A Residue in the S2 Subsite Controls Substrate
Selectivity of Matrix Metalloproteinase-2 and Matrix
Metalloproteinase-9*
Emily I.
Chen
§,
Weizhong
Li
,
Adam
Godzik
,
Eric W.
Howard¶, and
Jeffrey W.
Smith
From the
Cancer Research Center, The Burnham
Institute, La Jolla, CA 92037 and the ¶ Department of Pathology,
Oklahoma City, Oklahoma 73104
Received for publication, October 9, 2002, and in revised form, February 12, 2003
 |
ABSTRACT |
Matrix metalloproteinase (MMP)-2 and MMP-9 are
closely related metalloproteinases that are implicated in angiogenesis.
The two proteins have a similar domain structure and highly homologous catalytic domains, making them an excellent comparative model for
understanding the structural basis of substrate recognition by the MMP
family. Although the two MMPs exhibit some overlap in substrate
recognition, our recent work showed that MMP-2 can cleave a set of
peptide substrates that are only poorly recognized by MMP-9 (Chen,
E. I., Kridel, S. J., Howard, E. W., Li, W., Godzik, A.,
and Smith, J. W. (2002) J. Biol. Chem. 277, 4485-4491). Mutations at the P2 position of these peptide
substrates dramatically reduced their selectivity for MMP-2. Inspection
of the corresponding S2 pocket of the substrate-binding
cleft of the protease reveals that MMP-9 contains an Asp, whereas MMP-2
contains Glu. Here, we test the hypothesis that this conservative
substitution has a role in substrate selectivity. Mutation of
Glu412 in MMP-2 to Asp significantly reduced the hydrolysis
of selective substrates, with only a minor effect on hydrolysis of
non-selective substrates. The predominant effect of the mutation is at
the level of kcat, or turnover rate, with
reductions reaching as high as 37-fold. The residues that occupy this
position in other MMPs are highly variable, providing a potential
structural basis for substrate recognition across the MMP family.
 |
INTRODUCTION |
The matrix metalloproteinase
(MMPs)1 family consists of
over 25 secreted and cell surface proteases (1). The MMPs are involved in a wide range of normal biologic processes and have been linked to a
number of pathologic events (2, 3). Because of the associations with
disease, a number of synthetic MMP antagonists have been developed and
tested in human clinical trials (2, 4-7). Unfortunately, however,
these trials did not succeed (8). One reason for the lack of success
probably relates to the relatively broad spectrum of inhibition by the
compounds that were tested. For example, the first compound to enter
the clinic, Marimastat, has a similar affinity for at least five MMPs.
The lack of selectivity of most of the small molecule MMP inhibitors
can be traced to the fact that they were designed to exploit features
common to all MMPs. These include a zinc ion and the catalytic glutamic acid (9), and especially, the deep S1' pocket (10-15). With these ideas in mind, one of the objectives of the present study
was to identify regions of the MMP catalytic pockets that contribute to
substrate distinction and that could be exploited for the future design
of highly selective MMP antagonists.
Information on substrate selection by the MMPs could also help explain
the unique biological roles of these proteases. MMPs are no longer
looked upon as proteases whose action is limited to the destruction of
collagen and gelatin in basement membranes. Rather, the MMPs are
involved in the control of a number of events where proteolysis must be
exerted in a precise manner. For example, MMPs can activate other MMP
zymogens (proMMPs) by hydrolyzing propeptide bonds (16). They also
influence that activity of the serine proteases by selectively
degrading their macromolecular inhibitors such as serpin
1-proteinase inhibitor (17). MMPs can also influence that action of
growth factors by activating and releasing them for their functions
(18). Some MMPs potentiate the action of growth factors by selectively
degrading growth factor-binding proteins (19). All of these functions
are presumed to involve a relatively high degree of selectivity among
the MMPs for their physiologic substrates. There are also circumstances
in which closely related MMPs are present in the same locale but have
different roles. For example, both MMP-2 and MMP-9 are expressed within tumors, but only one participates in the angiogenic switch (20). Similar distinctions in the role of these MMPs have also been observed
in platelet function (21) and in cell migration (22). These findings
strongly suggest that MMP-2 and MMP-9 operate by cleaving distinct substrates.
Despite the expanding awareness of selective hydrolysis by the MMPs,
the structural basis for such selectivity is still not well understood.
