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J. Biol. Chem., Vol. 277, Issue 50, 48696-48707, December 13, 2002

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Utilization of a Novel Recombinant Myoglobin Fusion Protein Expression System to Characterize the Tissue Inhibitor of Metalloproteinase (TIMP)-4 and TIMP-2 C-terminal Domain and Tails by Mutagenesis

THE IMPORTANCE OF ACIDIC RESIDUES IN BINDING THE MMP-2 HEMOPEXIN C DOMAIN^*

Heidi S.-T. Kai, Georgina S. Butler, Charlotte J. Morrison, Angela E. King, Gayle R. Pelman^§, and Christopher M. Overall^§¶

From the  Canadian Institute of Health Research Group in Matrix Dynamics and the Department of Oral Biological and Medical Sciences, Faculty of Dentistry, and the ^§ Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada

Received for publication, September 6, 2002, and in revised form, October 7, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Tissue inhibitor of metalloproteinase (TIMP)-4 binds pro-matrix metalloproteinase (MMP)-2 and efficiently inhibits MT1-MMP, but unlike TIMP-2 neither forms a trimolecular complex nor supports pro-MMP-2 activation. To investigate the structural and functional differences between these two TIMPs, the C-terminal domains (C-TIMP-4 and C-TIMP-2) were expressed independently from their N domains and mutations were introduced into the C-terminal tails. Myoglobin was used as a novel recombinant fusion protein partner because spectroscopic measurement of the heme Soret absorbance at 408 nm readily enabled calculation of the molar equivalent of the red-colored recombinant protein, even in complex protein mixtures. Both C-TIMP-4 and C-TIMP-2 bound pro-MMP-2 and blocked concanavalin A-induced cellular activation of the enzyme. Measurement of k_on rates revealed that the inhibition of MMP-2 by TIMP-4 is preceded by a C domain docking interaction, but in contrast to TIMP-2, this is not enhanced by a C-terminal tail interaction and so occurs at a slower rate. Indeed, the binding stability of C-TIMP-4 was unaltered by deletion of the C-terminal tail, but replacement with the tail of TIMP-2 increased its affinity for pro-MMP-2 by ~2-fold, as did substitution with the TIMP-2 C-terminal tail acidic residues in the tail of C-TIMP-4 (V193E/Q194D). Conversely, substitution of the C-terminal tail of C-TIMP-2 with that of TIMP-4 reduced pro-MMP-2 binding by ~75%, as did reduction of its acidic character by mutation to the corresponding TIMP-4 amino acid residues (E192V/D193Q). Together, this shows the importance of Glu¹⁹² and Asp¹⁹³ in TIMP-2 binding to pro-MMP-2; the lack of these acidic residues in the TIMP-4 C-terminal tail, which reduces the stability of complex formation with the MMP-2 hemopexin C domain, probably precludes TIMP-4 from supporting the activation of pro-MMP-2.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Matrix metalloproteinases (MMPs)¹ are a family of important processing enzymes that can cleave and regulate the activity of an expanding degradome of bioactive molecules (1-5) as well as degrade extracellular matrix proteins in pathology (6). Gelatinase A (MMP-2) has been implicated in numerous biological processes, including the activation of cytokines such as tumor necrosis factor- (7), transforming growth factor-1 (8), and interleukin-1 (9). MMP-2 has also been shown to have anti-inflammatory actions by converting monocyte chemokine agonists to antagonists (10, 11) and causes loss of protection of CD4⁺ cells from human immunodeficiency virus-1 infection by processing stromal cell-derived factor-1 (12).² MMP-2 cleavage of type IV collagen, a major component of basement membranes, is important for tumor cell metastasis and angiogenesis (14-16). In view of these diverse and biologically important functions, it is not surprising that MMPs are under tight regulatory control, both at the transcriptional and post-transcriptional levels (17-19). Post-translational regulation is also pivotally important in regulating proteolytic activity in the pericellular and extracellular compartments and involves zymogen activation and inhibition by four endogenous inhibitors, the tissue inhibitor of metalloproteinases (TIMPs) (2). In contrast to the majority of MMPs, MMP-2 is constitutively expressed by a large number of cell types, indicating its importance during normal cellular functions, and is frequently overexpressed in metastatic tumors or reactive stroma (3, 20). Hence, activation of the MMP-2 zymogen is a critical control point in regulating its activity (21).

Pro-MMP-2 can be activated at the cell surface by membrane type 1 (MT1)-MMP (22-26) and by MT2-MMP (27). The MT1-MMP pathway requires TIMP-2 to tether pro-MMP-2 to MT1-MMP (21, 23, 24, 26). The inhibitory N-terminal domain of TIMP-2 binds the MT1-MMP catalytic domain, inhibiting its activity, and the TIMP-2 C-terminal noninhibitory domain docks with the pro-MMP-2 hemopexin C domain, generating a ternary complex (23, 26). Pro-MMP-2 is then activated on the cell surface after forming a quaternary activation complex with a free MT1-MMP molecule, a process particularly sensitive to TIMP-2 and TIMP-4 levels (24, 26, 27), clustering (28-30), and MT1-MMP interaction with native collagen (31). In contrast, the TIMP-2-independent activation of pro-MMP-2 by MT2-MMP is only inhibited by TIMP-2 (27). Although yeast two-hybrid analysis revealed that the isolated C domain of TIMP-2 stably interacts with the hemopexin C domain of MMP-2 (10, 28), this interaction has not been measured biochemically, nor has the relative importance of the molecular determinants critical for interaction with the hemopexin C domain been characterized in the absence of the N-domain, due in part to the difficulty in expressing small recombinant protein domains.

TIMP-4 also binds to pro-MMP-2 at the same site as TIMP-2 (26, 32), but it is unable to support pro-MMP-2 activation or to compete with TIMP-2 in the binding of pro-MMP-2 (26). However, TIMP-4 can inhibit the activation of pro-MMP-2 by inhibiting free MT1-MMP (26, 33) and MT2-MMP (27). Similarly, TIMP-3 has been shown to bind pro-MMP-2 and can inhibit MT-MMPs (34), but its ability to support activation by MT1-MMP is not reported. Hence, the role of TIMP-4 in binding pro-MMP-2 remains unknown, and the structural or sequence constraints that preclude the formation of functional trimolecular complexes remain to be characterized.

TIMP-2, TIMP-3, and TIMP-4 have nine additional C-terminal amino acid residues, termed the C-terminal tail, compared with TIMP-1 (Table I). Notably, TIMP-1 is the only member of the TIMP family that cannot interact with pro-MMP-2 through its C domain (35, 36) or inhibit MT-MMPs through its inhibitory N-domain (24). Mutagenic analysis has identified key amino acids in the A-B loop of the N-domain of TIMP-2, which are absent in TIMP-1, accounting for its lack of MT1-MMP-inhibitory properties (37). The TIMP-2 C-terminal tail was proposed to be critical for binding both active and pro-MMP-2 at the same site on the hemopexin C domain (38, 39). However, this docking site was mapped by mutagenesis to lie at the junction of modules III and IV on the lower rim of the hemopexin C domain (40). Hence, this indicated that the binding interaction to pro-MMP-2 differs from the inhibitory complex formed between TIMP-2 and active MMPs in which TIMPs adopt an elongated wedge morphology (41).

