X-ray Absorption Studies of Human Matrix Metalloproteinase-2 (MMP-2) Bound to a Highly Selective Mechanism-based Inhibitor

COMPARISON WITH THE LATENT AND ACTIVE FORMS OF THE ENZYME*

Oded KleifeldDagger , Lakshmi P. Kotra§, David C. Gervasi§||, Stephen Brown§, M. Margarida Bernardo§||, Rafael Fridman§||, Shahriar Mobashery§, and Irit SagiDagger **

From the Dagger  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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-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).

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 chi (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 chi (k) was constructed by adding the most important partial chi (k) values that contributed to the r-range of interest.

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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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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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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.

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---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 Delta 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 chi 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.

                              
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Table I
Curve fitting analysis of latent, active, and inhibited MMP-2
Results of EXAFS curve fitting analysis of pro-MMP-2, (A) active MMP-2 (B), and inhibited MMP-2 complex (C). The uncertainties are given in parentheses. The symbols F and V stand for "fixed" and "varied," respectively, and indicate how the respective parameter was treated in the fit model. R is distance of atoms from the zinc ion in Å. sigma 2 is the Debye-Waller factor. Note: footnote A indicates that both sigma 2s were treated as one.

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).


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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.

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 chi 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.

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).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    FOOTNOTES

* 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.

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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