(Received for publication, March 12, 1997, and in revised form, May 19, 1997)
From the Department of Biochemistry and Molecular
Biology, University of Miami School of Medicine, Miami, Florida
33101 and the § Department of Biochemistry and Molecular
Biology, University of Kansas Medical Center,
Kansas City, Kansas 66160
A bacterial expression system for the inhibitory
N-terminal domain of human tissue inhibitor of metalloproteinases 1 (N-TIMP-1) (Huang, W., Suzuki, K., Nagase, H., Arumugam, S., Van Doren,
S. R., and Brew, K. (1996) FEBS Lett. 384, 155-161)
has been used to produce 20 single- and double-site mutants that probe
the roles of different residues in its inhibitory action on
metalloproteinases. Mutations that produce the largest increases in the
Ki for a C-terminally truncated form of stromelysin
1, MMP-3(C), but do not disturb the conformation involve
substitutions of residues that are located in a ridge that is centered
around the disulfide bond between Cys1 and
Cys70. Specific residues that have a large influence on
activity include Cys1, Thr2, Met66,
Val69, and Cys70. Of the mutations introduced,
the greatest functional disturbances, reflected in
Ki increases of 2-4 orders of magnitude, are
generated by changes that disrupt the
Cys1-Cys70 disulfide bond and by substitution
of Ala for Thr2. Most mutations that perturb the
interaction with MMP-3 have parallel effects on the affinity of
N-TIMP-1 for MMP-1 (interstitial collagenase) and MMP-2 (gelatinase A).
However, the Thr2 to Ala mutation produces an inhibitor
that is 17-fold more effective against MMP-3 than MMP-1, suggesting
that it is feasible to engineer TIMP-1 variants that are more
specifically targeted to selected matrix metalloproteinases. The
reactive site identified by these studies is a structurally constrained
but elongated region of TIMP that can fit the matrix metalloproteinase
substrate-binding site.
Tissue inhibitors of metalloproteinases (TIMPs)1 are the only known protein inhibitors that are specific for the family of matrix metalloproteinases (MMPs), a group of endopeptidases that are responsible for the breakdown of components of connective tissue (1-3). Four paralogous TIMPs (TIMP-1, TIMP-2, TIMP-3, and TIMP-4), which differ in cellular expression, regulation, and localization, have been identified in mammals and birds (4-9). All four TIMPs consist of two domains, each stabilized by three disulfide bonds (10). The N-terminal domain is responsible for their inhibitory action against active MMPs, and the C-terminal domain modulates the interaction of TIMPs with pro-MMPs (11). TIMPs also have growth factor activity for erythroid and other cells (12-14).
MMPs play critical roles in biological processes associated with connective tissue turnover, and the balance between levels of TIMPs and active MMPs is important in normal processes such as tissue remodeling and wound healing. Imbalances in the activities of TIMPs and MMPs are associated with pathological conditions such as arthritis, tissue ulceration, and tumor cell invasion and metastasis. Because of their biomedical significance, the mechanisms by which TIMPs regulate the activity of MMPs in vivo and in vitro have been the focus of many previous studies. Aspects of the expression of TIMPs in different tissues or cells and its regulation have been investigated intensively (see Ref. 10 for a review) as well as the role of TIMPs in modulating the activation of MMPs (15). However, less progress has been made in determining the molecular basis of the inhibitory mechanism of TIMP.
Previously, mutagenesis has been carried out in attempts to identify key residues for the inhibitory activity of TIMP-2 (16). Since there was no information available on the three-dimensional structure of TIMP, the design of mutations was based on chemical modification data (17) and sequence conservation. The results suggested that the region between Cys3 and Cys13, particularly His7 and Gln9, of TIMP-1 is important for its interaction with MMPs. However, because amino acid substitutions at these sites increase Ki values by a factor of 3-7, reflecting a loss of <10% of the free energy of binding, they do not appear to account for the high affinity binding of TIMPs with MMPs.