The C-terminal tails of TIMP-4 and TIMP-2 differ by four residues, two of which (Glu¹⁹² and Asp¹⁹³) are acidic in TIMP-2, making this tail more anionic than that of TIMP-4 (Table I). The negatively charged tail is proposed to form salt bridges with cationic clusters located on the hemopexin C domain of MMP-2 (40). We hypothesized that the absence of the two anionic residues from the C-terminal tail of TIMP-4 reduces the binding affinity of TIMP-4 for the hemopexin C domain of MMP-2 compared with TIMP-2, rendering it incapable of participating in pro-MMP-2 activation. After our present mutagenesis studies were initiated to test this hypothesis, the very recent report of the three-dimensional structure of TIMP-2 bound to pro-MMP-2 (42) confirmed the importance of the seven basic residues in the hemopexin C domain that we had previously identified by mutagenesis as being important in the interaction with TIMP-2 (40). Further, the structure revealed the importance of hydrophobic interactions, hydrogen bond formation and several salt bridges in the C domain of TIMP-2 that do not involve the C-terminal tail in forming the large ~250-nm² interface between these two complexed proteins (42). However, the C-terminal two residues of the tail were not resolved, so any potential salt bridge formation involving Asp¹⁹³ could not be determined. Notably, the C-terminal tail binds the rim of hemopexin module III, starting at the interface with module IV (42), at an extended binding site that had also been previously identified by mutagenesis to be separate from the main binding site (40). This site of interaction of the C-terminal tail on module III is intriguing; it is spatially and structurally distinct from the main interaction site of the C domain of TIMP-2 on the rim of hemopexin module IV (42), but its relatively small surface area belies the importance ascribed to the C-terminal tail previously in kinetic (38, 39) and mutagenesis studies (28, 38, 40). Hence, unresolved from the structural studies is the relative importance played by the different contact elements of the binding surfaces to the total binding interaction which may be targeted by new anti-MMP drugs (18).

To investigate the interactions and differences in the C domains of TIMP-4 and TIMP-2 (C-TIMP-4 and C-TIMP-2) in the binding and activation of pro-MMP-2, C-TIMP-4 and C-TIMP-2 were expressed independently to avoid any effects of the inhibitory N-domains. To facilitate this, we developed a novel expression system that utilizes the red-colored protein myoglobin as a fusion partner. Myoglobin has spectrometric properties that readily enable accurate protein quantitation throughout protein purification, even in complex mixtures. Differences between C-TIMP-4 and C-TIMP-2 binding of pro-MMP-2 were explored by mutagenesis. We report that the acidic residues Glu¹⁹² and Asp¹⁹³ in the C-terminal tail of TIMP-2 are necessary for a stable interaction between TIMP-2 and pro-MMP-2; their absence from the C-terminal tail of TIMP-4 appears to preclude formation of a stable trimolecular complex with pro-MMP-2 and MT1-MMP, so TIMP-4 is unable to promote the activation of pro-MMP-2.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- TIMP-2-free human pro-MMP-2 and the catalytically inactive mutant pro-MMP-2 E375A (43) were expressed in Timp2/ embryonic fibroblasts and purified as previously described (12, 26). MMP-2 hemopexin C domain was expressed in Escherichia coli and purified as described before (44). Human TIMP-2 and TIMP-4 were expressed in Chinese hamster ovary and baby hamster kidney cells, respectively, and purified (26). Affinity-purified polyclonal antibodies used in Western blotting included a rabbit polyclonal antibody raised to horse heart myoglobin (-Mb) and two antipeptide antibodies raised to the C-terminal tails of human TIMP-4 and TIMP-2, designated -CT4-Tail and -CT2-Tail, respectively (26, 28). An affinity-purified rabbit polyclonal antibody raised to the His₆ tag (-His₆) was used in enzyme-linked immunosorbent assays (45). The quenched fluorescence general MMP substrate ((7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-[3-(2,4-dinitrophenyl)-L-2,3-diaminoproprionyl]-Ala-Arg-NH₂) (46) was supplied by Dr. C. G. Knight (University of Cambridge).

Construction of a Myoglobin Fusion Protein Expression Vector-- The E. coli expression construct, pGYMC-Mb, which expresses high amounts of recombinant horse heart myoglobin in the bacterial cytosol (47), was engineered to add at the C terminus of myoglobin a flexible hinge (Gly-Gly) and linker amino acid residues encoded by a multiple cloning site. Mutagenesis was performed (47, 48) using the oligonucleotide 5'-PGCCTGCAGTCATTAGCTAGCAGGCCTGCGGCCGCCCTGGAAACCCAG-3' to insert NheI, StuI, and NotI restriction sites (underlined) 3' to the coding sequence for horse heart myoglobin to make the pGYMC-MbMCS vector. The mutagenesis reaction was confirmed by DNA sequencing. Recombinant horse heart myoglobin (47) and myoglobin with the C-terminal linker extension was expressed to validate the new construct.

Cloning of C-TIMP-4 and C-TIMP-2-- The coding regions of human C-TIMP-4 and human C-TIMP-2, beginning with Gly¹²⁸ and Glu¹²⁷, respectively, which lie between the conserved disulfide bonds at the junction of the TIMP N and C domains, and ending with a stop codon, were amplified from human TIMP-4 (kindly provided by Dr. Y. E. Shi, Albert Einstein College of Medicine, New York) (49) and TIMP-2 cDNAs (generously provided by Prof. D. Edwards, University of East Anglia), respectively. The following primers were used to add 5' NheI and 3' HindIII (C-TIMP-4) or 3' PstI (C-TIMP-2) restriction sites (underlined): 5'C-TIMP-4 (5'- CGGGGGGCTAGCGGCTGCCAAATCACC-3'); 5'C-TIMP-2 (5'-CGGGGGGCTAGCGAGTGCAAGATCACGC-3'); 3'C-TIMP-4 (5'-GTCAAGCTTCTAGGGCTGAACGATGTC-3'); 3'C-TIMP-2 (5'- TGCCTGCAGTTATGGGTCCTCGATGTC-3'). PCR products were gel-purified and digested with the appropriate restriction enzymes before ligation; C-TIMP-4 was cloned directly into pGYMC-MbMCS, whereas C-TIMP-2 was cloned into PCRScript (Stratagene) prior to restriction digestion and cloning into pGYMC-MbMCS. Both clones were fully sequenced.

Mutagenesis-- Residues that were targeted by mutagenesis in the C-terminal tail sequences of TIMP-4 and TIMP-2 are shown in Table I. The following sites were mutated in C-TIMP-4 (Fig. 1): K187Stop (CT4T) to delete the C-terminal tail; V193E/Q194D (CT4+q) to swap in the homologous acidic residues of the tail of TIMP-2; and K187Q/V190L/V193E/Q194D (CT4T2) to change the tail of C-TIMP-4 to that of C-TIMP-2. Mutations made in C-TIMP-2 were Q186Stop (CT2T), to delete the C-terminal tail (Fig. 1); E192V/D193Q (CT2q) to replace the acidic residues with the homologous residues of TIMP-4; and Q186K/L189V/E192V/D193Q (CT2T4) to change the tail of C-TIMP-2 to that of C-TIMP-4. Amino acid numbering commences at the NH₂-terminal Cys¹ residue of TIMP-4 and TIMP-2. Mutations were made using the QuickChange® site-directed mutagenesis kit (Stratagene) using the oligonucleotides and templates presented in Table II, and all mutant cDNAs were fully sequenced.

