From the Department of Structural Biology, The
Weizmann Institute of Science, Rehovot 76100, Israel and the
§ Institute for Drug Design, the ¶ Department of
Chemistry, Pharmacology and of Biochemistry and Molecular Biology, and
the
Department of Pathology, Wayne State University,
Detroit, Michigan 48202
Received for publication, December 22, 2000
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ABSTRACT |
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Malignant tumors express high levels of
zinc-dependent endopeptidases called matrix
metalloproteinases (MMPs), which are thought to facilitate tumor
metastasis and angiogenesis by hydrolyzing components of the
extracellular matrix. Of these enzymes, gelatinases A (MMP-2)
and B (MMP-9), have especially been implicated in malignant processes,
and thus, they have been a target for drugs designed to block their
activity. Therefore, understanding their molecular structure is key for
a rational approach to inhibitor design. Here, we have conducted x-ray
absorption spectroscopy of the full-length human MMP-2 in its
latent, active, and inhibited states and report the structural changes
at the zinc ion site upon enzyme activation and inhibition. We have
also examined the molecular structure of MMP-2 in complex with SB-3CT,
a recently reported novel mechanism-based synthetic inhibitor that was
designed to be highly selective in gelatinases (1). It is shown that
SB-3CT directly binds the catalytic zinc ion of MMP-2. Interestingly,
the novel mode of binding of the inhibitor to the catalytic zinc
reconstructs the conformational environment around the active site
metal ion back to that of the proenzyme.
Zinc-dependent endopeptidases of the family of matrix
metalloproteinases (MMPs)1
serve important functions in tissue remodeling, organ development, ovulation, fetus implantation, embryogenesis, wound healing, and angiogenesis (2). Moreover, various members of the MMP family have been
implicated in a number of pathological conditions, including cancer
growth, tumor angiogenesis, metastasis and arthritis, connective tissue
diseases, inflammation, and cardiovascular and autoimmune diseases (3).
Due to the broad spectrum of pathological conditions associated with
disregulation of MMP activity, synthetic MMP inhibitors are highly
sought (1, 4-8). However, the molecular mechanisms of MMP activation
and inhibition are still not fully understood (9, 10).
X-ray crystal structures are available for the catalytic domains of
various MMPs (11, 12), including the full-length latent MMP-2
(pro-MMP-2) (13). In addition, structures of inhibitor-enzyme complexes
are also available (14-19).
Structural analysis of MMP-inhibitor complexes has mainly focused on
the study of the interactions of the catalytic domains of the enzyme
with sulfonamide or hydroxamic acid derivatives as zinc-cheating
ligands (7, 12, 17, 20). Most of the synthetic inhibitors are designed
to provide a bidentate chelating ligand to the catalytic zinc ion. It
is a general trend that these metal chelators largely lack selectivity
in inhibition of MMPs. This complicates the possibility for targeting
specific members of the MMP family in a particular pathological
condition. A recent report by Brown et al. (1) described a
novel concept for the selective inhibition of gelatinases (MMP-2 and
MMP-9) by the design and synthesis of the first mechanism-based MMP
inhibitor ("suicide substrate") for any MMP. This small molecule
inhibitor, designated SB-3CT, provides a potent and highly selective
inhibition of human gelatinases by the manifestation of both slow
binding and mechanism-based inhibition behavior in its kinetic profile
(1).
To gain insight into the mechanism of inhibition of MMP-2 by SB-3CT and
the local structure around the catalytic zinc ion in latent, active,
and inhibited MMP-2, the zinc ion coordination shell in all complexes
was studied by x-ray absorption spectroscopy (XAS). Our results show
that the catalytic zinc ion is directly coordinated to the sulfur atom
of the bound inhibitor in a monodentate manner to form a tetrahedral
coordination at the zinc ion. Interestingly, the inhibited enzyme
retains the conformation of the latent MMP-2 around the zinc
coordination shell, which may explain the remarkable selectivity of
SB-3CT for gelatinases for which it was designed.