The region of TIMP that is important for its inhibitory activity has also been investigated using synthetic peptides and antibodies to inhibit its interaction with interstitial collagenase, MMP-1 (18). The results were interpreted as indicating that the second "disulfide knot" (Cys13-Cys124 and Cys127-Cys174) plays a major role in activity. However, low molecular weight peptides generally have little structure in solution and can potentially inhibit TIMP-1 action by nonspecific binding or by acting as alternative substrates for the metalloproteinase. Such mechanisms appear likely since peptides were required at concentrations in the millimolar range to exert effects as compared with nanomolar concentrations of TIMP-1. The reliability of information obtained with antibodies is also suspect since immunoglobulins are very large as compared with TIMP-1 (Mr = 150,000 as compared with 25,000), and their binding can impede the access of the metalloproteinase to a large surface area around their epitope on TIMP.
The solution structure of the inhibitory amino-terminal domain of
TIMP-2 has been determined at low resolution by multidimensional NMR
(19). This shows that N-TIMP-2 has a fold similar to that of the
OB-fold family of oligonucleotide/oligosaccharide-binding proteins (see
Fig. 1) and gives a basis for designing and interpreting the results of
structure-function relationships in N-TIMP-1. With the goal of studying
structure-function relationships of TIMP, we have developed a bacterial
expression system for producing high yields of recombinant human
N-TIMP-1 that has been characterized as having a fully native fold by
CD spectroscopy and two-dimensional NMR (20). This system is convenient
for designed mutagenesis studies to probe structure-function
relationships and allows appropriate physical characterization of the
structural state of variants. We describe here 20 mutants of N-TIMP-1
that have been expressed as inclusion bodies in Escherichia
coli, refolded successfully in vitro, and characterized
with respect to activity and CD spectra. Their functional properties
emphasize the importance of a region that includes
Met66-Cys70 and
Cys1-Thr2 for TIMP inhibitory activity.
Substitution of Thr2 differentially influences the activity
of TIMP-1 for binding to MMP-1, MMP-2 (gelatinase A), and MMP-3
(stromelysin 1), suggesting that it may be possible to engineer TIMPs
that are selective inhibitors of MMPs.
Ultima DNA polymerase with proofreading function
was purchased from Perkin-Elmer. WizardTM PCR Preps purification kits
were purchased from Promega. Other reagents, separation media, and kits
were from the same sources as described in a previous work (20). Dr. R. Werner (Department of Biochemistry and Molecular Biology, University of
Miami School of Medicine) provided synthetic oligonucleotides. MMP-1
and MMP-2 were isolated as zymogens as described previously (21, 22).
C-terminally truncated pro-MMP-3(C) was expressed in E. coli as inclusion bodies, from which it was extracted, folded, and
purified.2 Pro-MMP-2 and
pro-MMP-3(
C) were activated with 4-aminophenylmercuric acetate and
pro-MMP-1 with MMP-3 in the presence of 4-aminophenylmercuric acetate
(21). The MMP-3 used for pro-MMP-1 activation was removed by anti-MMP-3
affinity chromatography (23).
Mutations were introduced using the polymerase chain reaction megaprimer method (24) with the pET3a-N-TIMP-1 expression vector as the template (20). The amplification to generate the megaprimer was performed in each case with the synthetic T7 promoter primer or the T7 terminator primer together with an appropriate mutagenic primer. Megaprimers were purified using the WizardTM PCR Preps purification kit and used in a second amplification with the cognate T7-based primer. After purification by agarose gel electrophoresis, the final amplification product was digested with BamHI and NdeI, and the product was cloned into pET3a as described for N-TIMP-1. The coding sequences of all mutants were checked by DNA sequencing of the expression vector. N-TIMP-1 mutants were expressed as inclusion bodies and extracted, folded, and purified as described previously (20).