Recombinant Protein Preparation-- C-TIMP recombinant proteins were expressed in the E. coli strain BL21 DE3 Gold. Cultures (4 × 700 ml) were incubated at 37 °C for 24 h before harvesting. The cells were lysed, and the inclusion bodies were solubilized in 6 M guanidine-HCl according to the protocol of Steffensen et al. (45). Hemin (Sigma) was added to the solubilized protein at 5 mg/ml prior to buffer exchange and refolding in refolding buffer (55 mM Na₂CO₃, 45 mM NaHCO₂, pH 10.0, 0.02% (w/v) NaN₃) and slow step dialysis into 10 mM Tris-HCl, pH 8.0. To prevent degradation of the fusion proteins, benzamidine-Sepharose resin (5 ml) (Amersham Biosciences) equilibrated in 10 mM Tris-HCl, pH 8.0, was added to the soluble refolded protein and incubated overnight at 4 °C to bind proteases. The resin was removed from the protein solution, and NaCl and benzamidine were added to the supernatant to 0.5 M and 10 mM, respectively. The Ni²⁺-binding properties of myoglobin were utilized to purify the recombinant fusion proteins using a Ni²⁺-charged chelating Sepharose column (Amersham Biosciences) with a total column bed volume (V_t) of 30 ml, equilibrated in 10 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 10 mM benzamidine. The C-TIMPs were eluted with a 0-300 mM imidazole gradient in 10 mM Tris-HCl, pH 7.0, 1 mM benzamidine. The C-TIMP-containing eluate was dialyzed into 10 mM Tris-HCl, pH 7.0, 1 mM benzamidine prior to chromatography on CM Sepharose (Amersham Biosciences; V_t = 5 ml) equilibrated in the same buffer. Elution was accomplished with a 0-500 mM NaCl gradient in 10 mM Tris-HCl, pH 8.0, 1 mM benzamidine. The fractions containing pure recombinant protein were pooled and dialyzed into 50 mM Tris-HCl, pH 8.0, 0.15 M NaCl and then snap frozen in liquid nitrogen for storage at 70 °C. Protein concentrations were determined by analysis of the Soret absorbance at 408 nm using a standard curve of known amounts of recombinant horse heart myoglobin (47). The molar equivalent of C-TIMP was quantified throughout purification, even in impure fractions, on the basis of the amounts of heme in the fusion proteins (1:1 molar ratio) calculated from the Soret peak of the recombinant myoglobin fusion protein samples.

Electrophoresis-- The recombinant proteins were electrophoresed on 15% polyacrylamide gels and visualized by Coomassie Brilliant Blue R-250 or silver nitrate staining and by Western blotting using the -Mb, -CT4-Tail, or -CT2-Tail antibodies and enhanced chemiluminescence. Samples analyzed by zymography were electrophoresed on 10% polyacrylamide gels copolymerized with 320 µg/ml or 1 mg/ml gelatin (Bio-Rad) and developed as previously described (50).

Mass Spectrometry and Absorbance Spectrometry-- To measure the masses of the recombinant proteins, electrospray ionization time of flight mass spectrometry was performed (11). To confirm the absence of apomyoglobin in the recombinant protein preparations, spectrophotometric analysis was performed to measure the Soret peak at 408-409 nm. Excess hemin was added, and the wavelength analysis was repeated. The absence of changes in the amplitude of the Soret peak verified that all of the myoglobin was fully reconstituted with heme and so the Soret absorbance could be used to quantitate protein quantities.

Hemopexin C Domain Binding Assays-- The binding interaction between full-length TIMPs or C-TIMPs and MMP-2 hemopexin C domain was measured by a microwell plate enzyme-linked immunosorbant assay (32, 40). TIMPs and C-TIMPs (0.2 µg/well) were immobilized in wells, and the MMP-2 hemopexin C domain was serially diluted in Tris-buffered saline (50 mM Tris-HCl, pH 8.0, 0.15 M NaCl). Binding of MMP-2 hemopexin C domain to TIMPs or C-TIMP domains was quantified using a rabbit polyclonal -His₆ antibody, which was detected using a goat anti-rabbit IgG antibody conjugated with alkaline phosphatase, developed with p-nitrophenyl phosphate substrate, and analyzed on a microplate reader at 405 nm. Curve fitting of the amount of protein bound was performed using SigmaPlot (45).

Binding to Active MMP-2-- Binding of C-TIMPs to the hemopexin C domain of active TIMP-2-free MMP-2 was measured by competition with TIMPs; pro-MMP-2 was activated with 2 mM 4-aminophenylmercuric acetate and active site-titrated against a standard preparation of TIMP-1 (kindly provided by Prof. G. Murphy, University of East Anglia, Norwich, UK) in fluorimetry assay buffer (FAB; 100 mM Tris-HCl, pH 7.5, 10 mM CaCl₂, 100 mM NaCl, 0.05% (v/v) Brij-35). TIMP-4 and TIMP-2 were prepared and active site-titrated as described before (26). The association rate constant (k_on) was measured for TIMP-4 (380 pM) or TIMP-2 (380 pM) with MMP-2 (15 pM) at 25 °C in FAB using 1 µM quenched fluorescence substrate as detailed previously (26). Increasing concentrations of C-TIMP-4 or CT4T were added to TIMP-4 prior to incubation with active MMP-2. Likewise, C-TIMP-2 or CT2T was premixed with TIMP-2 before assay.

Binding to Pro-MMP-2-- Apparent binding affinities of pro-MMP-2 to the C-TIMPs were determined by an enzyme capture assay with the bound MMP-2 being measured by the cleavage of gelatin in the linear range of zymograms and quantitated by densitometry. TIMP-4, TIMP-2, C-TIMP proteins, or the control proteins, recombinant myoglobin and ovalbumin (Sigma), were immobilized onto 96-well high protein binding fluorimetry plates (Dynex Microfluor® 2) at a concentration of 0.2 µg/well, prior to blocking with 1% bovine serum albumin (BSA). Following incubation with TIMP-2-free pro-MMP-2 in FAB, the unbound pro-MMP-2 was removed by thorough washing with Tris-buffered saline, 0.5% Tween 20. The bound pro-MMP-2 was then eluted in nonreducing SDS-PAGE sample buffer and analyzed by zymography. Where saturable binding was achieved, binding affinities of pro-MMP-2 were determined by measuring enzymic activity against protein amounts within the sensitivity limits of the assay. For weak binding mutants, the order of relative binding affinities of the different C-TIMP proteins was determined from amounts of enzyme captured in the linear response range of the assay.

Chemical Cross-linking-- TIMP-4 and TIMP-2 were incubated with MMP-2 hemopexin C domain at 6:1, 4:1, 3:1, 2:1, and 1:0 molar ratios of TIMP to MMP-2 hemopexin C domain for 1 h at room temperature and cross-linked with 0.5% glutaraldehyde as previously described (10). The TIMP-4·MMP-2 complex was identified by Western blotting using the -CT4-Tail antibody. C-TIMP-4, C-TIMP-2, CT4T, CT2T, and myoglobin were also incubated with or without pro-MMP-2 at 2.5:1 molar ratios and cross-linked as above. The reactions were analyzed by SDS-PAGE, silver staining, and zymography.