Human pro-MMP-2 was expressed in a recombinant vaccinia virus
mammalian cell expression system, as described earlier (21). Pro-MMP-2
was purified to homogeneity from the media of infected HeLa
cells by gelatin-agarose affinity chromatography, as described previously (21). The protein concentration of proMMP-2 was determined using the molar extinction coefficient of 122,800 M The synthetic mechanism-based inhibitor was synthesized for these
studies as described earlier (1). MMP-2 was inhibited for our studies
according to the procedure that has been reported previously (1).
Inductively Coupled Plasma Atomic Emission Spectroscopy
(ICP-AES)
The metal content in MMP-2 samples was analyzed by inductively
coupled plasma atomic emission spectroscopy using the ICP-AES model
"Spectroflame" from Spectro (Kleve, Germany). Prior to measurement, the samples were digested with nitric acid, and the volume was adjusted
to 6 ml (final concentration 10%). The zinc content in the protein
samples was determined relative to an equivalent amount of the enzyme
assay buffer.
XAS Studies
Sample Preparation--
All enzyme samples were subjected to
gelatin zymography before XAS data collection. The enzyme was
concentrated by ultrafiltration using a Millipore Centricon-30
(Bedford, MA) device to make a final concentration of 10 mg/ml. Samples
were loaded into copper sample holders (10 × 5 × 0.5 mm)
covered with Mylar tape and were frozen immediately in liquid nitrogen.
The frozen samples were then mounted inside a Displex closed-cycle
helium cryostat, and the temperature was maintained at 30 K, to
minimize the thermal disorder in the XAS data.
Data Collection--
XAS data collection was performed at the
National Synchrotron Light Source at Brookhaven National
Laboratory, beam line X9B. The spectra were recorded at the zinc
K-edge in fluorescence geometry at low temperature (30 K). The
beam energy was defined using a flat Si(111) monochromator crystal. The
incident beam intensity I0 was recorded using an
ionization chamber. The fluorescence intensity was recorded using a
13-element germanium detector. The transmission signal from a zinc foil
was measured with a reference ion chamber simultaneously with
fluorescence to calibrate the beam energy. Several scans of each sample
were collected for a total of 1 × 106 counts across
the edge. The samples were checked for burning marks after each scan,
and the beam position on the sample was changed before each scan to
minimize radiation damages.
Data Processing and Analysis--
The average zinc K-edge
absorption coefficient µ(E), which was obtained after
10-12 independent XAS measurements for each sample, was aligned using
the first inflection point of a reference zinc metal foil XAS data
(9659 eV). Subsequently, the absorption coefficients for different
samples were shifted in x-ray energy until their first inflection
points were aligned at the same energy.
The smooth atomic background was removed with the AUTOBK program of the
UWXAFS data analysis package, developed at the University of
Washington, Seattle (24). The same energy, E0 = 9659 eV, was chosen for the purpose of background removal as the origin of the photoelectron energy. The R-space region for minimizing the
signal below the first shell was chosen between 1.2 and 3 Å. After the
removal of background, the useful k-range in the resultant
k2-weighted
The theoretical XAFS signal was fitted to the experimental data using
the nonlinear least squares method, implemented in the program FEFFIT
(24) in R-space, by Fourier transforming both theory and data. Data and
theory were weighted by k and multiplied by a Hanning window
function in Fourier transforms.
Molecular Modeling
X-ray crystal structure of MMP-2 (RCSB Protein Data Bank Code
1CK7) was used to model the inhibitor in the active site of MMP-2 as
well as to model the covalent complex between the inhibitor and MMP-2.
The point charges on the inhibitor molecule were MNDO
electrostatic potential charges, and the charge on the zinc was
+1.62.2 The same
charge on the zinc ion was used in the covalent complex and the
noncovalent complex. The inhibitor molecule was docked into the active
site of MMP-2 by fitting the biphenyl moiety into the S1' pocket. The
complex, including crystallographic water molecules, was solvated using
TIP3 water molecules in a box of 10-Å thicknesses from the surface of
the enzyme. The entire complex was energy-minimized for 2500 iterations
using AMBER 6.0 (28). In the case of the covalent complex, a bond
between the zinc ion and the thiirane sulfur, and a bond between
Glu404 and the methylene moiety of the thiirane ring, were
defined. This complex was energy-minimized as described above. Only one enantiomer of the thiirane inhibitor is shown in Fig. 4, although we
carried out energy minimizations with both stereoisomers.
