Protein Engineering of the Tissue Inhibitor of Metalloproteinase 1 (TIMP-1) Inhibitory Domain

IN SEARCH OF SELECTIVE MATRIX METALLOPROTEINASE INHIBITORS*

Shuo WeiDagger , Ying Chen§, Linda Chung, Hideaki Nagase, and Keith BrewDagger ||

From the Dagger  Department of Biomedical Sciences, Florida Atlantic University, Boca Raton, Florida 33431 and the  Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College of Science, Technology and Medicine, 1 Aspenlea Road, Hammersmith, London W6 8LH, United Kingdom

Received for publication, November 19, 2002, and in revised form, December 11, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Studies of the structural basis of the interactions of tissue inhibitors of metalloproteinases (TIMPs) and matrix metalloproteinases (MMPs) may provide clues for designing MMP-specific inhibitors. In this paper we report combinations of mutations in the major MMP-binding region that enhance the specificity of N-TIMP-1. Mutants with substitutions for residues 4 and 68 were characterized and combined with previously studied Thr2 mutations to generate mutants with improved selectivity or binding affinity to specific MMPs. Some combinations of mutations had non-additive effects on Delta G of binding to MMPs, suggesting interactions between subsites in the reactive site. The T2L/V4S mutation generates an inhibitor that binds to MMP-2 20-fold more tightly than to MMP-3(Delta C) and over 400-fold more tightly than to MMP-1. The T2S/V4A/S68Y mutant is the strongest inhibitor for stromelysin-1 among all mutants characterized to date, with an apparent Ki for MMP-3(Delta C) in the picomolar range. A third mutant, T2R/V4I, has no detectable inhibitory activity for MMP-1 but is an effective inhibitor of MMP-2 and -3. These selective TIMP variants may provide useful tools for investigation of biological roles of specific MMPs and for possible therapy of MMP-related diseases.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Degradation of the extracellular matrix is essential for normal biological processes including embryonic development and morphogenesis (1, 2), reproduction (3) and wound healing (4), and enhanced turnover is associated with diseases including arthritis (5, 6), tumor angiogenesis and metastasis (7), multiple sclerosis (8), and cardiovascular diseases (9). The matrix metalloproteinases (MMPs)1 are a family of more than 20 zinc-dependent proteases that catalyze extracellular matrix turnover (10). Activity and zymogen activation in MMPs are regulated by a group of endogenous proteins named the tissue inhibitors of metalloproteinases (TIMPs) (11).

TIMPs are distributed in both invertebrates and vertebrates (11-13). The mammalian TIMPs are a family of four members (TIMP-1-4) that have about 40% sequence identity and fold into two domains, each containing three disulfide bonds (11). The isolated N-terminal domains (N-TIMPs) are able to form the correct native structure that carries the inhibitory activity against the MMPs (14). Although there are four TIMPs, their inhibitory activities toward different MMPs are not particularly specific. A notable exception is that TIMP-1 is a weak inhibitor of MT-MMPs, whereas TIMP-2 and TIMP-3 are much more effective (15-17).

Reported structures of TIMPs include crystal structures of TIMP-1 in a complex with the MMP-3 catalytic domain (18), TIMP-2 in a complex with the catalytic domain of membrane type MT1-MMP (19) and in a free form (20), and the solution NMR structures of N-TIMP-1 (21) and N-TIMP-2 (22). These structures show that the N-terminal inhibitory domain consists of a 5-stranded beta -barrel with three associated alpha -helices resembling the folds of members of the oligonucleotide/oligosaccharide binding (OB) protein family (23). The structure of the TIMP-1/MMP-3 complex reveals that about 75% of all intermolecular contacts are made by residues adjacent to the disulfide bond between Cys1 and Cys70, especially residues 1-5 and 66-70. These two sections of chain insert into the active site cleft of the MMP, thus blocking its accessibility to substrates (18). The N-terminal Cys1 coordinates the catalytic Zn2+ through the alpha -amino group and the peptide carbonyl group and is crucial for the inhibitory activity of TIMPs for MMPs, as shown by the complete loss of inhibitory activity for MMPs in TIMP-2 on carbamylation of the alpha -amino group of the NH2-terminal Cys1 (24) or mutation to append an alanine extension to the amino terminus (25).