Velocity Sedimentation-- Velocity sedimentation was performed using a protocol modified from Loewen and Molday (51). Samples consisted of 4.7 µg of TIMP-4, 4.7 µg of TIMP-2, 4.9 µg of C-TIMP-4, or 5.0 µg of C-TIMP-2 incubated for 7 h at 4 °C alone or with 2 mol equivalents (8.5 µg) of the catalytically inactive mutant pro-MMP-2 E375A. Marker proteins (1 µg of myoglobin (18.8 kDa), carbonic anhydrase (29 kDa), ovalbumin (45 kDa), BSA (66 kDa), and phosphorylase B (97 kDa)) were added to the samples, which were then applied to a 5-20% (w/v) sucrose gradient in 50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 0.05% Brij-35. Due to similarity in molecular mass, carbonic anhydrase was omitted from samples containing C-TIMP, and BSA was omitted from samples containing pro-MMP-2. The samples were centrifuged at 50,000 rpm for 16 h at 4 °C in a Beckman TLS-55 rotor. Fractions were collected as previously described (51), analyzed by SDS-PAGE, and quantitated by densitometry.

Cell Assays-- Early passage human gingival fibroblasts were seeded into 96-microwell plates at 1 × 10⁴ cells/well in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% Cosmic calf serum (Hyclone, Inc.). After 24 h, the cells were washed twice with phosphate-buffered saline (8 mM Na₂HPO₄, 1.5 mM KH₂PO₄, pH 7.4, 137 mM NaCl, 2.7 mM KCl), and the cultures were synchronized by incubation in serum-free Dulbecco's modified Eagle's medium for 24 h before adding up to 4.5 µM C-TIMP-4, C-TIMP-2, or myoglobin (as a control) in serum-free Dulbecco's modified Eagle's medium containing 20 µg/ml concanavalin A to induce the cellular activation of MMP-2 (50). Control cells were incubated in the absence of C-TIMP proteins plus or minus concanavalin A. Cells were incubated overnight at 37°C, and then the culture supernatants were harvested and analyzed by zymography.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Analysis of C-TIMP Fusion Proteins-- E. coli producing the myoglobin fusion proteins exhibited a rust red color. The recombinant proteins were located in inclusion bodies, which were solubilized under denaturing conditions and required refolding in the presence of heme to reconstitute the myoglobin fusion partner. The C-TIMPs and mutants (see Fig. 1) were purified to homogeneity as revealed by single protein bands that electrophoresed at ~28 kDa (Figs. 2 and 3). No disulfide cross-linked multimers were present, as shown by nonreducing SDS-PAGE analysis (Fig. 2). Electrospray ionization time of flight mass spectrometry analysis confirmed the fidelity of gene expression and protein translation and revealed that all C-TIMP recombinant proteins were present as both N-terminal methionine-processed and unprocessed forms (Table III). The identities of the C-TIMP recombinant proteins were confirmed by Western blotting with the -Mb, -CT4-Tail, and -CT2-Tail antibodies (Fig. 3, A and B). All C-TIMPs could be tracked during purification due to the red color of the myoglobin fusion partner (Fig. 4A). When analyzed by scanning spectrophotometry, the myoglobin C-TIMPs exhibited a characteristic Soret absorbance at 408 nm upon refolding, indicative of a protein complexed with heme (47). The addition of 1 mol equivalent of heme to reconstituted and refolded myoglobin C-TIMPs did not significantly affect the Soret absorbance but increased the absorbance at 380 nm where free heme absorbs (Fig. 4B). This demonstrated that the myoglobin fusion partner was completely reconstituted in the holo form after heme addition during refolding. Protein that had been refolded in the absence of heme did not display a Soret peak and was a light straw color (data not shown). Therefore, the Soret absorbance could be used for quantitation of the recombinant proteins, even in complex mixtures, using the calculated extinction coefficient of 149,415 M¹ cm¹.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Schematic representation of the C-TIMP domain recombinant proteins. The hatched section represents horse heart myoglobin (Mb). The white sections represent the C domain of TIMP-2. The black sections represent the C domain of TIMP-4. The amino acid sequences shown represent the C-terminal tail and the mutated residues introduced.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   SDS-PAGE analysis of C-TIMP-2 and C-TIMP-4. C-TIMP-2 (CT2), C-TIMP-4 (CT4), TIMP-2, TIMP-4, and horse heart myoglobin (Mb) (100 ng) were electrophoresed on a 15% polyacrylamide gel with or without reduction with dithiothreitol (DTT) and silver-stained. The positions of migration of molecular mass markers are as indicated.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   SDS-PAGE and Western blot analysis of the C-TIMP mutant proteins. The C-TIMP recombinant proteins, TIMP-2, TIMP-4, and horse heart myoglobin (Mb) (100 ng) were reduced with 65 mM dithiothreitol, electrophoresed on 15% polyacrylamide gels, and analyzed by silver staining (upper panel) and Western blotting (lower three panels) with antibodies raised against peptides corresponding to the C-terminal tails of TIMP-2 (-CT2-Tail) or TIMP-4 (-CT4-Tail) or an antibody raised against recombinant horse heart myoglobin (-Mb). The positions of molecular mass markers are indicated. A, C-TIMP-2 (CT2) variant proteins compared with myoglobin, full-length TIMP-2, and TIMP-4. B, C-TIMP-4 (CT4) variant proteins compared with myoglobin and full-length TIMP-2 and TIMP-4.

View larger version (34K): [in this window] [in a new window]   Fig. 4.   Spectrophotometric properties of the myoglobin C-TIMP fusion proteins. A, samples of the mutant protein CT4+q eluted from Ni2+-chelating Sepharose, representative of all C-TIMP proteins, showing in the color figure that the recombinant C-TIMPs can be visually tracked during purification due to the red coloring of the horse heart myoglobin fusion protein. B, following heme reconstitution and purification, spectrophotometric analysis of the C-TIMP-myoglobin fusion protein (CT2T, 1.32 nmol) revealed that the purified protein exhibited a characteristic Soret absorbance at 408 nm, confirming the presence of holomyoglobin (thin black line; Purified C-TIMP). The addition of a further mol equivalent of heme (1.32 nmol) did not significantly change the Soret absorbance, but it did increase the absorbance around 380 nm, the absorption wavelength for free heme (thick colored line; + 1.32 nmole heme). This indicates the presence of excess free heme and that the protein was essentially all in the holo form with no apomyoglobin fusion protein present. The analysis shown for CT2T was typical for all recombinant proteins.

Characterization of C-TIMP-4 and C-TIMP-2 Tail Mutant Proteins and Antibodies-- To analyze the role of the nine residues of TIMP-4 and TIMP-2 that comprise the C-terminal tail in binding pro-MMP-2, several mutations were made in both recombinant C-TIMP-4 and C-TIMP-2. Silver staining and Western blot analysis under reducing (Fig. 3, A and B) and nonreducing (data not shown) conditions confirmed the presence of the tail swap and deletion mutations in the expressed proteins. The -CT4-Tail antibody recognized TIMP-4, C-TIMP-4, and CT2T4, whereas the -CT2-Tail antibody recognized TIMP-2, C-TIMP-2, CT4T2, and CT4+q. The absence of recognition of C-TIMP-4 and CT2q by the -CT2-Tail antibody, but its ability to recognize CT4+q indicated that the specificity of this antibody is dominated by the acidic residues Glu¹⁹² and Asp¹⁹³. The absence of recognition for CT4+q by the -CT4-Tail antibody indicated that the epitope for this antibody includes the residues Val¹⁹³ and Gln¹⁹⁴ at the homologous positions of the acidic residues in TIMP-2. However, because the -CT4-Tail antibody also did not recognize CT2q, which contains Val¹⁹³ and Gln¹⁹⁴, this suggested that the epitope for the -CT4-Tail antibody includes flanking residues of the C-terminal tail of TIMP-4.