In a prior report, we used substrate phage display to gain an in-depth
understanding of the substrate recognition properties of MMP-2. We
identified a surprising number of highly selective substrates for MMP-2
(23). Interestingly, the selective substrates could be segregated into
three subfamilies based on their sequences. However, within each
family, the P2 residue of substrate was key to selectivity
for MMP-2 over MMP-9. The S2 pocket, which interacts with
the P2 residue in substrate, is remarkably similar in both
enzymes (14), save for the presence of Glu412 in MMP-2,
which is replaced by an Asp in MMP-9. In fact, our modeling work
suggested that Glu412 of MMP-2 forms a hydrogen bond with
the backbone of the selective substrates, a bond that cannot be formed
by the shorter Asp in MMP-9.
Here we test the importance of the Glu/Asp alteration in substrate
recognition. Mutation of Glu412 to Asp has a significant
influence on the substrate recognition by MMP-2. The substitution
causes a significant reduction in
kcat/Km for peptide
substrates that are selective for MMP-2 over MMP-9. The reduction in
kcat/Km is predominantly
driven by a reduction in kcat or the rate of turnover of
these substrates. Interestingly however, this Glu/Asp
substitution had far less of an effect on substrates that were
recognized equally well by the two MMPs. In these cases, only a minor
reduction in kcat (and correspondingly,
kcat/Km) was observed. The
deleterious effect of the Glu/Asp substitution on hydrolysis of
selective substrates was also observed on EphB1, a protein substrate
that we have found to be selectively cleaved by MMP-2 in
vitro. The results of the Glu412/Asp mutation provide
further proof that selective and non-selective substrates for MMP-2
dock via different contact points within the catalytic cleft.
Altogether, the findings of this report substantiate the hypothesis
that Glu412 in the S2 subsite of the MMP-2 is a
key residue in conferring hydrolysis of selective substrates. The study
also raises the interesting hypothesis that selectivity between MMP-2
and MMP-9 arises because MMP-9 is a "slower" protease than
MMP-2.
 |
EXPERIMENTAL PROCEDURES |
Source of Commercial Proteins and Reagents--
Ilomastat was
purchased from AMS Scientific (Concord, CA). Restriction enzymes were
from New England Biolabs. Oligonucleotides were synthesized by
Integrated DNA technologies, Inc. (Coralville, IA). Tissue culture
media and reagents were from Irvine Scientific (Irvine, CA). All other
reagents, chemicals, and plastic ware were from Sigma or Fisher.
Docking of the MMP-2 Peptide Substrates into the Catalytic
Domains of MMP-2 and MMP-9--
The complexes between MMP-2 and the
peptide substrates were modeled using the Sybyl package from
Tripos (www.tripos.com). The coordinates of MMP-2 are from the crystal
structure (Protein Data Bank accession number 1qib). For docking, the
initial structure of each peptide was built in an extended conformation within the catalytic cleft of the protease. Since the binding pocket of
the enzyme is known, and some interactions between the protein and site
around P1' of the substrate are obvious, these interactions
were used as restrictions in the modeling procedure. The anchoring
restrictions included a hydrogen bond between the NH group of
P1' and the oxygen atom of Ala165, the hydrogen
bond between the oxygen atom of P1' and the NH group of
Ala165, the interaction between the side chain of
P1', and the hydrophobic patch of MMP-2 comprised of
Val198 and its surroundings. The substrate was first placed
at the binding cleft at a position favoring the formation of bonds
making up these restrictions. Under these constraints, the initial
conformation of the substrate resembled an antiparallel
-strand
lying against the neighboring
-strand in the catalytic cleft of
MMP-2. Then a conformation search of the substrate was performed with
only two restrictions: (i) P1' was maintained as the anchor
point and (ii) the center (C
atom) of P1' remained fixed
during the modeling. Since there are no known correlations between the
conformation of substrate on opposite sides of the P1'
position (the anchoring point), the conformational searches of the N
terminus and C terminus were run separately. To reduce the
complexity of computation, the searches were done in torsion angle
space, where the bond length and bond angles of the whole substrate
remained fixed. The model of substrates within the cleft of MMP-9 were
generated in a similar manner, except that the structure of MMP-9 was
first modeled on the crystal structure of MMP-2 as described previously (23).