Kinetic StudiesThe synthetic fluorogenic substrates (7-methoxycoumarin-4-yl)acetyl-Arg-Pro-Lys-Pro-Val-Glu-norvalinyl-Trp-Arg-Lys-(2,4-dinitrophenyl)-NH2 (kindly provided by Dr. G. B. Fields, University of Minnesota) for MMP-3 (25) and (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-N-3-(2,4-dinitrophenyl)-L-2,3-diaminopropylAla-Arg-NH2 (Bachem Bioscience Inc., King of Prussia, PA) for MMP-1 and MMP-2 (26) were used to assay TIMP-1 activity as described previously (20). The inhibition of each of the above MMPs by N-TIMP-1 and its mutants was assayed by preincubating MMP (0.1-1 nM) and TIMP (at various concentrations) in 50 mM Tris-HCl, pH 7.5, containing 0.15 M NaCl, 10 mM CaCl2, and 0.02% Brij 35 at 37 °C for 1 h. The time required for equilibration was determined by following the progress of inhibition after mixing MMPs and TIMP at similar concentrations to those used in the assay. An aliquot (60 µl) of substrate (15 µM) was then added to 540 µl of preincubated MMP/TIMP mixture, and activity was determined at 37 °C by following product release with time. Inhibited rates were measured from the initial 10 min of the reaction profile where product release was linear with time. Ki values were calculated using a treatment of data for slow tight-binding inhibitors (27) or, in some cases, by a standard treatment for reversible lower affinity inhibitors.
CD SpectroscopyNear- and far-UV CD spectra of N-TIMP-1 and its mutants were determined with a Jasco J-710/720 spectropolarimeter as described previously (20). Near-UV CD spectra (250-320 nm) were determined using a cell with a 1-cm path length, and far-UV spectra (200-250 nm) were determined using a cell with a 0.1-cm path length. Proteins were dissolved in 20 mM Tris-HCl, pH 7.4, containing 0.2 M NaCl.
Our strategy has been to probe regions of TIMP-1 that have been implicated in its activity by chemical modification studies and by analogy with other OB-fold proteins that share structural similarities to N-TIMP-2. Trace labeling studies3 have indicated that groups of residues in the second disulfide loop between Cys13 and Cys70 are perturbed in reactivity toward acetic anhydride on binding to MMP-3. To investigate this region, a series of residues in this region (residues 16, 18, 22, 35, 38, 42, 45, 46, and 66) were mutated to Ala or to amino acids with similar polarity. Tyr35 and Tyr38 were chosen for replacement because, in the low resolution solution structure of TIMP-2, their equivalents are components of a surface loop that is adjacent to the binding site in other OB-fold proteins. Additional evidence that influenced our experimental design was the observation that human neutrophil elastase inactivates TIMP-1 by cleaving the Val69-Cys70 peptide bond, whereas this bond is protected and TIMP-1 activity is preserved by formation of the TIMP·MMP-3 complex.4 These results and the perturbed Ki, observed in the M66A mutant, prompted us to probe residues that are close to this region in covalent structure and in the solution NMR structure. This led us to construct mutants with substitutions of Val69, Cys70, Cys1, Thr2, and Pro5. The locations of the corresponding residues in N-TIMP-2 are shown in Fig. 1.
Preparation of N-TIMP-1 MutantsThe polymerase chain reaction method introduced some unwanted mutations adjacent to the sequence derived from the mutagenic primer, which can be attributed to the terminal transferase activity of Taq DNA polymerase. These were minimized by the use of a thermostable polymerase with proofreading activity (Ultima DNA polymerase) and by designing primers so that all substitutions at the susceptible site are silent. A few additional mutations were found outside the primer presumably because of errors made by the polymerase, which generated the double mutants C1S/F12Y, M45A/F49V, N14S/V69T, and V69A/A103V.
Wild-type and mutant forms of N-TIMP-1 were expressed in E. coli, solubilized, partially purified, and folded in vitro as described above. The final purification was carried out by cation-exchange chromatography with CM52. The yields ranged from 5 to 20 mg of purified protein/liter of bacterial culture, with the exception of the mutant Y38A, for which the yield was ~2 mg/liter of culture. Concentrations of the mutant proteins were calculated from the absorbance of solutions at 280 nm using extinction coefficients calculated from their contents of tryptophan, tyrosine, and cystine (28).
