From the
Department of Structural Biology, The
Weizmann Institute of Science, Rehovot, 76100, Israel and the Departments of
Chemistry, Pharmacology, Biochemistry, Molecular
Biology, and ¶Pathology, Wayne State University,
Detroit, Michigan 48202
Received for publication, February 3, 2003 , and in revised form, March 20, 2003.
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ABSTRACT |
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INTRODUCTION |
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Gelatinases, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), constitute a distinct subgroup of the MMPs family due to the incorporation of three repeats of the fibronectin-type II module in the catalytic domain. These enzymes have been shown to play a key role in tumor cell invasion, metastasis, and angiogenesis by promoting degradation of ECM and the processing of cytokines, growth factors, hormones, and cell receptors (5, 6). As with other MMPs, MMP-2 and MMP-9 are expressed as zymogenic latent enzymes (pro-MMPs) requiring activation. Both proteolytic and non-proteolytic mechanisms have been described for zymogen activation. However, the "cysteine-switch" hypothesis (7) proposes that upon activation, the zinc ion in the latent active site is converted to a catalytic zinc ion by the dissociation of the conserved cysteine thiolate within the active site. The cleavage or the dissociation of the propeptide makes available the catalytic zinc ion to the substrates (4).
Activation of pro-MMP-2 is mediated by members of the membrane-type MMP subfamily of membrane-tethered MMPs, in particular MT1-MMP. Pro-MMP-2 activation by MT1-MMP is a highly regulated process involving the action of the endogenous tissue inhibitor of metalloproteinase-2 (TIMP-2) (812). In this process, TIMP-2 binds the N terminus of the active MT1-MMP and the hemopexin-like domain of pro-MMP-2, forming a ternary complex. This results in the accumulation of pro-MMP-2 on the cell surface and its subsequent activation by an adjacent TIMP-2-free MT1-MMP (13). Thus, a delicate balance between MT1-MMP and TIMP-2 on the cell surface determines the ability of cells to activate pro-MMP-2.
Because of the central function that MMP-2 and MMP-9 play in many pathological processes, these MMPs constitute and remain major targets for therapeutic intervention (14). To date, the most common approach to target MMP activity, including gelatinase activity, has been the development of reversible hydroxamate-based peptidomimetic inhibitors such as Batimastat and Marimastat, which possess high affinity toward the catalytic site of the enzymes (15, 16).
Unfortunately, clinical trials using these broad spectrum MMP inhibitors have been disappointing. Several reasons were provided to explain these poor results including lack of specificity, toxicity, and the stage of cancer in the patient (17, 18). In addition, although the reversible hydroxamic acid-based inhibitors were selected for their high affinity inhibition in classical enzyme inhibition studies, their effects on MMP structure and function both at the protein and at the cellular level are poorly understood. We have shown that reversible synthetic MMP inhibitors like Marimastat can, under certain conditions, promote pro-MMP-2 activation by MT1-MMP in the presence of TIMP-2 (19). This paradoxical effect correlates with the extent of the synthetic inhibitor affinity toward MT1-MMP since an irreversible mechanism-based inhibitor selective for gelatinases had no such effect. Structurally, we have recently demonstrated that the mechanism-based inhibitor for gelatinases produces a conformational state in the active enzyme complexed with the inhibitor that resembles that of the latent inactive form (20). Together, these observations suggest that the binding mode of the inhibitor may exert profound effects in the structure, regulation, and activity of MMPs.
A recent report by Bernardo et al. (21) described the design, synthesis, and characterization of two new potent slow binding inhibitors that are selective for MMP-2 and MMP-9. Specifically, these dithiol inhibitors are 4-(4-phenoxphenylsulfonyl-)butane-1,2-dithiol (1) and 5-(4-phenoxphenylsulfonyl) pentane-1,2-dithiol (2) (see Scheme 1) exhibiting Ki values in the range of 46260 nM (21). In contrast, both 1 and 2 exhibit much lower affinity toward MMP-3. Based on their slow binding mode of inhibition, inhibitors 1 and 2 were expected to initiate subtle conformational changes in the active site of the enzyme, resulting in a stable complex that do not readily reverse to allow recovery of activity (21), as we reported for the interaction of the first mechanism-based inhibitor selective for the gelatinases (20).
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Herein we report on the nature of the interactions of these two novel MMP inhibitors within the active site of MMP-2. Specifically, we show that the dithiol moieties of these inhibitors are directly coordinated to the zinc ion in a bidentate manner, in a process that proceeds with a conformational change of the enzyme involving structural alternations in the nearest coordination shell of the zinc ion.
