weak Met interactions through its incompletely formed “active site region.” In this model the HGF ␤-chain interaction with Met would serve to properly orient the ␤-chain/␤-chain interaction site. Whereas this HGF ␤-chain/␤chain contact may be a crystallization artifact, the presence of the identical contact in the crystal lattice of the HGF ␤䡠Met complex offers some support (22). A dimeric arrangement of HGF ␤ modules in the HGF䡠Met signaling complex would favor a 2:2 model in which two individual HGF䡠Met complexes form a higher order signaling complex consisting of two HGF and two Met molecules (9). If this model is correct, then amino acid changes in this putative dimer interface may adversely affect Met-dependent functions. More extensive studies are needed to unequivocally support or reject this hypothesis. Comparison of HGF ␤ with Plasmin and Other Proteins— Among proteins with reported molecular structures, the amino acid sequence of HGF ␤ is most homologous with that of plas-

min/plasminogen, having 37% identity. Superimposition (33) of the plasmin protease domain 1BUI (41, 42) with HGF ␤ yields a root mean square deviation of 1.2 Å for 192 C␣ pairs (out of 227 in our HGF ␤ structure). A structure-based sequence alignment with plasmin shows that HGF ␤ has single amino acid deletions immediately before and after the sequence 505IGWMVSLRYR514 (Fig. 6B), another single amino acid deletion following QCF536 (Gln534 is homologous with His [c57]), and a two-amino acid insertion between His554 [c74] and Gly557 [c75]. The deletions following Arg514 [c36] and Phe536 [c59] and the insertion after His554 [c74] are in loop regions where length heterogeneity among homologous proteins is common. However, the deletion preceding Ile505 [c27] is unusual. To our knowledge, it appears only in HGF and its closest relative MSP among homologous human protein sequences. In comparison with plasmin, the trace of HGF ␤ in this segment is more direct between Thr503 [c24] and Gly506 [c28]. Furthermore, the plasmin structure (42) includes the C-terminal fragment from the plasmin 〈-chain, which is connected to the protease domain

39924


with two disulfide bonds. In HGF, the ␣-chain to ␤-chain link homologous to plasmin Cys567/Cys685 [c122] has been proposed to form between Cys487 and Cys604 [c128] (9). However, this may not be the case since Cys561 [c79] could also form a disulfide with Cys487 as suggested recently (22). The nonenzymatic “catalytic triad” of HGF is shared by the acute phase plasma protein haptoglobin (43), the Trypanosome lytic factor binding protein haptoglobin-related protein (44), and the blood coagulation cofactor protein Z (45). Like HGF, they retain the intact “catalytic triad residue” Asp [c102], but have changes in residues [c57] (Lys) and [c195] (Ala or Gly). MSP, the other plasminogen-related growth factor, also has a nonenzymatic “catalytic triad” in which residues [c57] and [c102] are each changed to Gln. Except for MSP, which uses the ␤-chain for a high affinity interaction with its receptor tyrosine kinase Ron, the role of these other nonenzymatic protease-like domains is not well understood. It is tempting to speculate that their function may involve activation-dependent formation of a protein binding site similar to that found on the ␤-chains of HGF and MSP. Although zymogen forms of proteases are generally not catalytically competent, some are still capable of binding and even cleaving substrates. For example, single-chain forms of tissue plasminogen activator and urokinase-type plasminogen activator still have catalytic activity, albeit somewhat reduced from their activated forms (46, 47). Thus, binding of the zymogenlike ␤-chain of pro-HGF to Met would not be without precedent; our binding data of scHGF ␤ to Met supports this idea. Another HGF ␤-chain region with the potential for proteinprotein interactions corresponds to exosite I of thrombin (fibrinogen-binding exosite). Exosite I is present in zymogen and active forms of thrombin (48) and contains a positively charged patch centered at the [c70 – 80] loop, which is involved in interactions with substrates, cofactors, and inhibitors (35). HGF ␤ also has a positively charged surface in this region, suggesting a potential role in protein interactions. Although two mutational changes introduced in this region (I550-E559 [c70-c77]) did not affect HGF function in cell migration assays, the possibility of this region interacting with cell surface co-stimulatory factors of Met signaling remains. Conclusions—In conclusion, the results presented herein show that the ␤-chain of HGF contains a new interaction site with Met, which is similar to the active site region of serine proteases. Thus HGF is bivalent, having a high affinity Met binding site in the ␣-chain and a low affinity site in the ␤-chain. Other important interactions may occur between two HGF ␤-chains, two HGF ␣-chains (9), and as found with MSP/Ron (49), between two Met Sema domains (50). Furthermore, heparin also plays a key role in HGF/Met receptor binding. The identification of a distinct Met binding site on the HGF ␤-chain may further the design of new classes of Met inhibitors with therapeutic potential for cancer. Acknowledgments—We thank Jenny Stamos for the Met ECD construct; Laura DeForge for KIRA support; Sokunthea Chen for HGF molecular biology; Ignacio Aliagas for a preliminary model of HGF ␤; Genentech support groups (oligonucleotide synthesis, DNA sequencing, protein sequencing, and mass spectrometry); and Bart de Vos for helpful discussions and support. We are grateful to Gerry McDermott for assistance with x-ray data collection. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, Materials Sciences Division, of the United States Department of Energy under Contract DE-AC03-76SF00098 at Lawrence Berkeley National Laboratory. REFERENCES 1. Nakamura, T., Nishizawa, T., Hagiya, M., Seki, T., Shimonishi, M., Sugimura, A., Tashiro, K., and Shimizu, S. (1989) Nature 342, 440 – 443

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