The Zinc Stoichiometry in the Full-length Recombinant Human
MMP-2--
Given the importance of the zinc ion in the proenzyme
latency and in catalysis by the mature enzyme, quantitative analyses of
the zinc stoichiometry in MMPs have given puzzling results (29-31).
Direct measurements of the zinc ion content in various MMPs show
variations between 1 and 2 equivalents of metal ions per enzyme.
Studies by Willenbrock et al. (34), Springman et al. (33), and recently by Kleifeld et al. (32) suggest
that full-length intact MMPs (for the ones tested) contain a single zinc ion, while truncated enzymes (lacking the C-terminal domain) contain two zinc ions. In addition, it was suggested that the zinc content in MMPs might be dependent on the procedure used for
enzyme purification (32, 33). Nevertheless, the crystal structure of an
inactive full-length pro-MMP-2 mutant clearly shows the
presence of both structural and catalytic zinc ion sites (13).
The zinc content in our protein preparation has been determined by
ICP-AES and XAS. The ICP-AES analysis gave a zinc stoichiometry of
1.93 ± 0.1 zinc ions per protein, as determined in four separate determinations.
The zinc-protein ratio was further investigated by XAS edge step
analysis following previous procedures (32). Briefly, the edge
step of the XAS coefficient should be proportional to the concentration
of the absorbing element. Therefore, the number of zinc ions per
protein can be determined by comparing the edge step intensity measured
in the enzyme absorption coefficient data with an edge step calibration
curve obtained for standard compounds, where the edge step is measured
as a function of zinc concentration. This calibration curve is linear
throughout its range where linearity in the lower concentrations of the
calibration curve was obtained by extrapolating the line to zero. To
obtain the estimated concentration of zinc ion in the enzyme as
measured by XAS, we crossed the experimental edge step intensity value
of recombinant human pro-MMP-2 with the calibration curve. The
concentration of zinc ion obtained by this analysis was 1.75 ± 0.05 zinc ions per enzyme (data not shown).
The results obtained from the ICP-AES and XAS support the presence of
two zinc ions per enzyme molecule. Similar ICP studies on the
full-length recombinant pro-MMP-2 (purified from NSO mouse myeloma cells) showed a one-to-one ratio for zinc ion and protein (34),
as opposed to two-to-one ratio reported by x-ray crystallography (13). Based on our studies of the natural human pro-MMP-9 (where only one zinc ion per protein was found), we cannot
rule out the possibility that the zinc ion content in MMPs is dependent
on the overall stability of the enzyme and the purification procedures.
Nevertheless, we have treated our x-ray structural analysis with two
zinc ions according to the stoichimetric ratio seen in the preparation
used in this study.
X-ray Absorption Studies of the Latent, Activated, and Inhibited
MMP-2--
The active site structures of the catalytic zinc ion in the
recombinant human MMP-2 in its latent, active, and inhibited states were studied by x-ray absorption near edge structure (XANES), and by
XAFS spectroscopy. XAFS refers to modulations in x-ray absorption
coefficient around an x-ray absorption edge of a given atom. XAFS is
divided into EXAFS (extended x-ray absorption fine structure) and XANES
that provide complementary information. EXAFS is a valuable technique
for elucidating the structure of a variety of metal-binding
sites in metalloproteins (36). Information available from XAFS includes
average bond distances, mean square variation in distance, coordination
numbers, and atomic species. Fig. 1 shows
the proposed mechanism for inhibition of gelatinases by the
mechanism-based inhibitor (adopted from Brown et al. (1)). Fig. 2 shows the normalized raw x-ray
absorption edge spectra of recombinant human MMP-2 in the latent,
active, and inhibited states of the enzyme. The proenzyme edge spectrum
has three distinct peaks at 9665, 9710, and 9738 eV. Activation of
MMP-2 results in a slight increase in peak intensity at 9665 eV and in
drastic reduction in peak intensity at 9738 eV. The changes in the
spectral features are consistent with the raw x-ray absorption spectra observed for the latent and active natural human MMP-9, respectively (32). Similar to the natural human MMP-9, a slight edge shift to
a higher energy is observed upon activation. Interestingly, we observed
that MMP-2 in complex with SB-3CT exhibited an edge spectrum similar to
that observed with pro-MMP-2 alone with an increase in peak intensity
at 9738 eV. In addition, the edge position is shifted back to lower
energy and overlaps with the proenzyme edge position. This may indicate
that the total atomic charge of the zinc ion in the inhibited enzyme is
consistent with the atomic charge of the zinc ion in the proenzyme.