C-TIMP-4 and C-TIMP-2 Bind to the MMP-2 Hemopexin C Domain-- Binding of the C domain of TIMP-4 and TIMP-2 in the absence of the N-domain to the MMP-2 hemopexin C domain has not previously been shown. In a solid phase binding assay, both C-TIMP-4 and C-TIMP-2 bound the MMP-2 hemopexin C domain to saturation (Fig. 5). TIMP-4 and C-TIMP-4 exhibited similar binding affinities, whereas C-TIMP-2 exhibited reduced binding to the MMP-2 hemopexin C domain by an order of magnitude when compared with full-length TIMP-2.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Binding of C-TIMP-4 and C-TIMP-2 to MMP-2 hemopexin C domain. Full-length TIMP-4 and C-TIMP-4 (A) or full-length TIMP-2 and C-TIMP-2 (B) were coated onto a 96-well enzyme-linked immunosorbent assay plate, blocked with 1% BSA, and overlaid with a serial dilution of MMP-2 hemopexin C domain. Bound MMP-2 hemopexin C domain was quantified using an affinity-purified rabbit polyclonal -His6 antibody, which was detected using a goat anti-rabbit IgG antibody conjugated with alkaline phosphatase and developed with p-nitrophenyl phosphate substrate.

To confirm that the C-TIMPs could bind the MMP-2 hemopexin C domain in the context of the full-length proenzyme, we examined these interactions in solution by glutaraldehyde cross-linking, because solid phase assays may denature proteins coated on plastic. C-TIMP-2 (Fig. 6A) or C-TIMP-4 (Fig. 6B) formed complexes with pro-MMP-2 as revealed by C-TIMP/pro-MMP-2 heterodimeric bands corresponding to a combined mass of the enzyme and recombinant protein (~98 kDa). Zymography identified MMP-2 in the heterodimer bands, which were shifted to a higher apparent molecular weight as compared with the samples incubated in the absence of glutaraldehyde or MMP-2 incubated with buffer alone. The fuzziness of the MMP-2 bands in the samples containing the cross-linker is probably due to multiple coupling and intramolecular cross-linkage, which reduced the activity of the samples. When myoglobin was incubated with pro-MMP-2 prior to cross-linking as a control (Fig. 6C), no heterodimeric bands were generated, and there was no change in the apparent molecular weight of MMP-2, confirming the specificity of the interaction observed with the myoglobin C-TIMP fusion proteins. As positive controls, full-length TIMP-2 (Fig. 6D) or TIMP-4 (Fig. 6E, upper panel) incubated with MMP-2 hemopexin C domain were glutaraldehyde cross-linked, resulting in TIMP/MMP-2 hemopexin C domain heterodimer bands. Increasing amounts of hemopexin C domain added to the TIMP proteins before cross-linking resulted in a decrease in TIMP monomer and an increase in TIMP/hemopexin C domain heterodimers. The identity of the TIMP-4/MMP-2 hemopexin C domain band was confirmed by Western blotting with -CT4-Tail antibody (Fig. 6E, lower panel).

View larger version (53K): [in this window] [in a new window]   Fig. 6.   Demonstration of C-TIMP/pro-MMP-2 interactions in solution using glutaraldehyde cross-linking. C-TIMP-2 (CT2) (A), C-TIMP-4 (CT4) (B), or horse heart myoglobin (Mb) (C) were incubated with pro-MMP-2 (0.25 mol equivalents) for 1 h prior to the addition of glutaraldehyde (GA) to 0.5%. After 30 min, the reaction was stopped with an equal volume of 2× SDS-PAGE sample buffer and analyzed on SDS-polyacrylamide gels (10%) stained with silver nitrate. A and B, pro-MMP-2 incubated with C-TIMP-2 or C-TIMP-4 formed pro-MMP-2/C-TIMP heterodimer bands that were identified as containing MMP-2 in the zymogram. C, pro-MMP-2 incubated with myoglobin in the presence of glutaraldehyde did not form heterodimer bands as analyzed by either silver-stained gels or zymograms. The myoglobin monomer band was electrophoresed off the gel. Small amounts of C-TIMP-4, C-TIMP-2, and myoglobin homodimers were sometimes detected by silver staining after extended incubation times at these concentrations. Full-length TIMP-2 (D) and TIMP-4 (E) were incubated with MMP-2 hemopexin C domain (HexCD) and cross-linked as described. The samples were analyzed on 15% polyacrylamide gels. For MMP-2 hemopexin C domain, + denotes 0.25 mol equivalent, ++ denotes 0.5 mol equivalent, and +++ denotes 1 mol equivalent versus TIMP-2 and TIMP-4. The positions of migration of molecular mass markers (M) are indicated.

To examine the interaction of the C-TIMP domain with pro-MMP-2 in solution in the absence of cross-linker, velocity sedimentation was performed. Like TIMP-2 (Fig. 7A) and TIMP-4 (Fig. 7B), the peak elution fractions of C-TIMP-2 (Fig. 7C) and C-TIMP-4 (Fig. 7D) shifted, corresponding to an increased molecular weight, when incubated with the catalytically inactive mutant pro-MMP-2 E375A, indicating complex formation. This inactive mutant was employed to avoid autoactivation and autodegradation of pro-MMP-2 over the long incubation and centrifugation time employed in this method.

View larger version (33K): [in this window] [in a new window]   Fig. 7.   Demonstration of C-TIMP domain/pro-MMP-2 interactions using velocity sedimentation. The catalytically inactive mutant pro-MMP-2 E375A (pE375A) (0.12 nmol) was incubated alone or with 0.22 nmol of TIMP-2 (A), TIMP-4 (B), C-TIMP-2 (C), or C-TIMP-4 (D), or TIMPs and C-TIMPs were incubated in the absence of pE375A. Samples plus markers were then layered on a 5-20% sucrose gradient and centrifuged for 16 h at 4 °C. The protein layers were collected by needle perforation and then analyzed by SDS-PAGE on 10% polyacrylamide gels and silver staining. Densitometric analysis was performed on the protein bands in the elution fractions and plotted with arbitrary units. The arrows indicate the peak elution fraction of co-incubated protein markers, which also confirms the specificity of the interaction between the TIMP and MMP-2 proteins.

C-TIMP-4 and C-TIMP-2 Inhibit MMP-2 Activation-- The binding assays showed that the C domain of TIMP-4 and TIMP-2 can bind to the MMP-2 hemopexin C domain in the absence of the TIMP N-domain, and this was confirmed using MMP-2 in the full-length zymogen form. To investigate whether this binding interaction affected pro-MMP-2 activation, C-TIMP-4 or C-TIMP-2 were added to concanavalin A-stimulated human fibroblasts. Above 1 µM, both C-TIMP-4 and C-TIMP-2 inhibited the cellular activation of pro-MMP-2 (Fig. 8), whereas the control protein, myoglobin, had no effect. This suggests that at high concentrations, the C-TIMP proteins could compete for the low levels (<100 nM, quantified by Western analysis) of endogenous TIMP-2 binding to pro-MMP-2 and so prevent formation of the ternary activation complex.

View larger version (13K): [in this window] [in a new window]   Fig. 8.   Recombinant C-TIMP-4 and C-TIMP-2 block MMP-2 activation by concanavalin A-stimulated fibroblasts. Human gingival fibroblasts were cultured for 24 h in serum-free medium containing 4.5 µM C-TIMP-2 (CT2), C-TIMP-4 (CT4), or horse heart myoglobin (Mb) (n = 4 for each treatment) in the presence or absence of 20 µg/ml concanavalin A as indicated. Controls were cultured in the absence of recombinant protein. The cell culture supernatants were analyzed by gelatin zymography (10% polyacrylamide gels). The proenzyme (pro), activation intermediate (intermediate), and active (active) MMP-2 enzyme bands are indicated.