Site-directed Mutagenesis of MMP-2--
The mutations of
Glu412 to Asp in the MMP-2 catalytic domain was
generated with the QuikChange site-directed mutagenesis kit from
Stratagene (San Diego, CA). Oligonucleotides used for generating mutated MMP-2 were 5'-AAG CTT ATG GAG GCG CTA ATG GCC CG-3' and 5'-CGC
CAT GGG GCT GGA TCA CTC CCA AGA CCC-3'. Each pair of oligonucleotides was annealed to the denatured template plasmid encoding the catalytic domain of MMP-2. Subsequently, the mutated MMP-2 was generated by PCR
amplification of cDNA from these complexes. The mutations were
verified by sequencing the newly generated catalytic cDNA. The
mutated MMP-2 was subcloned into the pCDNA3 expression vector (Invitrogen).
Expression and Purification of the Catalytic Domain of Mutated
MMP-2--
Briefly, the cDNA encoding the mutated catalytic domain
of MMP-2 was used to transfect HEK 293 cells. Individual
antibiotic-resistant clones were isolated with cloning discs, expanded,
and then screened by reverse transcription-PCR and zymography. The
mutated catalytic domains of MMP-2 were purified from conditioned
medium by the method described previously (23). The purity of the
mutant MMP-2 was greater than 90% judging by silver-stained acrylamide
gels. The purified mutant MMP-2 was stored at
70 °C at
concentrations of ~0.3 mg/ml. For kinetic studies, the mutated MMP-2
was activated and active site-titrated as described previously
(23).
Quantifying the Kinetic Parameters of Peptide
Hydrolysis--
The kinetic parameters of substrate hydrolysis were
measured using a fluorescamine incorporation assay (24). The method for
determining the Km and kcat
of peptide hydrolysis was described previously (23).
Assessing the Extent of Hydrolysis of EphB1 by Mutated
MMP-2--
Recombinant fusion proteins between EphB1, EphB2, and the
Fc domain of IgG were purchased from R&D systems Inc. The fusion proteins (1.8 µM) were incubated for 4 h at
37 °C with 280 nM of wild type MMP-2, wild type MMP-9,
or MMP-2E412D. Following incubation, samples were resolved
by 10% SDS-PAGE, and samples were visualized by Coomassie staining.
 |
RESULTS |
Mutagenesis of Glu412 to Asp in the Catalytic Domain of
MMP-2--
Previously, we observed that the residue at the
P2 position within peptide substrates has a major role in
determining the selectivity of the peptide for MMP-2 versus
MMP-9 (23). A key distinction between MMP-2 and MMP-9 is the
substitution of a Glu for an Asp within the S2 subsite, and
we suggested that this substitution could account for differences in
substrate recognition. To test this hypothesis, we mutated
Glu412 to Asp in a construct encoding the catalytic domain
of MMP-2. The mutated MMP-2 was expressed in HEK 293 cells as described under "Experimental Procedures." The mutated MMP-2 was expressed at
levels similar to wild type MMP-2 and could be purified by affinity
chromatography on gelatin-Sepharose. To ensure that kinetic parameters
were accurately calculated, the amount of properly folded MMP was
quantified by active site titration with the small molecule inhibitor,
Ilomastat, as described under "Experimental Procuedures." Both
proteins had similar levels of catalytic activity per mg of purified protein.
Assessing the Ability of Mutated MMP-2 to Cleave the Non-selective
and Selective Peptide Substrates--
We measured the ability
of wild type and MMP-2E412D to hydrolyze non-selective
peptides substrates that contain the PXXL motif (Table
I). Only minor changes were observed in
the kcat/Km values for
peptides such as C15 and m1A11, which display no
selectivity between MMP-2 and MMP-9 (Table I). In contrast, however,
the E412D mutation had significant and consistent effects on the
hydrolysis of a panel of peptides that were found previously to be
selective for MMP-2 (Table II). Each of
the selective peptides has a higher kcat/Km ratio for MMP-2 than
for MMP-9. In all cases, the mutation of Glu to Asp caused a decrease
in the kcat/Km such that the
ratio for the mutant falls between the value for MMP2 and MMP-9. This
observation is consistent with the fact that the E412D mutation alters
the structure of the catalytic pocket to more closely resemble that of
MMP-9.