Our previous mutagenesis studies of N-TIMP-1 (26, 27), together with work with N-TIMP-2 by others (28), suggest that the affinity and specificity of TIMP for MMPs can be modified by site-directed mutagenesis. A major determinant of the affinity of N-TIMP-1 for different MMPs is the residue at position 2 in the sequence (threonine 2 in the wild-type protein) which interacts with the S1' pocket of MMPs, a key to MMP substrate specificity (27). Based on this, it is reasonable to hypothesize that other residues that make contact with MMPs also contribute to the binding affinity and specificity of N-TIMP-1 and that selective variants can be generated by combining suitable mutations at these sites. In the present study, we show that N-TIMP-1 mutants with substitutions at positions 4 and 68 showed changes in affinity and specificity for MMPs. Combinations of mutations in these positions with those in position 2 led to the discovery of N-TIMP-1 variants with higher binding affinity and specificity for individual MMPs.

    EXPERIMENTAL PROCEDURES
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Materials-- Vectors and cell lines used for expression of N-TIMP-1 and variants were from the same sources as in previous studies (26, 27, 29). Miniprep and gel extraction kits were purchased from Qiagen. Restriction enzymes and Vent DNA polymerase were obtained from New England BioLabs. PCR reactions were performed in a PCR Sprint HYBAID system from Midwest Scientific. C-terminally truncated MMP-3 (MMP-3(Delta C)) and active forms of MMP-1 and MMP-2 were generated as described previously (29). The Biologic DuoFlow medium-pressure chromatography system was purchased from BioRad, and the cation exchange Mono S HR 5/5 HPLC column was from Amersham Biosciences. Synthetic fluorogenic peptide substrates Knight ((7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-N-(3-{2,4-dinitrophenyl}-L-2,3-diaminopropyl)-Ala-Arg-NH2) and NFF-3 ((7-methoxycoumarin-4-yl)acetyl-Arg-Pro-Val-Glu-norvalinyl-Trp-Arg-Lys-(2,4-dinitrophenyl)-NH2) were from Bachem. DNA sequencing was carried out in the laboratory of Dr. R. Werner, Dept. of Biochemistry and Molecular Biology, University of Miami School of Medicine. Synthetic oligonucleotides were synthesized by Sigma Genosys.

Construction and Expression of N-TIMP-1 Mutants-- Plasmid pET-3a containing the N-Timp-1 gene was used as the template for site-directed mutagenesis by PCR. Val4 mutations and double mutations at positions 2 and 4 were introduced by direct PCR with a forward primer containing the mutations and a T7 terminator primer as reverse primer. The primers used (mutated codons are underlined) are as follows: 5'-GGAGATATACATATGTGCACCTGTGCCCCACCCCAC-3' for V4A; 5'-GGAGATATACATATGTGCACCTGTATCCCACCCCAC-3' for V4I; 5'-GGAGATATACATATGTGCACCTGTAAACCACCCCAC-3' for V4K; 5'-GGAGATATACATATGTGCACCTGTTCCCCACCCCAC-3' for V4S; 5'-GGAGATATACATATGTGCCTCTGTTCCCCACCCCAC-3' for T2L/V4S; 5'-GGAGATATACATATGTGCTCCTGTGCCCCACCCCAC-3' for T2S/V4A; and 5'-GGAGATATACATATGTGCCGCTGTATCCCACCCCAC-3' for T2R/V4I.

Ser68 mutations and triple mutations were introduced by mega-primer PCR as described previously (26, 27) using a T7 promoter primer (for Ser68 single mutation) or primers introducing the corresponding double mutations at positions 2 and 4 (for triple mutations) as the forward primer, a reverse primer that introduces the corresponding mutation at position 68, and a T7 terminator primer as the other reverse primer. The following primers were used for mutagenesis (the mutated anti-codons are underlined): 5'-GAAGTATCCGCAGACAGCCTCCATGGCGGGGGT-3' for S68A; 5'-GAAGTATCCGCAGACCTCCTCCATGGCGGGGGT-3' for S68E; 5'-GAAGTATCCGCAGACACGCTCCATGGCGGGGGT-3' for S68R; and 5'-GAAGTATCCGCAGACATACTCCATGGCGGGGGT-3' for S68Y.

PCR products were cloned into pET-3a vector using the NdeI and BamHI restriction sites. All constructs were confirmed by DNA sequencing using T7 promoter primer. The N-Timp-1 mutants were expressed in Escherichia. coli BL21(DE3) cells. Protein was purified from inclusion bodies and folded in vitro as described previously (29).

Further Purification of N-TIMP-1 and Mutants-- N-TIMP-1 and mutant proteins purified by cation exchange chromatography with CM-52 were dialyzed overnight against 15 volumes of 20 mM bis-Tris-HCl, pH 5.5 (pH 6.0 for T2R/V4I) and applied to a cation exchange Mono S HR 5/5 column previously equilibrated with the same buffer and connected to a Biologic DuoFlow medium pressure chromatography system. The protein was eluted with a linear salt gradient of 0-0.5 M NaCl over 60 min at a flow rate of 1 ml/min. The activity of different fractions was estimated by the fluorescence assay method using MMP-3(Delta C) and NFF-3 substrate as described (26).