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EXPERIMENTAL PROCEDURES |
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X-ray Absorption (XAS) Studies
Sample PreparationAll 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. All samples were loaded into copper sample
holders (10 x 5 x 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 CollectionXAS data collection was performed at the National Synchrotron Light Source at Brookhaven National Laboratory, beam-line X9B. The spectra were recorded at the Zn 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 in order to calibrate the beam energy. Several scans of each sample were collected for a total of 1 x 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 AnalysisThe average Zn k-edge absorption coefficient µ(E), which was obtained after 1012 independent XAS measurements for each sample, were 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 (k) was between 2.0
and 9 Å1. 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 (PDB code 1CK7
[PDB]
). Using the computer code FEFF7
(26,
27), 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.
The theoretical XAFS signal was fitted to the experimental data using the non-linear 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.
Circular Dichroism (CD)
CD was measured using an Aviv spectrophotometer, model 202. The data was
collected using quartz cells with a light path 0.1 cm for far UV
(180240 nm). In the inhibition studies, all samples contained 6.6
µM active MMP-2, 66 µM 1 or 2, in
0.5% ethanol. All samples were prepared in 50 mM Tris, pH 7.6, 75
mM Na2SO4, 5 mM CaSO4,
0.02% Brij.
Computer Modeling
A recently published x-ray structure of MMP-2
(4) (PDB code 1CK7
[PDB]
) was used
for the molecular modeling carried out in this study. Crystallographic water
molecules present in the x-ray structure were retained, and the program
"protonate," a component of the AMBER 6 package
(28) was used to add hydrogen
atoms to the enzyme. The two enantiomers of the inhibitor 1 were then
constructed and docked into the active site of MMP-2 using SYBYL (Tripos Inc.,
St. Louis, MO). The distances between the active site zinc ion and its ligand
atoms were constrained to values determined experimentally. Atomic charges for
1 were determined using the RESP fitting procedure
(29). This consisted of first
optimizing the molecules at the HF/321G* level of theory and basis set,
followed by a HF/631G* single-point energy calculation to determine the
electrostatic potential around the molecule, which was subsequently used in
the two-stage RESP fitting procedure. All ab initio calculations were
carried out using the Gaussian 98 suite of programs
(30). Following the RESP
fitting procedure, the R- and S-1/MMP-2 complexes were fully solvated
in a box of TIP3P waters such that no atom in the complex was less than 10
Å from any face of the box. This resulted in a total of 53,587 atoms.
The particle mesh Ewald (PME) method was used to treat long range
electrostatics (31). The AMBER
6 software package using the Cornell et al. force field
(32) based on the Parma99 data
set of parameters was used to carry out the energy minimization. Two
individual 20,000 energy minimization steps were carried out for the fully
solvated R- and S-1 stereoisomers bound to the
MMP-2 active. The first 500 steps consisted of steepest descent energy
minimization, followed by 19,500 steps of conjugate-gradient energy
minimization.
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RESULTS AND DISCUSSION |
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XANES Studies of Pro-MMP-2, Active MMP-2, and of the Complexes of MMP-2:1 and MMP-2:2Fig. 1 shows the normalized zinc k near-edge spectra of pro-MMP-2, active MMP-2, and inhibited states of the enzyme. Although difficult to interpret quantitatively, near-edge spectra are very sensitive to the geometry and the nature of the ligands. Thus, they can be very useful as fingerprints of particular coordination environments. The near-edge spectrum of pro-MMP-2 has two characteristic peaks at 9714 eV and at 9738 eV (Fig. 1). As we reported previously, the peak intensity at 9738 eV changes upon activation or inhibition of the enzyme due to alternation in the local structure around the catalytic zinc ion. The dissociation of the cysteine residue from the catalytic zinc ion during activation of MMP-2 results in the reduction of the peak intensity at 9738 eV. Interestingly, binding of both 1 and 2 further reduce this intensity (Fig. 1), while binding of the mechanism-based inhibitor 3 (also referred to as "SB-3CT" in earlier publications), which interacts with the zinc ion by one thiolate upon the requisite reaction within the active site that results in the formation of the thiolate and the attendant inhibition of the enzyme, largely restores it (20). In addition, the changes in the edge energy upon binding of 1 and 2 to MMP-2 are more pronounced (Fig. 1, inset). Specifically, a significant shift of the edge position to higher energy is observed. As we reported earlier, such shifts to higher energy may be associated with changes in coordination number or ligand exchange at the metal ion (20, 34). Overall, these results, suggest that the interactions of 1 and 2 with the catalytic zinc ion of MMP-2 are different than the one observed for 3.