View this table:
[in this window]
[in a new window]
|
Table I
Hydrolysis of non-selective peptide substrates by MMP-2E412D
The hydrolysis of synthetic peptides by each MMP was quantified using
procedures outlined under "Experimental Procedures." For each
protease, hydrolysis was measured across a concentration range of
peptide. Values for kcat and Km
were derived from Lineweaver-Burk plots. Each measurement was repeated
at least three times and the relative error of
kcat/Km in these measurements was
less than 10%.
|
|
View this table:
[in this window]
[in a new window]
|
Table II
Hydrolysis of a panel of selective peptide substrates by
MMP-2E412D
The hydrolysis of a panel synthetic peptides previously shown to be
selective for MMP-2 (23) was quantified using procedures outlined in
"Experimental Procedures." For MMP-2 and mutant MMP-2, hydrolysis
was measured over a concentration range of peptide such that values for
kcat and Km were derived from
Lineweaver-Burk plots. For MMP-9, the
kcat/Km ratio was measured as
described in the previous paper (23). Each measurement was repeated at
least three times and the relative error for
kcat/Km and for
kcat independently less than 10%.
|
|
Interestingly, the primary effect of the mutation seemed to be at the
level of kcat, a parameter that was decreased
for all of the selective peptides. Decreases in
kcat ranged from 3 to 36-fold, in most cases
approximating the values we have measured for MMP-9. In contrast, the
effects of the E412D mutation on Km were less
consistent. We observed this parameter to increase and decrease
depending on the peptide being tested. Consequently, the E412D mutation
in the S2 pocket appears to primarily affect the rate of
substrate turnover.
One peptide was identified that has an increased
kcat/Km ratio for the E412D
mutant of MMP-2. This peptide, A13R, was originally synthesized and
characterized in our prior report on substrates selective for MMP-2
(23). The parent peptide, A13, contains the SX
L motif
that is selective for MMP-2, but A13R contains the RX
L
sequence and is cleaved better by MMP-9. Here, we compared the rate of
hydrolysis of the A13R peptide with all three of the MMPs (Fig.
1). In three independent replications of
this experiment, the rate of peptide hydrolysis by MMP-9 and by
MMP-2E412D was higher than that of wild type MMP-2. This
finding provides further support for the idea that mutation of
Glu412 to Asp shifts the substrate recognition of MMP-2
closer to that of MMP-9. Because the hydrolysis of this peptide by
MMP-2E412D is better than that of wild type MMP-2, the
observation also excludes the possibility that the mutation within
MMP-2 simply causes general diminution in proteolytic activity.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 1.
Effect of the E412D mutation on
hydrolysis of peptide substrates. A13R peptide was cleaved by
MMP-2 ( ), MMP-2E412D ( ), and MMP-9 ( ), and the
extent of hydrolysis for individual protease was measured by relative
fluorescent units (RFUs) at 2-min intervals up to 10 min.
Hydrolysis of A13R showed that the activity of MMP-2E412D
resembles MMP-9 rather than the wild type MMP-2. Four independent
replicates were performed for each time point, and standard deviation
was less than 10% as indicated by the error bars.
|
|
Comparing the Hydrolysis of EphB1 by Wild Type and Mutated
MMP-2--
The EphB1 receptor tyrosine kinase contains the
SXL motif and is selectively cleaved by MMP-2 (23). In fact,
we found that this protein is generally resistant to hydrolysis by
MMP-9. Here we tested the ability of MMP-2E412D to
hydrolyze EphB1 (Fig. 2). The EphB1
fusion protein was incubated with equimolar amounts (280 nM) of wild type MMP-2, mutated MMP-2, and MMP-9 for 4 h. The quantity of each protease was measured by active site titration
prior to initiating the experiment. The extent of hydrolysis of EphB1
was gauged by SDS-PAGE (Fig. 2). The EphB1-Fc fusion protein was almost
quantitatively cleaved by MMP-2 (Fig. 2, lane 2). As
expected, MMP-9 had no effect on the migration of the protein,
indicating a lack of hydrolysis (Fig. 2, lane 4).
Interestingly, the hydrolysis of the EphB1-Fc fusion protein by
MMP-2E412D fell between that of wild type MMP-2 and MMP-9
(Fig. 2, lane 3). This result
is consistent with the observations made with the panel of peptide
substrates and corroborates the conclusion that the E412D mutation
shifts the phenotype of the protease away from that of MMP-2 and toward
that of MMP-9.

View larger version (9K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of the E412D mutation on hydrolysis of
EphB1. The effect of E412D substitution in MMP-2 on hydrolysis of
EphB1 was tested using a recombinant fusion protein of EphB1 and the Fc
domain of IgG. The EphB1-Fc fusion protein (1.8 µM) was
incubated for 4 h at 37 °C with 280 nM of MMP-2,
MMP-2E412D, or MMP-9. Following this incubation, samples
were resolved by 10% SDS-PAGE, and the proteins were visualized by
Coomassie Blue staining. The position of the fragment of EphB1
generated by MMP-2 is shown by an arrow.
|
|

View larger version (123K):
[in this window]
[in a new window]
|
Fig. 3.