The Effect of the C-TIMP Tail on MMP-2 Binding-- C-TIMP-4 without the tail, CT4T (Fig. 9A), and C-TIMP-2 without the tail, CT2T (Fig. 9B), were found to complex with pro-MMP-2 by chemical cross-linking. Binding was then quantitated using a competition assay as follows. Increasing concentrations of C-TIMP proteins were added with a constant amount of TIMP-4 or TIMP-2 to active MMP-2, and the rate of association (k_on) was measured. Increasing the concentration of C-TIMP-4 from 0 to 157 nM reduced the k_on of TIMP-4 for MMP-2 from 4.51 × 10⁶ M¹ s¹ to 1.85 × 10⁶ M¹ s¹ (Fig. 10A). Similarly, the k_on for TIMP-2 with MMP-2 was reduced from 4.97 × 10⁶ M¹ s¹ to 1.33 × 10⁶ M¹ s¹ at 120 nM C-TIMP-2 (Fig. 10B). The steady state rate (v_s) also increased with increasing concentrations of C-TIMPs (data not shown). The C-TIMP proteins alone did not inhibit MMP-2 proteolytic activity. This indicates that C-TIMP-4 and C-TIMP-2 bind to the hemopexin C domain of active MMP-2, preventing the C domain docking interaction of full-length TIMP-4 and TIMP-2, respectively. Hence, C-TIMP binding retards the interaction of full-length TIMP to the catalytic domain, with similar effects observed for CT4T and CT2T.

View larger version (26K): [in this window] [in a new window]   Fig. 9.   Glutaraldehyde cross-linking analysis of pro-MMP-2 binding by the C-terminal tail deletion mutants of C-TIMP-4 and C-TIMP-2. CT4T and pro-MMP-2 (A) or CT2T and pro-MMP-2 (B) were incubated for 1 h and cross-linked upon the addition of 0.5% glutaraldehyde (GA). Complexes were analyzed by 10% polyacrylamide gel electrophoresis and silver staining. The position of molecular mass markers (M) is indicated.

View larger version (16K): [in this window] [in a new window]   Fig. 10.   Competition of C-TIMPs with TIMPs for binding to the hemopexin C domain of MMP-2. The association rate constant (kon) for the interaction of 380 pM TIMP-4 (A) or TIMP-2 (B) with MMP-2 (15 pM) was measured at 25 °C as described under "Experimental Procedures." Increasing amounts of C-TIMP-4 (closed circles) or CT4T (open circles) (A), or C-TIMP-2 (closed circles) or CT2T (open circles) (B) were mixed with TIMP-4 or TIMP-2, respectively, prior to the addition to MMP-2 and measurement of kon.

In the enzyme capture assay at identical pro-MMP-2 concentrations, TIMP-4 captured ~80% less enzyme than TIMP-2 (Fig. 11A), consistent with the K_d values for TIMP-4 and TIMP-2 (32, 40). Deletion of the C-terminal tail of C-TIMP-2 greatly reduced binding to pro-MMP-2 in this assay, with CT2T having an apparent K_d >~10⁶ M (Fig. 11, B and C). In contrast, deletion of the C-terminal tail of C-TIMP-4 had only a minor effect on its binding affinity (Fig. 11C). Exchanging the tails of C-TIMP-4 and C-TIMP-2 showed the importance of the C-terminal tail sequence; CT4T2 resulted in increased binding of pro-MMP-2 to levels similar to that found for C-TIMP-2 under identical conditions (Fig. 11, B and C). In contrast, replacing the tail of C-TIMP-2 with that of TIMP-4 (CT2T4) markedly reduced the amount of pro-MMP-2 captured (apparent K_d of >~10⁶ M) to similar levels seen for the CT2T protein described above. Hence, the C-terminal tail of TIMP-4 plays a less important role in binding to the MMP-2 hemopexin C domain docking site than the TIMP-2 C-terminal tail.

View larger version (25K): [in this window] [in a new window]   Fig. 11.   Zymographic analysis of pro-MMP-2 enzyme capture assay. TIMP-2, TIMP-4, C-TIMP mutant recombinant proteins, and horse heart myoglobin (Mb) (0.5 µg) were coated onto a high protein-binding 96-well plate and blocked with 1% BSA. A serial dilution of pro-MMP-2, as indicated, was incubated with each coating protein for 2 h. The captured pro-MMP-2 was analyzed by gelatin zymography on 10% polyacrylamide gels. A, pro-MMP-2 captured by TIMP-2, TIMP-4, and myoglobin controls. B, pro-MMP-2 captured by C-TIMPs. C, relative binding affinities of C-TIMP variant domains. For all mutant proteins, the quantities of bound enzyme were quantified in the linear response range of the assay (250 ng) by densitometric analysis and expressed as band density normalized to the band density of MMP-2 captured by C-TIMP-2. For domains to which pro-MMP-2 could be bound to saturation, apparent Kd values were determined from curve fits.

The Critical Role of Glu¹⁹² and Asp¹⁹³ in the TIMP-2 C-terminal Tail-- To define in molecular detail the important elements in the TIMP-2 C domain interaction that strengthen binding compared with TIMP-4, the role of Glu¹⁹² and Asp¹⁹³ in the C-terminal tail was examined. Mutating V193E and Q194D in the C-TIMP-4 variant protein CT4+q (Fig. 1) increased pro-MMP-2 binding (apparent K_d of ~2 × 10⁹ M) and approached levels found for C-TIMP-2 in this assay (apparent K_d of ~1 × 10⁹ M) (Fig. 11B). The importance of Glu¹⁹² and Asp¹⁹³ in the C-terminal tail of TIMP-2 was confirmed by their replacement with the corresponding residues of the C-terminal tail of TIMP-4 (CT2q). Like replacement of the entire C-terminal tail with that of TIMP-4, loss of these negative charges reduced the amount of pro-MMP-2 captured to levels similar to that found for C-TIMP-4 (Fig. 11B). Because assay sensitivity set detection limits in these experiments, the rank order of binding affinities of the variant proteins was established by comparison of the amount of pro-MMP-2 bound at the same protein levels in the linear response range of the assay in experiments performed under identical conditions (Fig. 11C). Consistent results were obtained when the zymograms were quantified at other values in the linear range of the assay. The relative binding affinities of the C-TIMP variant proteins were in accordance with the apparent K_d values that could be determined. Hence, these mutagenesis studies demonstrated that the C-TIMP domains can still bind to pro-MMP-2 independent of the C-terminal tail. However, unlike TIMP-4, where the C-terminal tail plays a lesser role in this interaction, in TIMP-2 the tail greatly enhances binding stability. In particular, our studies revealed the pivotal importance of the unique acidic residues at positions 192 and 193 in stabilizing the TIMP-2/pro-MMP-2 interaction.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The different hemopexin C domain and TIMP interactions that occur on forming the noninhibitory complexes of TIMP-4 and TIMP-2 with pro-MMP-2 compared with those made in the binding and inhibition of active MMP-2 render interpretation of TIMP binding studies complex. The MMP-2, TIMP-2, and MT1-MMP ternary complex has been extensively studied by many groups (21). The catalytic domain of pro-MMP-2 alone is unable to bind to full-length TIMP-2 (24), and since N-TIMP-2 inhibits MT1-MMP and MMP-2 in the absence of the TIMP-2 C domain (37-39) but does not support MMP-2 activation, it was concluded that the TIMP-2 C domain binds to the hemopexin C domain of pro-MMP-2. These deletion mutant studies were supported by MMP-2 hemopexin C domain binding studies (28, 34-36), mapping the TIMP-2 docking site on the MMP-2 hemopexin C domain by mutagenesis (40), yeast two-hybrid analysis (10, 28), and very recent crystallographic studies (42). TIMP-4 was also found to bind the TIMP-2 docking site of pro-MMP-2 (32), but when so bound cannot also bind the catalytic domain of MT1-MMP, despite its potent inhibitory properties for this MMP (26) and so cannot form a ternary complex.