Thus, in both the latent and the inhibited active form of the enzyme, a
thiolate completes the coordination sphere. The great similarity in XAS features between natural MMP-9, which was shown to have one zinc ion
(32), and MMP-2 points out to the near identity of the structure around the catalytic zinc ion in both enzymes. In addition, the binding
of the inhibitor does not influence the backscattering contribution of
the structural zinc. This observation is also supported by the
MMP-2-SB-3CT binding ratio.
To further study the structure of the zinc site in the enzyme, we have
performed a rigorous EXAFS analysis. EXAFS analyses of MMP-2, in its
latent, active, and inhibited states, were conducted by fitting the
data to theoretical phase shifts and amplitudes. Theoretical models of
the proposed structural and catalytic zinc sites were constructed from
the crystal structure of pro-MMP-2 (13). The crystal structure reports
the presence of two zinc ions with proposed catalytic and structural
functions. The catalytic zinc ion in pro-MMP-2 is bound to three
histidines and one cysteine, whereas the structural zinc ion is bound
to three histidines and one aspartate. The theoretical models that were
constructed from these sites were used to modulate the catalytic and
structural zinc sites of MMP-2 in the latent, active, and inhibited
enzyme EXAFS data in our data analysis procedures. The conformational variations at the structural zinc (37) site were addressed by fitting
the zinc site models obtained from the crystallographic data to the raw
EXAFS data. Fig. 3, a
EXAFS analysis of the active enzyme (Table IB, Fit 1) revealed three
Zn-N(His) contributions at 1.97 ± 0.02 Å, one Zn-O contribution at 2.01 ± 0.05 Å and, five/six Zn-C contributions at 3.09 ± 0.03 Å. All attempts to successfully resolve a Zn-S phase and
amplitude in the active enzyme data failed. The absence of a Zn-S(Cys)
ligand at the zinc site indicates that a complete cleavage of the
propeptide took place upon enzyme activation. In addition, good fits
were obtained by using the "one zinc ion model," which means that
the first coordination shell of both zinc ions in the active enzyme has
similar atomic environments. The bond distances in this fit represent
the average distances of both zinc centers. We have assigned the
Zn-O contribution to the binding of a water molecule to the catalytic
zinc ion. These results are consistent with the proposed "cysteine
switch hypothesis" in MMPs (13, 38) where the intact propeptide
maintains the latency of the proenzyme by shielding the active site
from the milieu.
The EXAFS fitting results of the inhibited enzyme are in excellent
agreement with its edge spectra. The first coordination shell of the
inhibited enzyme is consistent with a tetrahedral coordination, which
is similar in its type of ligation to the proenzyme. The zinc-ligand
distances (Table IC, Fits 1-2) are consistent with three Zn-N(His) at
2.06 ± 0.03 Å, one Zn-S(inhibitor) at 2.22 ± 0.03 Å, and
seven Zn-C contributions where three distances are at 3.05 ± 0.05 Å and four distances are at 3.31 ± 0.04 Å. These results are
consistent with the reported molecular mechanical calculations (1),
which predict a direct binding of the mechanism-based inhibitor to the
catalytic zinc ion via the sulfur atom (Figs. 1 and
4). Interestingly, the EXAFS fitting
analysis clearly shows that the Zn-S(SB-3CT) bond distance in the first
coordination shell of the catalytic zinc atom in the inhibited enzyme
is somewhat shorter than the Zn-S(Cys) in the proenzyme. This suggests
that the mechanism-based inhibitor is bound to the catalytic zinc ion via a thiolate group. Essentially, initial coordination of the thioether group in the inhibitor has been transformed to that of the
thiolate coordination, as would be expected for the mechanism of enzyme
inhibition (Figs. 1 and 4).