Arg at P2 supports
favorable docking to both MMP-2 and MMP-9. Peptide substrate B74R,
which is not selectively cleaved, is shown in the cleft of MMP-2
(A) and MMP-9 (B). The extension of the guanidino
group of the Arg into the S2 pocket is the key feature of
this substrate because it is positioned to interact favorably with the
acidic side of chain of either Glu412 in MMP-2 or
Asp410 in MMP-9 (circled).
|
|
 |
DISCUSSION |
MMP-2 and MMP-9 are considered to be close homologs because of
their unique domain structure and their high sequence similarity and
because they both have gelatinase activity. Nevertheless, the two
proteases clearly have distinct biological functions. In many
instances, the two proteases have different effects even when they are
analyzed in the same biological system (21, 22). Consequently, it
follows that MMP-2 and MMP-9 are likely to cleave different substrates.
We have recently begun to examine differences in the way that MMPs
recognize peptide substrates. This analysis places focus on structural
and functional distinctions at the catalytic cleft, a region that has
generally been overlooked as a feature that can distinguish one MMP
from another. In fact, early comparisons of peptide substrates for
MMP-2 and MMP-9 showed that they each cleaved the same set of
collagen-like peptide substrates (25), a finding that indicates
structural and functional similarity at the catalytic pocket.
Nevertheless, we recently made the observation that MMP-2 and MMP-9
recognize distinct sets of peptide substrates (23). We found that both
MMP-2 and MMP-9 hydrolyze peptides with the canonical
recognition motif PXXXHy, as originally
described by Netzel-Arnett et al. (25), but we also found
MMP-2 to uniquely recognize three additional sets of peptide
substrates. Like the canonical motif, the three other families of
substrates all contain a hydrophobic residue at the P1'
position, but they lack the characteristic proline at P3.
In an extension of this study, we found that the substrates selective
for MMP-2 could be converted to substrates for MMP-9 by inserting Arg
into the P2 position (23). This finding underscored the
significance of the P2 position within substrate and
strongly suggested that the corresponding S2 subsite within the catalytic clefts of MMP-2 and MMP-9 must interact differently with
substrate. Interestingly, the S2 subsite contains one of the few differences between the catalytic clefts of the two MMPs; Glu412 of MMP-2 is replaced by Asp410 in MMP-9.
The objective of the present study was to test the hypothesis that this
conservative substitution accounts for the distinct substrate
recognition profiles of the two enzymes.
We swapped this residue by mutating each protease. Although we were
able to express the mutated MMP-2, the mutant MMP-9 could not be
expressed (see below). Consequently, most of our discussion focuses on
results obtained with the mutant of MMP-2. The mutated MMP-2 was
expressed to high levels in HEK 293 cells, could be purified on
gelatin-agarose affinity columns, and exhibited gelatinase activity in
zymography gels. In addition, the E412D mutant hydrolyzed substrates
with the canonical PXXXHy recognition motif to
essentially the same degree as wild type MMP-2. This is to be expected
since MMP-9, which displays the Asp rather than Glu at this position, also hydrolyzes this subfamily of substrates with high efficiency. In
conjunction with the fact that the mutation is conservative and
represents a sequence found in the closest homolog, these observations
allow us to exclude the possibility that substitution of Glu for Asp at
position 412 causes a general diminution of catalysis by forcing
improper folding.
The E412D mutation had substantial effects on the hydrolysis of peptide
substrates that are selective for MMP-2. The mutation significantly
reduced the kcat/Km ratio for
all of the MMP-2-selective substrates. Reductions to the
kcat/Km ratio were primarily
driven by reductions to the turnover rate, kcat,
which was reduced in every case. Changes in both directions were
observed for Km, so the E412D mutation has no
uniform influence on binding affinity. The E412D mutation also extended to larger protein substrates. We recently demonstrated that the extracellular domain of the EphB1 receptor tyrosine kinase is cleaved
by MMP-2, but not by MMP-9. The ability of the E412D mutant of MMP-2 to
cleave EphB1 fell in between that of wild type MMP-2 and MMP-9,
indicating the mutation shifted the function of the protease closer to
that of MMP-9. This is taken as another indication that the
Glu412/Asp410 alteration has a role in
substrate distinction.