Two similarly sized peaks, which slightly overlap, were obtained in this separation, and fractions corresponding to the second peak, which contained the MMP-3 inhibitory activity, were pooled and titrated with MMP-3(Delta C). Various concentrations of the inhibitors were incubated with MMP-3(Delta C) (300 nM) for 4 h at 37 °C, diluted 300-fold with TNC buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM CaCl2 and 0.02% Brij 35) and immediately assayed with 1.5 µM NFF-3 substrate as described (26). These results showed that this protein is >85% active for MMP inhibition.

Inhibition Kinetic Studies-- Ki(app) of N-TIMP-1 and mutants against MMP-1, -2, and -3(Delta C) were determined using fluorescence assay as described previously (26) with slight modifications. The incubation time for the inhibitors with MMP-1 and MMP-3(Delta C) was 2 and 4 h, respectively. To calculate Ki(app), the following formulas were used (30) as seen in Equations 1 and 2,


<FR><NU>I<SUB>t</SUB></NU><DE>1−<FR><NU>v<SUB>i</SUB></NU><DE>v<SUB>0</SUB></DE></FR></DE></FR>=E<SUB>t</SUB>+K<SUB>i</SUB><SUP>(app)</SUP><FR><NU>v<SUB>0</SUB></NU><DE>v<SUB>i</SUB></DE></FR> (Eq. 1)

K<SUB>i</SUB><SUP>(app)</SUP>=K<SUB>i</SUB><FENCE><FR><NU>A<SUB>t</SUB>+K<SUB>m</SUB></NU><DE>K<SUB>m</SUB></DE></FR></FENCE> (Eq. 2)
where It is the total inhibitor concentration, Et is the total enzyme concentration, At is the total substrate concentration, and Km is the Michaelis constant. In our assays the value of Et/Ki(app) does not exceed 100 so that the inhibitor is distributed in both free and bound forms, and Ki(app) can be calculated by fitting inhibition data to Equation 1 (30). Because the inactive portion of the N-TIMP-1 does not interfere with the binding of the active inhibitor with MMPs, as shown by isothermal titration by isothermal titration,2 the true Ki values can be determined by multiplying the Ki, calculated as described above, by the fraction of active TIMP determined by titration. Because the substrate concentration is very low relative to the estimated Km, the apparent Ki values are essentially identical to the true Ki values.

    RESULTS
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INTRODUCTION
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Substitutions for Val4 of N-TIMP-1-- In the crystal structure of the N-TIMP-1/MMP-3 complex, the side chain of Val4 interacts with the S3' site of MMP-3, forming contacts at the edge of the interaction interface (Fig. 1, and Ref. 18). Four mutants with substitutions at position 4 of N-TIMP-1 were constructed and expressed in E. coli. As compared with the Thr2 mutations, which generally have strong effects on MMP binding (27), these mutations produce more moderate changes (Table I). Mutations of Val4 into isoleucine, lysine, and serine cause significant increases in the Ki(app) for MMP-1 while having only a small effect on the affinity for MMP-2.


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Fig. 1.   Surface structures of TIMP-1 and MMP-3 catalytic domain in complex. A, surface of TIMP-1. B, surface of MMP-3 catalytic domain with contact residues from TIMP-1. The binding region in TIMP-1 adjacent to Cys1-Cys70 disulfide bond is highlighted. Colors used are as follows: Cys1, Cys3, and Cys70, brown; Thr2, yellow; Val4, red; Ser68, purple; Val69, blue; and the catalytic Zn2+ of MMP-3, cyan.

                              
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Table I
Ki(app) (nM) of N-TIMP-1 position 4 and 68 mutants against MMP-1 (5 nM), MMP-2, and MMP-3(Delta C) (1 nM each)

Mutants V4K and V4S are more selective for MMP-2 than the wild-type N-TIMP-1 as a result of minor changes in affinity for MMP-2 and larger reductions in binding to MMP-1 and MMP-3. The V4S mutant has an unchanged Ki for MMP-2 but 5-fold and 8-fold increased Ki values for MMP-1 and -3, respectively. The other two mutants also have modified inhibition activities; V4A has an increased affinity for MMP-3(Delta C), whereas V4I results in a 15-fold reduced affinity for MMP-1 but unchanged activity with MMP-2 and MMP-3(Delta C).