Activity StudiesInitially, all mutants were assayed for
their activities as inhibitors of a C-terminally truncated form of
MMP-3 (MMP-3(C)). The assay was developed to provide a facile means
of comparing different mutants. In this assay, preincubation at
37 °C is performed for 1 h to ensure that the binding of
inhibitor and metalloproteinase reaches equilibrium, but the addition
of substrate results in a 10% dilution of the two proteins.
Re-equilibration after this dilution will be slow and is expected to
have little effect on the results. Since the substrate concentrations
are very low relative to the Km (1.5 µM compared with 25 µM), any correction of
the apparent Ki determined directly from the kinetic study for competition by substrate is expected to be negligible. Table
I lists the apparent
Ki values mostly calculated using the treatment for
tight-binding inhibitors (27), except with mutants for which very large
reductions in affinity became apparent; the data for the latter group
were analyzed as low affinity reversible inhibitors. Substitutions of
Asp16, Val18, and Lys22 produced
only minor changes in affinity compared with the wild type.
Substitutions of the methionines at positions 42 (M42T) and 45 (M45A/F49V) and of the tyrosines at positions 35 (Y35A) and 38 (Y38F)
produced significant but not major (4-6-fold) reductions in affinity,
whereas substitution of Ala for Met66 had a larger effect
(14-fold loss of binding). Mutations at positions 2 (T2A) and 38 (Y38A)
reduced the affinity for MMP-3(
C) by >2 orders of magnitude.
Substitutions of either of the two disulfide-bonded cysteines,
Cys1 and Cys70, produced a decrease of >3
orders of magnitude in the affinity for MMP-3(
C). To put these
mutations into perspective in relation to the proportion of molecular
contacts between the inhibitor and protease affected by the
substitution, the change in the Gibbs free energy change for the
protein-protein interaction produced by the mutation
(
G) was calculated for each mutation using the relationship
G =
RT
ln(Ki(wild-type)/Ki(mutant)); these values are shown in Fig. 2. C70S,
C1S/F12Y, T2A, and Y38A lost 3-4 kcal/mol of their free energy of
interaction with MMP-3(
C).
|
Mutants with moderate to large decreases in affinity toward MMP-3(C)
were selected for further investigation with MMP-1 and MMP-2 (Table
II). Interestingly, whereas most
mutations had parallel effects on the affinity of N-TIMP-1 for the
three proteinases, T2A was more selective than other mutations, showing
in particular a 17-fold higher affinity for MMP-3(
C) as compared
with MMP-1 (Fig. 3).
|
CD Spectroscopy
The mutants that displayed major changes in
Ki were further characterized by UV CD spectroscopy
to see if the loss of activity was a direct result of the sequence
change or the indirect consequence of a global change in secondary or
tertiary structure in N-TIMP-1. The only feature of the far-UV CD
spectrum of N-TIMP-1 is a trough with a minimum at 208 nm (20), which is not characteristic of any known type of secondary structure. Consequently, changes in the CD spectrum in this wavelength range do
not provide useful information about the structural effects of
mutations. In contrast, N-TIMP-1 has a complex near-UV CD spectrum with
multiple peaks that reflect the fixed environments of aromatic side
chains and disulfide bonds in the native structure. The spectrum of
this region in N-TIMP-1 is therefore a preferable indicator of changes
in tertiary structure introduced by mutations and can help to identify
mutants with partially folded conformations (29). The near-UV CD
spectra of mutants that show large changes in functional properties are
shown in Fig. 4. Although the mutations
affected the magnitude of the largest peak centered at 290 nm, the
multiple peaks and inflections between 255 and 285 nm were preserved in most of the mutants, including M66A/C70F, which had the lowest activity
of all of the mutants constructed in this study. In contrast, the T2A
mutant showed some signs of being structurally perturbed, whereas Y38A
and the C1S/F12A double mutant lost much of the detail in their CD
spectra. It seems likely that some of the loss of activity in these
variants results from a loss of fixed tertiary structure.