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EXAFS Data Analysis of Pro-MMP-2, Active MMP-2, and MMP-2:1 and MMP-2:2 ComplexesThe results of the EXAFS data analysis of MMP-2, in its latent, active, and inhibited states complemented our XANES studies. The EXAFS analysis were conducted by fitting the data to theoretical phase shifts and amplitudes that were constructed by feeding the coordinates of the structural and catalytic zinc sites of pro-MMP-2 (PDB code 1CK7 [PDB] ) into the FEFF7 program (26). 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 zinc site of the various enzyme complexes.
Standard curve-fitting procedures were used to fit the FEFF7 theory data to
the real and imaginary parts of the Fourier-transformed (k). The
k2 weighting factor and the Hanning window function,
defined between 2 and 9 Å1 were used in the
Fourier transforms of all data sets. During the fitting procedure, the
corrections to the energy origin (
E0), bond
distances (
R), and mean square disorders of the distances
(
2) were varied until the best fit was obtained. The number
of relevant independent data points Nidp in the data was
calculated using Equation 1 (35),
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Fig. 2 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 ZnN, ZnO,
ZnS, and ZnC 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. In order to account for two zinc ions in our fitting
procedures, we have used the following strategy, which was specifically
developed, in our laboratory for the EXAFS analysis of MMPs
(20). Briefly, 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 results.
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Stable and reproducible fits of pro-MMP-2 were consistent with a tetrahedral coordination of the zinc ion with three ZnN(His) at 1.90 ± 0.01 Å, one ZnS(Cys) at 2.24 ± 0.01 Å contributions (in the first coordination shell), and seven ZnC contributions were two ZnC at 2.63 ± 0.05 Å and five at 3.01 ± 0.04 Å (in the second coordination shell). The zinc-ligand distances derived from our EXAFS analysis for the pro-enzyme are in agreement (within the experimental error) with both the crystal structure (4) and the EXAFS data analysis of pro-MMP-2 reported before (20). Using our non-linear curve fitting analysis methods, we could not detect the presence of activated enzyme, which may be present in small portions (1015%) and in equilibrium with the pro-enzyme in our enzyme preparations. Similarly, the EXAFS fitting analysis of the active enzyme was consistent with the previously reported results (20) and revealed four ZnO/N contributions at 1.99 ± 0.02 Å, which are consistent with three ZnN (His) and one ZnO (H2O) binding ligands, and four/five ZnC contributions at 3.00 ± 0.01 Å at the second coordination shell (Table I).
The EXAFS curve fitting parameters and the quality of the fits of the catalytic zinc site of MMP-2 in the inhibited complexes are represented in detail in Table I and Fig. 2. The first coordination shell of the inhibited enzyme in both MMP-2:1 and MMP-2:2 complexes is consistent with pentavalent geometries. Specifically, the catalytic zinc ion is coordinated to two sulfur atoms of the inhibitor dithiol moieties, and three nitrogen atoms of the active site residues His403, His407, and His413. The zinc-ligand distances in the MMP-2:1 complex are consistent with three ZnN(His) at 1.91 ± 0.01 Å, two ZnS(of 1) at 2.24 ± 0.01 Å, and eight ZnC contributions where four distances are at 2.76 ± 0.04 Å and four distances are at 3.05 ± 0.05 Å. The EXAFS curve fitting analysis of the local structure around the catalytic zinc ion in the MMP-2:2 shows an expansion of the zinc-ligand bond distances of the first coordination shell comparing to the MMP-2:1 complex. Specifically, the zinc-ligand distances in MMP-2:2 are consistent with three ZnN (His) ligands at 1.95 ± 0.01 Å, and two ZnS (inhibitor 2) ligands at 2.29 ± 0.01 Å. Attempts to fit the catalytic zinc ion in the inhibited complexes with one or three sulfur ligands resulted in unacceptable Deby-Waller factors, instability of the fits, and higher chi-squares (Table I).
Interestingly, the second shell zinc-ligand distribution is different than
the ones observed for the pro and active forms of the enzyme
(Table I). The second shell
zinc-ligand distances in MMP-2:1 are consistent with four Zn-C
contributions at 2.76 ± 0.04 Å and four Zn-C contributions at
3.05 ± 0.05 Å. The second shell zinc-ligand bond distances in
MMP-2:2 are consistent with two Zn-C contributions at 3.34 ±
0.09 Å and five Zn-C contributions at 2.96 ± 0.09. The
alternation in structure in the first and second nearest shells around the
catalytic zinc ion, among the various forms of the enzyme, is also reflected
in the raw Fourier transform data (Fig.
2). Specifically, Fig.
2 shows the radial distribution of the nearest neighboring atoms
of the first second and third shell from the absorber (the zinc ion). The most
pronounced changes are observed in the second shell atoms, which may be
assigned in part to the and
carbons of the corresponding three
histidine rings. While the peak intensities of these contribution is mildly
affected upon activation of pro-MMP (Fig.