Substitutions for Ser68-- Residues 66-70 of the C-D loop also form part of the core of the TIMP/MMP contact site (Fig. 1 and Ref. 18), and previous studies have shown that substitutions for Met66 or Val69 affect TIMP activity (26). Here we mutated the Ser68 to Ala, Glu, Arg, and Tyr. These mutations have large effects on MMP binding (Table I). Three of the four mutants have improved selectivity for MMP-2 relative to the other two MMPs, whereas the fourth mutant, S68Y, inhibits MMP-3 much more strongly than MMP-1 and -2.

Combined Mutations of Thr2, Val4, and Ser68-- Based on the mutagenesis studies of Thr2, Val4, and Ser68, we constructed double and triple mutants containing combinations of the more selective single-site mutations. Characterization of these mutants shows that effects of the individual mutations are generally additive but with some exceptions (Table II), suggesting that these residues do not always contribute independently to the stability of the TIMP/MMP complex.

                              
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Table II
Ki(app) (nM) of N-TIMP-1 combined mutants against MMP-1 (5 nM), MMP-2, and MMP-3 (1 nM each)
Data of the Thr2 mutants were taken from our previous study (30).

The T2L mutation was combined with V4S to generate a N-TIMP-1 variant that is more selective for MMP-2. As predicted, with MMP-2 the resulting double mutant inhibits 20-fold more strongly than with MMP-3(Delta C) and about 470-fold more strongly than with MMP-1. Introduction of a third mutation, S68A, produced a mutant binding very weakly to MMP-1 while retaining a good activity for MMP-2. However, this inhibitor is much more effective with MMP-3 than is predicted based on additive effects on the free energy of binding (Table II). This triple mutant, T2L/V4S/S68A, is less selective against MMP-3(Delta C) than the double mutant T2L/V4S.

The double mutant, T2S/V4A, has an increased inhibitory activity for MMP-3. Binding with MMP-2 is unchanged, whereas that with MMP-1 is weaker than the wild-type protein. Interestingly, the triple mutant T2S/V4A/S68Y has the highest inhibitory activity for MMP-3(Delta C) among all mutants characterized so far, with a Ki of 50 pM. Unexpectedly, it also has a greatly improved affinity for MMP-2, whereas binding to MMP-1 was reduced over 36-fold.

The T2R/V4I mutant was purified as a ~95% active form using a Mono S column at pH 6.0 instead of pH 5.5, which is used for the wild-type protein. We could not detect any inhibition of MMP-1 by this mutant even at a concentration of 10 µM, yet it retains good activity as an inhibitor of MMP-2 and MMP-3(Delta C).

    DISCUSSION
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The TIMPs are important regulators of extracellular matrix metabolism (11), principally through their primary activities as endogenous inhibitors of MMPs. Imbalances between the levels of TIMPs and active MMPs are linked to disease processes such as tumor metastasis, arthritis, and atherosclerosis (31, 32). Low molecular weight MMP inhibitors have been developed and extensively studied, and a few have been used in clinical trials but with little success (33). Nonspecific inhibition of housekeeping MMP functions has been proposed as the major drawback of these inhibitors (34). We proposed previously that N-TIMP-1 can be engineered to produce more selective MMP inhibitors that could be applied in gene therapy of MMP-related diseases (35, 36), and residue 2 of TIMP-1 was found to be a major determinant of affinity and specificity for MMPs (27). In this paper we report the effects of mutagenesis of two other residues that are important in TIMP/MMP binding; combinations of mutations in these positions with Thr2 mutations generate N-TIMP-1 variants with increased selectivity and/or affinity for specific MMPs.

The side chain of Val4 occupies a site similar to the substrate P3'-subsite in currently known TIMP/MMP complexes (18, 19). In the complex of TIMP-1 with MMP-3, the side chain of Val4 sits in a shallow groove at the margin of the interaction site close to the side chains of Gly161, Asn162, Leu164, and Tyr223 of the protease (15). These residues are >4 Å from Val4, and the insignificant effect of truncating the side chain through the Ala substitution on the affinity for all three MMPs suggests that interactions between the side chain of Val4 and the protease contributes little to the free energy of binding. The Val4 side chain projects away from the protease surface in the TIMP-1/MMP-3 complex (18), and the more extended Ile side chain can be accommodated in MMP-2 and -3 without perturbing the protein-protein interaction, but in MMP-1 it produces a 14-fold loss in affinity. The basis for this is uncertain, because the structures of complexes of TIMP-1 with MMP-1 and -2 have not been determined, but this result suggests that the S3' site of MMP-1 does not readily accommodate a larger side chain; it is also affected more than the other proteases by the Lys substitution (Table I). Differences between the MMPs in the residues that form the P3' site do not readily account for these effects; residues Leu164 and Tyr223 of MMP-3 are conserved in MMP-1 and -2, whereas residues corresponding to Gly161-Asn162 are Gly-Gly in MMP-1 and Asp-Gly in MMP-2, so changes in side chain size are not responsible for the reduced steric tolerance in this subsite in MMP-1. The binding of TIMP-1 to MMP-2 is least affected by substitutions for Val4, suggesting that there is greater separation between MMP-2 and N-TIMP-1 in this part of the interaction site.