The zinc ion coordination shell was found to be tetrahedral in the
zymogenic, active, and inhibited enzymes. Attempts to fit additional
Zn-O/N contribution resulted in high Inhibition of Recombinant Human MMP-2 by SB-3CT--
Fig. 4 shows
the modeling results of the binding of SB-3CT to the catalytic zinc ion
in MMP-2. The design of the molecule commenced with the x-ray structure
of MMP-2. The molecule was built based on the knowledge of binding of
other inhibitors to the active site of the MMPs. The biphenyl group was
designed to fit in the conserved hydrophobic P1' pocket in
gelatinases, and the thiirane group was intended to coordinate to the
catalytic zinc ion. Fig. 4A depicts the energy-minimized
complex for the MMP-2-inhibitor complex prior to covalent bond
formation. This coordination would promote the thiirane for
nucleophilic addition by the active-site glutamate, which results in
irreversible enzyme inhibition. The energy-minimized model for the
covalently modified MMP-2 is given as Fig. 4B. The
irreversibly modified enzyme was predicted to have a thiolate
coordinated to the catalytic zinc ion, and the inhibitor would be
covalently tethered to the active-site glutamate (1).
In this work we present the local structure of the catalytic site
zinc ions in MMP-2. The first and second coordination environments of
the zinc ions were studied in the latent, active, and inhibited forms
of MMP-2 using XANES, EXAFS spectroscopy, and modeling studies. Our
EXAFS data analysis of the local structures around both the structural
and catalytic zinc ions in pro-MMP2 is consistent with the reported
crystal structure (13). The EXAFS fitting analysis of active MMP-2
shows that the Zn-S contribution at the catalytic zinc ion is replaced
by Zn-O contribution. These results are consistent with the proposed
"cysteine switch" mechanism where the propeptide cysteine residue
is cleaved off the active site and substituted by a water molecule (13,
38).
Interestingly, great similarities in XANES and EXAFS features between
pro-MMP-2 and inhibited MMP-2 can be observed (Figs. 2 and 3). The
enzyme was inhibited with SB-3CT, which is a newly designed
mechanism-based inhibitor of MMPs (Fig. 1) and highly selective for
gelatinases (1). Overall, our results suggest that the molecular
coordination at the catalytic zinc site in the presence of SB-3CT is
similar in its type of ligation, coordination number, and conformation
to the proenzyme structure. In addition, the XAS analysis of
MMP2-SB-3CT is consistent with our modeling studies (Fig. 4) and the
original proposed inhibition mechanism of MMPs by SB-3CT (1).
On the basis of these results, we suggest that the design of
mechanism-based inhibitors provides a novel approach for MMP inhibition
by imparting or re-establishing the proenzyme structural motifs. In
essence, the covalently attached inhibitor mimics the binding of the
propeptide segment in MMPs, which is coordinated via a cysteine residue
to the catalytic zinc ion, by forming similar binding via the thiolate
group. This fact, along with the provisions that incorporated excellent
shape complementarity for the enzyme to the active site during the
design of the inhibitor, are important factors for the mimicry of the
proenzyme metal coordination on the onset of inhibition by our inhibitor.
Detailed structures are available for the complexes between TIMP-1 and
MMP-3 and TIMP-2 (16, 39, 40). These structures provide information
about the critical residues involved in MMP inhibition by the natural
inhibitors. Interestingly, the catalytic zinc ion in the MMP3-TIMP-1
complex is coordinated in a bidentate fashion to the Cys1
residue of the TIMP molecule via the N-terminal amino acid and the
carbonyl group. The architectures of the inhibited active sites of
these complexes have provided a model for the design of putative
synthetic MMP inhibitors. The vast majority of these inhibitors utilize
a hydroxamic acid to chelate the catalytic zinc ion by imposing a
bidentate interaction between the zinc ion and the inhibitor
hydroxamate group. A similar binding mode was proposed recently for
thiol-based inhibitors (41).