Altogether the results of this study suggest a structural basis for the
role of the S2 subsite in substrate recognition and catalysis. In large part, our results support the idea that the S2 subsite can engage in two types of interactions that
depend on the composition of the substrate (Figs. 3 and
4). When Arg is present at
P2, substrates show selectivity for MMP-2 and MMP-9 over
other MMPs (26). In this case, the Arg in the substrate extends far
enough into the S2 subsite to interact favorably with the
side chains of both Asp in MMP-9 or the Glu in MMP-2 (Fig. 3). Because
Arg at this position decreases Km (26), we suggest
that the salt bridge that Arg forms with the side chains of Glu or Asp
increases binding affinity for the protease (Fig. 3). A second type of
interaction at the S2 subsite is likely with the peptides
that lack Arg and are selectively cleaved by MMP-2. In the absence of
Arg at P2, only the Glu of MMP-2 extends far enough into
the S2 pocket to form a hydrogen bond with the backbone of
bound substrate (Fig. 4A), a concept that is generally
supported by our mutational studies. The corresponding Asp in MMP-9
fails to extend far enough into the pocket to make a similar contact (Fig. 4B). Interestingly, our findings also suggest that a
switch to this binding mode in MMP-2 changes the overall effect of the S2 subsite on substrate recognition. Rather than playing a
role in binding affinity, the hydrogen bond between Glu412
and the backbone of bound substrate influences
kcat, indicating that it helps to properly
position the substrate for optimal catalysis.

View larger version (120K):
[in this window]
[in a new window]
|
Fig. 4.
Substrates lacking Arg at P2 are
selective for MMP-2. The model shows MMP-2 (A) and
MMP-9 (B) bound to the B74 substrate, which is selective for
MMP-2. The distinct interactions at the S2 subsite are
highlighted with a circle. Glu412 within MMP-2
is capable of forming a hydrogen bond with the backbone of substrate.
In contrast, the side chain of Asp410 in MMP-9 cannot
extend far enough into the cleft to make this bond.
|
|
Although our findings strongly support the role of the
Glu412/Asp410 substitution in determining
substrate recognition by the two MMPs, this mutation alone was not
sufficient to completely convert the recognition profile of MMP-2 to
that of MMP-9. Consequently other factors must be involved. These are
likely to include cooperativity among other subsites where subtle
differences exist or a global difference in the shape of the catalytic
pocket that is governed by sequence differences outside of the
catalytic cleft. A resolution to this particular issue will require a
three-dimensional structure for MMP-9.
The importance of the residue corresponding to Glu412 in
the S2 subsite may extend across the MMP family. Most of
the residues surrounding the S2 subsite are strictly
conserved among all MMPs (Fig. 5). The
two histidines that flank the subsite and the glycine in the center of
the subsite are conserved across all family members. In contrast, the
position taken by Glu412 in MMP-2 is highly variable. It is
occupied by acidic residues, large hydrophobic residues, and in some
cases, glycine (Fig. 5). The distinctions among these residues are
likely to have an influence on substrate recognition by each
MMP.

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 5.
Sequence variability in the
S2 subsite of the MMPs. The alignment of residues that
comprise the S2 subsite of the MMP catalytic domains are
shown. The residues corresponding to Glu412 of MMP-2 are
noted in bold text. (CMMP corresponds to chicken
MMP-22; XMMP corresponds to Xenopus laevis
MMP.)
|
|
Certainly an analysis of the analogous substitution within MMP-9 would
be informative. However, we were unable to express this mutant,
although we tested a number of different host cell types. Although the
inability to express this mutation may seem surprising given the
extensive homology between MMP-2 and MMP-9, there may be a mechanistic
basis for this observation. It is well established that the MMPs
require the propeptide, which inserts into the catalytic cleft via a
cysteine switch, for proper folding (27, 28). We suspect that this
mutant of MMP-9 cannot be expressed because it interferes with the
insertion of the propeptide into the catalytic cleft during
synthesis. This hypothesis is based on inspection of the crystal
structure of MMP-2 with the propeptide inserted into the cleft. In
MMP-2, Glu412 extends up from the floor of the cleft to
within 3-4 angstroms of the side chain of Asn109. In
MMP-9, the corresponding residues are Arg (longer than Asn) and Asp
(shorter than Glu). The Arg in the propeptide of MMP-9 would be
expected to extend further into the catalytic cleft than Asn. Such
positioning would likely be favorable with Asp in the S2
subsite. However, mutation of Asp410 to Glu, which would
extend further into the cleft, is likely to interfere with insertion of
Arg in the MMP-9 propeptide. Such interference would be likely to
prevent proper folding of the mutated MMP-9 and cause its degradation.