The TIMP C domains had not previously been expressed and characterized in the absence of the N-domain, which would simplify the analysis of TIMP binding interactions; nor had the differences between TIMP-4 and TIMP-2 been explored by mutagenesis to define the crucial molecular determinants that form the pro-MMP-2 binding site and that determine binding affinity. Therefore, to dissect the differences in the domain interactions that occur in the activation and inhibition of MMP-2, the C domains of TIMP-4 and TIMP-2 were expressed with an N-terminal horse heart myoglobin fusion protein in E. coli. Due to myoglobin's red color, which results from the bound heme cofactor, the recombinant fusion proteins could be visually tracked and quantified spectrophotometrically by measuring the Soret absorbance at 408 nm. This is a decided advantage over fusion partners such as -galactosidase, which cannot be monitored in this manner. We found that C-TIMP-4 and C-TIMP-2 bind to the MMP-2 hemopexin C domain as an isolated domain or in the context of full-length pro-MMP-2. However, C-TIMP-2 binds less avidly than full-length TIMP-2. The lack of the N-terminal domain may partially destabilize the C-TIMP domain, leading to a weaker interaction with the MMP-2 hemopexin C domain. It is unlikely that myoglobin sterically hinders this interaction, since C-TIMP-4 binds pro-MMP-2 with only slightly less affinity than TIMP-4. Hence, the present studies using TIMP C domain proteins biochemically confirm that this domain is necessary and sufficient for the noninhibitory TIMP-4 and TIMP-2 interaction with pro-MMP-2 on the hemopexin C domain.

TIMP binding to the hemopexin C domain of pro-MMP-2 is distinct from the more dynamic interactions that occur during binding and inhibition of active MMP-2. Following mapping of the noninhibitory TIMP-2 docking site on the hemopexin C domain of pro-MMP-2 by mutagenesis studies, it was evident that topographically this site cannot be used for inhibitory complex formation with active MMP-2 (40). This prompted alternate explanations involving two structurally and functionally distinct binding sites for TIMP-2 on the hemopexin C domain of pro- and active MMP-2, termed the docking and stabilization sites, respectively (5, 26, 28, 40), to account for the critical observations of Willenbrock et al. (38, 39) that the C-terminal tail of TIMP-2 enhances inhibition of active MMP-2. The TIMP stabilization site on the hemopexin C domain increases the affinity of TIMP binding to the active site cleft (24, 26, 34, 36, 52), forming stabilizing contacts with all TIMPs when bound in the elongated wedge orientation (41). Homologous stabilization sites are predicted on all MMP hemopexin C domains for all inhibitory TIMP interactions (5). Our present k_on data shows that C-TIMP-4 and C-TIMP-2 slowed the inhibition of MMP-2 by full-length TIMP-4 or TIMP-2, respectively. Since the C-TIMP proteins are noninhibitory as expected, this suggests that binding of the C-TIMP proteins to the MMP-2 hemopexin C domain prevents full-length TIMP-4 or TIMP-2 binding at the hemopexin C domain docking site. This initial docking interaction has only been characterized for TIMP-2 inhibition of active MMP-2 where TIMP-2 was demonstrated to utilize its anionic C-terminal tail (38). Competition of C-TIMP-4 with TIMP-4 suggests that a C domain docking interaction also initiates and enhances the rate of inhibition of active MMP-2 by TIMP-4, although, unlike TIMP-2, this is not driven by a C-terminal tail interaction and consequently occurs at a slower rate.

Although a structure has not been reported for any full-length TIMP in an inhibitory complex with a full-length active MMP, modeling predictions suggest that the stabilization site lies on the rim of the hemopexin C domain at the junction of modules I and II that are proximal to the active site (5). Hence, in the inhibitory interaction, the TIMP-2 C domain cannot bind in the same orientation that occurs at the docking site mapped by mutagenesis and crystallography to the rim of the hemopexin C domain on the opposite side of the molecule along the edge of modules III and IV (40, 42). Therefore, how does binding at the docking site enhance inhibition where contact with the catalytic domain cannot occur? We have suggested that, by initial tethering of TIMP-2 to the enzyme, the docking interaction enhances the rate of inhibition by increasing the probability of productive inhibitory complex formation that subsequently occurs (5, 28, 40). Inhibition, therefore, requires disengagement from the docking site and rebinding at the stabilization site to adopt the inhibitory elongated wedge topology. The three-dimensional structure of the TIMP-2·pro-MMP-2 complex adds support to this proposal; the orientation of TIMP-2 at the docking site precludes simultaneous interaction with the catalytic domain (42). The residual association rates of about 1.8 × 10⁶ M¹ s¹ for TIMP-4 and 1 × 10⁶ M¹ s¹ for TIMP-2 at higher concentrations of C-TIMPs in the competition experiments could reflect inhibition of MMP-2 by the full-length TIMPs binding directly to the catalytic domain in the elongated wedge topography. In other words, TIMP-4 and TIMP-2 may bind the hemopexin C domain stabilization site without prior binding to the docking site, the majority of which would be blocked by the excess C-TIMP present, so inhibition occurs at a slower rate resembling that for TIMP-1 (26). These data imply that in the inhibition of active MMP-2 two molecules of TIMP-2 or TIMP-4 may be simultaneously bound: one at the stabilization site and one at the docking site, as must occur for inhibition of active MMP-2 in the ternary complex.

Since TIMP-4 is homologous to TIMP-2 and can bind to pro-MMP-2 at the docking site (26, 32), it was proposed that TIMP-4 might also support or modulate the activation of pro-MMP-2 (32). We found here that high concentrations of C-TIMP-4 and C-TIMP-2 block the cellular activation of pro-MMP-2. We interpret this to occur by C-TIMP binding pro-MMP-2 in solution and thereby preventing the formation of trimolecular activation complexes on the cell surface. Recombinant hemopexin C domain of MMP-2 also inhibits the activation of pro-MMP-2, but in this case by competing for the available TIMP-2 (23, 27, 28). These results also demonstrate in a cellular context that the C domain alone of TIMP-4 and TIMP-2 can bind pro-MMP-2. The kinetic competition data confirm that in binding pro-MMP-2 the C-TIMP proteins block TIMP-2 binding to the docking site. Although we previously reported that TIMP-4 does not compete with TIMP-2 for binding of pro-MMP-2 (26), TIMP-4 was not used at the high concentrations that could be used for C-TIMP-4. Since TIMP-4 levels in vivo are unlikely to reach those of the C-TIMP proteins effective here (1 µM), this is unlikely to represent a physiological mechanism whereby TIMP-4 can modulate MMP-2 activation.