In contrast, our EXAFS analysis shows that the binding of the
mechanism-based inhibitor to the catalytic zinc ion via its sulfur atom
is clearly monodentate. We recently reported that the kinetic profiles
of MMP inhibition by SB-3CT and their natural protein inhibitors are
similar. Specifically, we showed that the interaction of MMP-2 and
MMP-9 with SB-3CT followed a slow binding pattern (42), which
ultimately resulted in covalent modification of the enzyme in the
active site. It is important to note that slow binding inhibition of
gelatinases is also seen with TIMP-2 and TIMP-1 (43). Furthermore,
it is most interesting that both the affinities of the inhibitor for
the enzyme and the kinetic parameters for the onset of the slow binding
inhibition by SB-3CT closely followed those for the binding of TIMPs at
the high affinity site (43). The kinetic parameters for the inhibition
showed a remarkable selectivity for both MMP-2 and MMP-9, since the
molecule was specifically designed for gelatinases (1). The data
presented here further indicate that inhibition of MMP-2 by SB-3CT
restores the metal coordination environment to that seen for the
zymogen form of the enzyme. These observations collectively argue for this inhibitor to be a nearly ideal inhibitor from the perspective of
the design paradigms.
MMPs play an essential role in the turnover and remodeling of
extracellular matrix on both normal and pathological processes (3, 27,
35). For example, MMP-2 has been shown to play a key role in
tumor angiogenesis and metastasis. Therefore, MMP-2 represents an
obvious target for the development of selective inhibitors that would
block tumor cell invasion and tumor angiogenesis. Understanding the
detailed molecular structures and the conformational changes of MMP-2
in its zymogen, active, and inhibited states can aid in the rational
design of anti-cancer drugs. Here we define the mode of binding of a
selective MMP inhibitor to the catalytic site of MMP-2 and provide
insights on the basis for its exceptional properties in selective
inhibition of gelatinases. In addition, we show that XAS can be used as
a tool to screen and/or to design active site modifications in MMPs (in
solution) upon binding to inhibitors via the catalytic zinc ion.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 cm
1
(22) and amino acid analysis. Pro-MMP-2 was activated by 1 mM
p-aminophenylmercuric acetate (dissolved in 200 mM Tris) for 30 min at 37 °C. The protein concentration
of the active MMP-2 was determined by titration with TIMP-2, as
described previously (23).
(k) was between 2.0 and 9 Å
1. Model data for the
fitting procedure were constructed by extracting the catalytic zinc
site coordinates (in a radius of 6 Å from the crystallographic
coordinates of gelatinase A (RCSB Protein Data Bank Code 1CK7).
Using the computer code FEFF7 (25, 26), we calculated the theoretical
photoelectron scattering amplitudes and phase shifts. Total theoretical
(k) was constructed by adding the most important partial
(k) values that contributed to the r-range of interest.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Proposed mode of MMPs inhibition by
SB-3CT. Schematic representation of the proposed binding of
SB-3CT, a mechanism-based inhibitor, to the catalytic zinc ion in MMPs
(adopted from Brown et al. (1)). The inhibitor is
coordinated to the zinc ion via the thiolate of the thiirane
group.
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[in a new window]
Fig. 2.
Edge spectra of pro, active, and
inhibited forms of MMP-2. Normalized raw XAS data in the
zinc K-edge region of pro-MMP2 (red), active
(green), and MMP-2-SB-3CT complex (black). The
overall shape of the edge spectra of the pro and the active enzyme
differs. The most distinct feature is the decrease in the peak at 9738 eV at the XANES region upon activation of the enzyme. Interestingly,
this peak is restored in the inhibited MMP-2-SB-3CT complex.
c,
shows the best fitting results of the EXAFS analysis of the various
structures. The fitting parameters and the quality of the fits are
listed in Table I. The zinc sites in the
various forms of the enzyme were fitted to the Zn-N, Zn-O, Zn-S, and
Zn-C paths using different combinations of varied and constraint
parameters. In addition, different initial conditions of distances,
Debye-Waller factors, and
E0 shifts were
applied in the fitting procedure. To account for two zinc ions in our
fitting procedures, we have used the following strategy. In the first
stage, the EXAFS data were fitted to a relevant theoretical model
containing only one zinc ion, and in the second stage, the fits were
refined by constraining and fixing the structural zinc contributions in
the fits and fitting the residual phases and amplitudes with the
appropriate model. This procedure was repeated in an iterative way
until a stable solution was achieved. Furthermore, final refinement of
the data included the repeat of the second stage by fixing the
catalytic zinc contributions. Using this fitting procedure allowed us
to better estimate the goodness of the fits and to refine the final
results. Stable and reproducible fits of pro-MMP-2 (Table IA, Fits
2-3) were consistent with a tetrahedral coordination of the zinc ion
with three Zn-N(His) at 2.07 ± 0.03 Å, one Zn-S(Cys) at
2.30 ± 0.03 Å contributions (in the first coordination shell),
and seven Zn-C contributions where three Zn-C at 2.87 ± 0.05 Å and four at 3.18 ± 0.04 Å (in the second coordination shell).