 |
FOOTNOTES |
*
This study was supported by National Institutes of Health
Grants AR42750, CA82713, and CA69306 and Grant 5JB003 from the
California Breast Cancer Research Program (to J. W. S.). Additional
support was derived from a Breast Cancer Center of Excellence
Grant from the Department of Defense (DAMD 17-02-1-0693). Additional
support was derived from National Institutes of Health Grant GM60049
(to A. G.) and Cancer Center Support Grant CA30199.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.
§
Supported by a predoctoral fellowship from the Department of
Defense Breast Cancer Research Program.
To whom correspondence should be addressed. E-mail:
jsmith@burnham.org.
Published, JBC Papers in Press, February 18, 2003, DOI 10.1074/jbc.M210324200
 |
ABBREVIATIONS |
The abbreviation used is:
MMP, matrix
metalloproteinase.
 |
REFERENCES |
1.
|
Sternlicht MD, W. Z.
(1999)
in
Guidebook to the Extracellular Matrix, Anchor and Adhesion Proteins
(T Kreis, R. V., ed)
, pp. 503-562, Oxford University Press, Oxford
|
2.
|
Greenwald, R. A.,
Zucker, S.,
and Golub, L. M.
(1999)
Inhibition of Matrix Metalloproteinases: Therapeutic Applications
, Vol. 878
, Annals of the New York Academy of Sciences, New York
|
3.
|
Sternlicht, M. D.,
and Werb, Z.
(2001)
Annu. Rev. Cell Dev. Biol.
17,
463-516[CrossRef][Medline]
[Order article via Infotrieve]
|
4.
|
Arap, W.,
Valtanen, H.,
Rainisalo, A.,
Medina, O. P.,
Heikkila, P.,
Kantor, C.,
Gahmberg, C. G.,
Salo, T.,
Konttinen, Y. T.,
Sorsa, T.,
Ruoslahti, E.,
Pasqualini, R.,
and Wojtowicz-Praga, S.
(1999)
Nat. Biotech.
17,
768-774[CrossRef][Medline]
[Order article via Infotrieve]
|
5.
|
Millar, A. W.,
Brown, P. D.,
Moore, J.,
Galloway, W. A.,
Cornish, A. G.,
Lenehan, T. J.,
and Lynch, K. P.
(1998)
Br. J. Clin. Pharmacol.
45,
21-26[CrossRef][Medline]
[Order article via Infotrieve]
|
6.
|
Nemunaitis, J.,
Poole, C.,
Primrose, J.,
Rosemurgy, A.,
Malfetano, J.,
Brown, P.,
Berrington, A.,
Cornish, A.,
Lynch, K.,
Rasmussen, H.,
Kerr, D.,
Cox, D.,
and Millar, A.
(1998)
Clin. Cancer Res.
4,
1101-1109[Abstract]
|
7.
|
Parsons, S. L.,
Watson, S. A.,
and Steele, R. J.
(1997)
Eur. J. Surg. Oncol.
23,
526-531[Medline]
[Order article via Infotrieve]
|
8.
|
Eckhardt, S. G.,
and Hidalgo, M.
(2001)
J. Natl. Cancer Inst.
93,
178-193[Abstract/Free Full Text]
|
9.
|
Birkedal-Hansen, H.,
Moore, W. G.,
Bodden, M. K.,
Windsor, L. J.,
Birkedal-Hansen, B.,
DeCarlo, A.,
and Engler, J. A.
(1993)
Crit. Rev. Oral Biol. Med.
4,
197-250[Abstract]
|
10.
|
Grams, F.,
Reinemer, P.,
Powers, J. C.,
Kleine, T.,
Pieper, M.,
Tschesche, H.,
Huber, R.,
and Bode, W.
(1995)
Eur. J. Biochem.
228,
830-841[Abstract]
|
11.
|
Bode, W.,
Reinemer, P.,
Huber, R.,
Kleine, T.,
Schnierer, S.,
and Tschesche, H.
(1994)
EMBO J.
