From the 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
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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 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 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.
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( 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(
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( Inhibition Kinetic
Studies--
Ki(app) of N-TIMP-1 and
mutants against MMP-1, -2, and -3( 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.
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( 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.
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(
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(
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( 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 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( 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.
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(
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(
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.
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-barrel with three associated
-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
-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
-amino group of the NH2-terminal Cys1 (24)
or mutation to append an alanine extension to the amino terminus
(25).
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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.
C) and NFF-3 substrate as described (26).
C). Various concentrations of the inhibitors were incubated
with MMP-3(
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.
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(
C) was 2 and 4 h, respectively. To calculate
Ki(app), the following formulas were
used (30) as seen in Equations 1 and 2,
(Eq. 1)
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.
(Eq. 2)
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View larger version (79K):
[in a new window]
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.
Ki(app) (nM) of N-TIMP-1 position 4 and 68 mutants against MMP-1 (5 nM), MMP-2, and MMP-3(C) (1 nM each)
C), whereas V4I results in a 15-fold
reduced affinity for MMP-1 but unchanged activity with MMP-2 and
MMP-3(
C).
Ki(app) (nM) of N-TIMP-1 combined
mutants against MMP-1 (5 nM), MMP-2, and MMP-3 (1 nM each)
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(
C)
than the double mutant T2L/V4S.
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.
C).
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G of binding. For example, the T2L/V4S/S68A mutant
has a >100-fold higher affinity for MMP-3(
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.
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).
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FOOTNOTES |
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* 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.
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ABBREVIATIONS |
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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.
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