The zinc-ligand distances derived from our EXAFS analysis for the
proenzyme are in agreement (within the experimental error) with the
crystal structure (13). Fitting the data to a one zinc site model
resulted in a high
2 and unstable fits (Table IA,
Fit 1).
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Fig. 3.
EXAFS fitting results for latent, active, and
inhibited forms of MMP-2. The results are presented in the
R-space of the experimental data (black) to simulated
theoretical zinc-ligand contributions (red). The
experimental data were extracted and normalized using the UWXAFS
analysis package. The theoretical XAFS signal was constructed based on
the MMP-2 crystal structure code 1CK7 from Protein Data Bank
and processed using FEFF7. The experimental data were fitted to
the theoretical data using the nonlinear least squares method
implemented in the program FEFFIT in R-space, by Fourier transforming
both theory and data. a, best fit of pro-MMP-2 to 3 Zn-N, 1 Zn-O, and 7 Zn-C contributions. b, best fit of active MMP-2
to 3 Zn-N, 1 Zn-O, and 5/6 Zn-C contributions. c, best fit
of the inhibited MMP-2-SB-3CT complex to 3 Zn-N, 1 Zn-S, and 7 Zn-C
contributions. A great similarity in the Fourier-transformed features
can be observed for pro-MMP-2, and inhibited enzyme can be observed.
The structural zinc-ligand contributions were fixed in the fitting
procedure in all fits. The fitting parameters for the various fits are
listed in Table I.
Curve fitting analysis of latent, active, and inhibited MMP-2
2 is the Debye-Waller factor. Note:
footnote A indicates that both
2s were treated as one.
View larger version (138K):
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Fig. 4.
Modeling of the catalytic zinc ion in the
MMP-2-SB-3CT complex. Stereo views of the noncovalent
(A) and covalent (B) complexes between the
thiirane inhibitor and MMP-2. The active site is represented as a
Connolly surface in green, and the catalytic zinc ion is
shown in orange as a sphere. The inhibitor
molecule and Glu404 are shown in ball-and-stick
representation (carbon, white; oxygen, red;
sulfur, yellow). Backbone of the enzyme is shown in
cyan as a capped-stick representation.
2 values and
high Debye-Waller factors. For the inhibited enzyme, these results
suggest that the mechanism of inhibition of MMP-2 by SB-3CT includes a
replacement of the Zn-O (water) at the catalytic zinc site with a
monodentate ligation of a sulfur atom (from the inhibitor, see Fig. 1),
as was anticipated in the design aspect of the inhibitor. The binding
stoichiometry (i.e. a one-to-one ratio) of SB-3CT to MMP-2
provides us with the confidence that the differences observed in the
mode of ligation in the XAFS spectra upon inhibition can be attributed
to the catalytic zinc ion.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported by Grants 6602/2 from the Binational Scientific Foundation and the Maurizio and Clotilde Pontecorvo Fund (to I. S.), Grant DAMD17-97-1-7174 from the United States Army (to S. M.), and Grants CA-61986 and CA-82298 from the National Institutes of Health (to R. F.).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.
** To whom correspondence should be addressed: Dept. of Structural Biology, The Weizmann Institute of Science, Rehovot 76100, Israel. Tel.:/Fax: 972-8-934-2130; E-mail: irit.sagi@weizmann.ac.il.
Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M011604200
2 L. P. Kotra, Y. Shimura, R. Fridman, H. B. Schlegel, and S. Mobashery, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: MMP, matrix metalloproteinase; XAS, x-ray absorption spectroscopy; ICP-AES, inductively coupled plasma atomic emission spectroscopy; XANES, x-ray absorption near edge structure; EXAFS, extended x-ray absorption fine structure; TIMP, tissue inhibitor metalloproteinase.
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