13,
1263-1269[Abstract]
|
12.
|
Stocker, W.,
Grams, F.,
Baumann, U.,
Reinemer, P.,
Gomis-Ruth, F. X.,
McKay, D. B.,
and Bode, W.
(1995)
Protein Sci.
4,
823-840[Abstract/Free Full Text]
|
13.
|
Gomis-Ruth, F. X.,
Maskos, K.,
Betz, M.,
Bergner, A.,
Huber, R.,
Suzuki, K.,
Yoshida, N.,
Nagase, H.,
Brew, K.,
Bourenkov, G. P.,
Bartunik, H.,
and Bode, W.
(1997)
Nature
389,
77-81[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
Morgunova, E.,
Tuuttila, A.,
Bergmann, U.,
Isupov, M.,
Lindqvist, Y.,
Schneider, G.,
and Tryggvason, K.
(1999)
Science
284,
1667-1670[Abstract/Free Full Text]
|
15.
|
Lovejoy, B.,
Cleasby, A.,
Hassell, A. M.,
Longley, K.,
Luther, M. A.,
Weigl, D.,
McGeehan, G.,
McElroy, A. B.,
Drewry, D.,
and Lambert, M. H.
(1994)
Science
263,
375-377[Medline]
[Order article via Infotrieve]
|
16.
|
Nagase, H.,
and Woessner, J. F., Jr.
(1999)
J. Biol. Chem.
274,
21491-21494[Free Full Text]
|
17.
|
Liu, Z.,
Zhou, X.,
Shapiro, S. D.,
Shipley, J. M.,
Twining, S. S.,
Diaz, L. A.,
Senior, R. M.,
and Werb, Z.
(2000)
Cell
102,
647-655[Medline]
[Order article via Infotrieve]
|
18.
|
Yu, Q.,
and Stamenkovic, I.
(2000)
Genes Dev.
14,
163-176[Abstract/Free Full Text]
|
19.
|
Imai, K.,
Hiramatsu, A.,
Fukushima, D.,
Pierschbacher, M. D.,
and Okada, Y.
(1997)
Biochem. J.
322,
809-814[Medline]
[Order article via Infotrieve]
|
20.
|
Bergers, G.,
Brekken, R.,
Mcmahon, G.,
Vu, T. H.,
Itoh, T.,
Tamaki, K.,
Tanzawa, K.,
Thorpe, P.,
Itohara, S.,
Werb, Z.,
and Hanahan, D.
(2000)
Nat. Cell Biol.
2,
737-744[CrossRef][Medline]
[Order article via Infotrieve]
|
21.
|
Fernandez-Patron, C.,
Martinez-Cuesta, M. A.,
Salas, E.,
Sawicki, G.,
Wozniak, M.,
Radomski, M. W.,
and Davidge, S. T.
(1999)
Thromb. Haemostasis
82,
1730-1735[Medline]
[Order article via Infotrieve]
|
22.
|
Giannelli, G.,
Falk-Marzillier, J.,
Schiraldi, O.,
Stetler-Stevenson, W. G.,
and Quaranta, V.
(1997)
Science
277,
225-228[Abstract/Free Full Text]
|
23.
|
Chen, E. I.,
Kridel, S. J.,
Howard, E. W.,
Li, W.,
Godzik, A.,
and Smith, J. W.
(2002)
J. Biol. Chem.
277,
4485-4491[Abstract/Free Full Text]
|
24.
|
Ding, L.,
Coombs, G. S.,
Strandberg, L.,
Navre, M.,
Corey, D. R.,
and Madison, E. L.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
7627-7631[Abstract]
|
25.
|
Netzel-Arnett, S.,
Sang, Q. X.,
Moore, W. G.,
Navre, M.,
Birkedal-Hansen, H.,
and Van Wart, H. E.
(1993)
Biochemistry
32,
6427-6432[Medline]
[Order article via Infotrieve]
|
26.
|
Kridel, S. J.,
Chen, E.,
Kotra, L. P.,
Howard, E. W.,
Mobashery, S.,
and Smith, J. W.
(2001)
J. Biol. Chem.
276,
20572-20578[Abstract/Free Full Text]
|
27.
|
Springman, E. B.,
Angleton, E. L.,
Birkedal-Hansen, H.,
and Van Wart, H. E.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
364-368[Abstract]
|
28.
|
Van Wart, H. E.,
and Birkedal-Hansen, H.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
5578-5582[Abstract]
|
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.