The side chain of Ser68 of TIMP-1 interacts with the S2 subsite of MMP-3 in the crystal structure of their complex and is in contact with Ala167 of the metalloproteinase (18). It is also near (<4.5 Å distance) to His166, Tyr168, Ala169, and His205. Mutation of Ser68 to Ala, Glu, and Arg reduces the affinity of N-TIMP-1 for all three MMPs, but the effect is less for MMP-2 than for MMP-1 and MMP-3. The Tyr mutant has a major effect on inhibitor specificity, producing a 150-fold loss in affinity for MMP-1, a 7-fold reduction in affinity for MMP-2, but essentially no change in binding to MMP-3. The side chain of Ser68 of TIMP-1 projects toward Ala169 of MMP-3 in their complex (18), and it appears that the Tyr side chain can be accommodated without major perturbation of the interaction interface. Ala is conserved at this site in MMP-2, but in MMP-1 it is replaced by Gln. A steric conflict between the Gln side chain and Tyr68 in the N-TIMP-1 mutant is a possible explanation of the effects of this mutation on inhibitor selectivity.

Substitutions for residues 2, 4, and 68 with similar selectivity were combined in an attempt to engineer N-TIMP-1 variants with higher selectivity and/or affinity for specific MMPs. In some cases, the effects of these mutations are essentially additive (Table II), indicating that interactions with the S1', S3', and S2 subsites of MMPs contribute independently to the stability of the TIMP/MMP complex. However, some mutants containing combinations of mutations have much lower Ki values for particular MMPs than predicted based on the assumption that individual substitutions have additive effects on the Delta G of binding. For example, the T2L/V4S/S68A mutant has a >100-fold higher affinity for MMP-3(Delta C) than expected, yet its Ki values for MMP-1 and MMP-2 are in good agreement with predictions. Similarly, the T2S/V4A/S68Y is a 60-fold better inhibitor of MMP-1 and also a 68-fold better inhibitor of MMP-2 than predicted based on additivity. These discrepancies greatly exceed the compounded errors of the single-site mutations and suggest interactive effects between the sites in these triple mutants. Because of the complexity of protein-protein interactions, there are many possible explanations for this, such as changes in relative orientation of TIMP and protease, structural changes introduced by the mutations, and changes in dynamics (11). Structural studies are in progress to address this question.

Several combined mutants have interesting and potentially useful properties. T2L/V4S is selective for MMP-2, providing a possible tool for gene therapy of gelatinase-related diseases. Another mutant, T2S/V4A/S69Y, is the best inhibitor for stromelysin-1 among all mutants discovered so far, although it also exhibits excellent inhibitory activity for MMP-2. The apparent Ki of this inhibitor for MMP-3(Delta C) is 50 pM, providing the most potent inhibitor of stromelysin-1 among all N-TIMP-1 mutants. The third mutant, T2R/V4I, has no detectable activity for MMP-1 while retaining good inhibition for MMP-2 and -3. This mutant is of special interest, because the failure of many general MMP inhibitors in clinical trials was the result of muscular-skeletal disorders that are thought to be caused by nonspecific inhibition of MMP-1 (38).

Our mutational studies have demonstrated the feasibility of generating selective MMP inhibitors by engineering TIMP. Using high throughput screening methods, it should be possible to identify TIMP variants that are specific inhibitors of individual MMPs for application in future clinical trials.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant AR40994.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: BioStratum, Inc., 4620 Creekstone Dr., Suite 200, Durham, NC 27703.

|| To whom correspondence should be addressed: Dept. of Biomedical Sciences, Florida Atlantic University, 777 Glades Rd., Boca Raton, FL 33431. Tel.: 561-297-0407; Fax: 561-297-2221; E-mail: kbrew@fau.edu.

Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M211793200

2 S. Arumugam, G. Gao, B. L. Patton, V. Semechenko, K. Brew, and S. R. Van Doren, submitted for publication.

    ABBREVIATIONS

The abbreviations used are: MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; N-TIMP-1, N-terminal domain of tissue inhibitor of metalloproteinases-1; PCR, polymerase chain reaction.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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REFERENCES

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37. Deleted in proof
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