Our C-TIMP data are consistent with previous reports using TIMP-2 having a modified N terminus, which eliminates the ability of the amino group of Cys¹ to coordinate with the MMP active site Zn²⁺ ion. This resulted in a molecule unable to bind MT1-MMP and therefore unable to form trimolecular complexes or facilitate pro-MMP-2 activation (53, 54). It has also been reported that TIMPs bind to cell surface receptors in a noninhibitory manner, potentially via their C domains, to modify cell behavior such as growth and differentiation (13, 20). It cannot be discounted that the C-TIMP proteins may have triggered such a pathway, leading to modulation of cell behavior and reduced MMP-2 activation. Indeed, in ongoing studies, the recombinant proteins generated here will prove extremely valuable for exploring the growth factor effects of TIMP-4 and TIMP-2 in the absence of any MMP-inhibitory activity.

TIMP-4 down-regulates MMP-2 activation by inhibiting MT1-MMP in a manner that does not support binding and trimolecular complex formation with pro-MMP-2 (26, 33). Since TIMP-4 and TIMP-2 have a similar inhibition constant (K_i) and k_on for MT1-MMP (26), this indicates that the difference between TIMP-4 and TIMP-2 in effecting MMP-2 activation resides within the TIMPC domain. Due to the similar binding properties found for C-TIMP-4 and CT4T, the C-terminal tail of C-TIMP-4 does not appear to be as important as the TIMP-2 tail in its interaction with pro-MMP-2. This was also evident, since CT2T4 exhibited as marked a reduction in binding to pro-MMP-2 as CT2T. Conversely, the importance of the C-terminal tail of C-TIMP-2 in strengthening binding to pro-MMP-2 was confirmed by the increase in pro-MMP-2 captured by CT4T2 and by the reduction in pro-MMP-2 binding by CT2T.

In deletion experiments, Willenbrock et al. first revealed the importance of the TIMP-2 C-terminal tail in binding to the docking site of the MMP-2 hemopexin C domain of the active enzyme (38, 39), but the exact residues involved in the interaction had not been defined. Several residues in the unique cationic clusters of the MMP-2 hemopexin C domain have been shown to be involved in TIMP-2 binding (40). Because the net charge of the TIMP-2 C-terminal tail is 4, compared with 1 for TIMP-4, we hypothesized that the stronger negative character of the TIMP-2 C-terminal tail plays an important role in promoting and stabilizing the interaction between TIMP-2 and pro-MMP-2, thereby allowing TIMP-2 but not TIMP-4 to form a stable trimolecular complex with pro-MMP-2 and MT1-MMP. We found that the removal of the acidic residues Glu¹⁹² and Asp¹⁹³ from C-TIMP-2 reduced binding by ~60%. Conversely, the addition of these residues to the tail of C-TIMP-4 resulted in an increase in the amount of pro-MMP-2 bound, reflecting the reduced apparent K_d of CT4+q, which was similar to the increased affinity found for CT4T2. Together, these data show that the additional anionic character of the C-terminal tail of TIMP-2 is responsible for its tighter binding to pro-MMP-2 compared with TIMP-4 and that this largely accounts for the deficiency in the ability of TIMP-4 to participate in the formation of a functional ternary complex.

TIMP-3, like TIMP-4, can bind pro-MMP-2, although it has not been shown to participate in the MT1-MMP-mediated activation of pro-MMP-2 (34). Notably, the C-terminal tail of TIMP-3 has a net charge of 0 with a positively charged lysine residue at the position homologous to Lys¹⁸⁷ found in the C-terminal tail of TIMP-4 but not in TIMP-2. This charge may further weaken binding to the positively charged MMP-2 hemopexin C domain docking site. Although the C-terminal tail appears to be important in the interaction with the MMP-2 hemopexin C domain docking site, our data show that the presence of acidic residues at positions 192 and 193 in TIMP-2 specifically leads to greatly added stability. Nonetheless, other elements of the TIMP C domain also contact the docking site as evidenced by the ability of CT4T and CT2T to be cross-linked with pro-MMP-2 and to compete for TIMP-4 and TIMP-2 binding to active MMP-2. This is consistent with the inability of TIMP-2 C-terminal tail peptide analogues to compete or prevent TIMP-2 binding (28) and with the structure of the TIMP-2·pro-MMP-2 complex (42). In particular, a hydrophobic pocket centered around Phe⁶²¹ on the hemopexin C domain that accommodates Met¹⁴⁹ in the C domain of TIMP-2 is believed to be a major contributor to binding stability (42). In TIMP-4, the residue at position 149 is a threonine and not a methionine as found in TIMP-2. Hence, an aliphatic hydroxyl that reduces the hydrophobic character of this part of the TIMP-4 contact face should also contribute to the lower binding affinity of TIMP-4 compared with TIMP-2 (42). Hydrophobic interactions are also proposed to be important in stabilizing the interaction of the C-terminal tail of TIMP-2, where Phe¹⁸⁸ closely packs with hemopexin C domain residues Tyr⁵⁵², Phe⁵⁵⁹, Phe⁵⁷³, Ala⁵⁸⁰, and Trp⁵⁸¹. Other hydrophobic interactions and hydrogen bonds were also revealed in the structure of the large interface of the TIMP-2·pro-MMP-2 complex (42).

Although the structure confirmed that several salt bridges were formed between TIMP-2 and the hemopexin C domain (42) that involved the cationic residues identified previously by mutagenesis (42) as having important roles in this interaction (Lys⁵⁴⁷, Lys⁵⁵⁰, Arg⁵⁶¹, Lys⁵⁶⁶, Lys⁵⁶⁸, Lys⁶¹⁰, Lys⁶¹⁷) (40), structural information alone does not reveal the relative contribution to the total binding energy of the individual interactions that make up the large ~250-nm² contact surface. Moreover, Asp¹⁹³ in the C-terminal tail of TIMP-2 was not resolved in the structure, so its contribution to binding could not be ascertained. Our present experimental data reveal that a potential salt bridge involving Asp¹⁹³ together with the salt bridge formed between Glu¹⁹² of TIMP-2 and Lys⁵⁵⁰/Lys⁵⁶⁶ of MMP-2 (42) is critical for the binding stability of TIMP-2. In view of the multiple and diverse forces that contribute to the overall binding energy, it is not obvious which are the important amino acid residues and forces that contribute most to the unique ability of TIMP-2 to form ternary complexes. Accordingly, it was somewhat surprising that two negatively charged residues alone of the C-TIMP-2 tail had such a strong influence on the stability of the TIMP-2 interaction with pro-MMP-2 and could exert a similar effect on C-TIMP-4 following their introduction into its C tail.

Overall, the present work demonstrates a clear and important role for the unique negatively charged residues Glu¹⁹² and Asp¹⁹³ in the anionic tail of TIMP-2 in binding pro-MMP-2. Physically, this tail binding site is quite distant and runs perpendicular to the main C domain contact surface (42) and thus appears to "bracket" TIMP-2 in place on the hemopexin C domain, potentially explaining the profound effects on binding stability that the replacement of Glu¹⁹²/Asp¹⁹³ shows. We suggest that the lack of these charges in the C-terminal tail of TIMP-4 is largely responsible for its weaker interaction with pro-MMP-2 and therefore its inability to participate in the activation of pro-MMP-2; not only is there an energy cost from the loss of salt bridge partners, but there is an additional energy penalty imposed by the introduction of nonpacking side chains at the end of the TIMP-4 tail. Identification of this site also suggests a new target to develop antagonists of TIMP-2 binding (18) that may block MMP-2 activation in disease. The role of TIMP-4 binding the hemopexin C domain of pro-MMP-2 remains unclear, but it may be to recruit this inhibitor to the vicinity of proteolytic activity that i