Tyr38, in particular, is an improbable candidate as a
component of the interaction site because substitution with
phenylalanine has little effect on activity (Table I).
In the absence of a three-dimensional structure for the complex of
a TIMP with a MMP, site-directed mutagenesis provides an effective
approach for investigating the roles of individual residues in the
inhibitory action of TIMP-1. Mutation of a series of sites between
Cys13 and Met45 produced relatively small
reductions in affinity for MMP-3. As discussed above, the Y38A variant,
which has a greatly reduced affinity for MMP-3(C), also has a
disturbed tertiary structure that may account for the reduction in
activity. The fact that substitution with Phe at this site has little
effect on inhibitory activity supports this hypothesis. In the solution
structure of TIMP-2, the side chain of the residue corresponding to
Tyr38 of TIMP-1 is partially buried (Fig. 1). Substitution
of a large hydrophobic side chain with a small methyl group (Y38A) will
introduce a cavity in the interior of the protein and destabilize the
structure. The low yield of this protein obtained after folding is
consistent with the effect of the mutation on folding and
stability.
Substitution of Ala for Met66 close to the C-terminal
section of the same disulfide loop produced a 14-fold increase in
Ki. Evidence for the location of the interaction
site in TIMP-1 derived from a "footprinting" experiment correlates
well with this observation. TIMP-1 loses its inhibitory function upon
incubation with human neutrophil elastase as a result of cleavage of
the peptide bond between Val69 and
Cys70.4 Cleavage of this peptide bond by
neutrophil elastase and the loss of TIMP activity are prevented when
TIMP-1 is preincubated with MMP-3. Although this does not prove that
Val69 and Cys70 are in direct contact with
MMP-3, it does suggest that they are sufficiently close for MMP-3 to
block access of the peptide bond by MMP-3.4 In the
three-dimensional structure of TIMP-2, two sections of polypeptide
chain, from Met66 to Cys70 and from
Cys1 to Pro5, are connected by a
Cys1-Cys70 disulfide bridge and form a
continuous surface ridge (19). Because the disulfide bond arrangements
are conserved in all known TIMPs, a similar structure must also be
present in TIMP-1. The mutational and protection results discussed
above suggest that this ridge forms part of the binding site. The
sequence of this region is well conserved in different TIMPs; besides
the conserved disulfide-bonded cysteines (Cys1 and
Cys70 of TIMP-1), the only amino acids found at the
position corresponding to Val69 of TIMP-1 are Val and Leu,
and only Thr and Ser are found at position 2. Proline is conserved as
residue 5 throughout the TIMP family. To probe the roles of these
conserved residues, the effects of substitutions of Cys1,
Thr2, Pro5, Val69, and
Cys70 were investigated. Replacement of either cysteine,
which prevents the formation of the disulfide bond between these
residues, produces a loss of affinity of >3 orders of magnitude. The
near-UV spectra of the disulfide bond mutants are qualitatively
different from but similar in magnitude to those of the wild-type
protein. The change in CD spectrum may be partly attributable to the
loss of the disulfide bond since disulfides contribute to the spectrum in this wavelength range (29), but some localized change in structure
may also be introduced by the mutation. However, the change in CD
spectrum is small compared with that of the misfolded Y38A mutant,
suggesting that the disulfide bond between Cys1 and
Cys70 is not essential for the overall fold of N-TIMP-1;
mutants lacking this bond probably have structures that are slightly
distorted versions of the wild-type structure. Treatment with Ellman's
reagent indicates that no free thiol groups are present in the folded disulfide bond mutants (data not shown), so the remaining cysteinyl residue is present as a mixed disulfide with mercaptoethanol or has
become oxidized during the purification process. Various substitutions of Val69 produce moderate changes in affinity, but
replacing the totally conserved Pro5 with Ala had little
effect. A striking functional change arose from substitution of Ala for
Thr2, which reduced the affinity for MMP-3 by 2 orders of
magnitude. Since this residue is conserved as Thr or Ser in different
TIMPs, it is possible that the hydroxyl group is important for the
formation of complexes between N-TIMP-1 and MMP-3(C). The CD
spectrum suggests that substitution with Ala perturbs the tertiary
structure. However, the effects of this substitution on TIMP-1
specificity (see below) support the view that residue 2 plays a key
role in TIMP action.
There are indications that different TIMPs vary quantitatively in
inhibitory activity toward different MMPs (11, 30). We were therefore
interested to determine if mutations that affect the affinity of
N-TIMP-1 for MMP-3(C) have similar effects on its interactions with
other metalloproteinases. Table II shows that most substitutions have
approximately parallel effects on the affinity for MMP-1, MMP-2, and
MMP-3 even when the Ki values are increased by >3
orders of magnitude, with the exceptions of T2A and, to a much lesser
extent, V69A/A103V. The former has a particularly striking 17-fold
discriminatory inhibitory action on MMP-3 over MMP-1 (see Fig. 3) as
well as a 7-fold higher affinity for MMP-2 relative to MMP-1. The
structural basis of this selectivity is currently unclear; a simple
explanation could be that the interaction with MMP-1 involves a
hydrogen bond between the Thr2 hydroxyl group and the
enzyme that is less important in binding to MMP-2 and MMP-3. However,
this result lends support to the proposal that a region that includes
residues 66-70 and residues 1 and 2, linked by the
Cys1-Cys70 disulfide bond (TIMP-1 numbering),
forms part of the inhibition site in TIMP. Bodden et al.
(31) have previously suggested that this disulfide bond is particularly
important for structure and/or activity in TIMP. Figs. 1 and
5 show that the disulfide bond links the
N-terminal region and residues 66-70 to form a distinct ridge on the
surface of the TIMP-2 solution structure.
Analyses by Jones and Thornton (32) show that, in high affinity heterologous protein-protein interactions that are comparable in nature and strength to the TIMP-MMP interaction, a large surface area of each component is buried on complex formation (785 ± 75 Å2 for other protease-inhibitor systems). The interaction site defined by our current results has a surface area of <200 Å2, smaller than expected for an interaction with a nanomolar dissociation constant. Although the extended ridge defined by our studies represents a suitable structure for fitting the MMP substrate-binding groove and appears to be a major component of the interaction site, additional residues in N-TIMP-1, adjacent to this region, are likely to contribute to the interaction with MMPs. A contribution to binding by a larger part of the N terminus is consistent with the previously published observation (16) that substitutions of His7 and Gln9 in TIMP-1 produce moderate 2-6-fold reductions in the Ki for MMP-7 (matrilysin). Our results also indicate that similar modest reductions in affinity are produced by substitutions of Tyr35, Tyr38, Met42, and Met45.
Amino-terminal disulfide-bonded cysteines are uncommon in proteins. The bond between Cys1 and Cys70 appears to be functionally significant as a linker between the two key sections of the reactive site. Additional residues N-terminal to Cys1 could potentially disrupt the substrate mimetic character of this region. Cys1 and Cys3 together with their associated disulfide links will also constrain the flexibility of the N-terminal region of TIMP. Structural rigidity in the reactive site may be important for the inhibitory strategy possibly for arresting the catalytic action of MMPs and preventing proteolysis of the inhibitor by its target. The properties of the T2A mutant provide an initial indication that residue 2 of TIMP is a key to specificity and that TIMP-1 variants that are specifically targeted to individual MMPs can be potentially constructed. Such proteins could be used therapeutically against pathological processes (such as tumor cell metastasis) that are associated with the activity of specific MMPs. Mutational studies are in progress to further investigate this possibility.
We thank Vera Ondricek, Tom Simmons, and Adriana Vasquez for technical assistance. We are grateful to Dr. Richard Williamson (University of Kent, Canterbury, United Kingdom) for providing the coordinates of the NMR structure of the amino-terminal domain of TIMP-2.