2, a and b), a strong reduction in these peak
intensities is observed upon inhibition with compounds 1 and 2.
This may result from the elongation of the ZnC bond distances or from
the occurrence of greater distribution of bond lengths, which may be induced
upon interaction of the metal site with the inhibitors. Although, XAS analysis
provides extremely accurate local structural information on the type of
ligation, electronics, and bond distances, it fails to resolve the bond angles
and the exact orientation of each atom in the active site. Yet, the observed
"subtle" conformational changes of the second shell ZnC
ligand orientations seem to be at the detectible limit of both crystallography
and NMR methods. In this respect, using XAS to probe such changes is
advantageous.
CDIn order to examine if binding of 1 or 2 to the active site of MMP-2 induces some local conformational changes at the protein backbone that would account for the slow binding behavior, we performed CD spectroscopy studies on active MMP-2 before and after its interaction with 1 or 2. CD spectroscopy refers to a form of light absorption spectroscopy that measures the difference in absorbance of right- and left-circularly polarized light (rather than the commonly used absorbance of isotropic light) by a substance (36).
Fig. 3 shows the far-UV CD
spectra of active-MMP-2, MMP-2:1, and MMP-2:2. The spectra of
the various complexes exhibit a positive ellipticity at 229 nm. This
corresponds mainly to the relatively large content of -turn structures
within the MMP-2 enzyme. A distinct change in spectral features around 210 and
225 nm is observed upon inhibition of MMP-2 by both 1 and 2,
this change corresponds to subtle reduction in the content of
-helixes,
-sheets, and
-turns. The CD spectra shown herein, is in agreement
with previous CD studies (37).
However, the evaluation of the changes in secondary structure should be
treated with extreme caution in view of the relatively low molar ellipticities
exhibited by MMP-2 (which is not dependent on protein concentration). The
interactions of 1 and 2 induce small but distinct conformational
changes at the catalytic site of the enzyme. In addition, the interaction of
2 seems to have a lesser effect on the induced local conformational
changes relative to 1 (Fig.
3). Nevertheless, these results suggest that the induced
conformational changes may affect the inhibition mechanism of MMPs as was
proposed before (21). In this
respect, both XAS and CD spectroscopy provide structural information of the
mode of interactions of these inhibitors with the catalytic zinc site of MMPs.
Although, the CD spectroscopy provides only gross properties of the observed
structural changes, together with XAS our results show that binding of either
1 or 2 induces distinct conformational changes in the
microenvironment of the catalytic site. These experimental measurements were
put in structural context by computational model building with the x-ray
structure for MMP-2.
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An interesting observation from Fig.
4A is that the sulfur atom located on the terminal
methylene of the dithiol moiety is within hydrogen bonding distance of
Glu404. A proton transfer from the dithiol moiety to active site
Glu404 is very likely to occur given that the positively charged
zinc ion further reduces the pKa of the thiol
moieties upon coordination. Recently, it was shown that a similar process
occurs in the active site of tumor necrosis factor- converting enzyme
(TACE), which also contains a catalytic zinc ion coordinated to three
histidine residues and a nearby glutamate
(39). The molecular orbital
study found that inhibitors with a hydroxamate moiety lost their proton to the
active site glutamate upon complexation with the zinc ion. Given the
similarities in the microenvironment surrounding zinc in TACE and in MMP-2, it
is likely that abstraction of proton from ligand by active site Glu also
occurs in MMP-2. A root mean-square superimposition of the structures of MMP-2
(PDB code 1QIB
[PDB]
) to that of TACE (PDB code 1KBC
[PDB]
) using the C
and C
from the side chains of active site glutamate and histidine residues resulted
in a root mean-square deviation of only 0.262 Å.
To further study the effect of proton transfer, an energy minimization was
carried out on the complex shown in Fig.
4A except that the proton was now bound to
Glu404 instead of the thiol of compound 1.
Fig. 4B shows that a
small conformational change ensued, where the terminal thiol sulfur is now
3.69 Å away from protonated O atom of Glu404. This new
conformation adopted by the dithiol moiety makes it less likely for proton
transfer back to the thiol sulfur atom to occur, a process that would be a
prerequisite for dissociation of the ligand from the active site.
A rational approach to MMP inhibition would benefit from understanding the molecular interactions that mediate the binding of the inhibitors within the active site of the enzyme and their effect on enzyme structure and function. Such information would be valuable for fine-tuning inhibitor structures that eventually will possess a predictable kinetic behavior and optimal inhibition rates. Our approach to MMP inhibition has been a point of departure from conventional approaches based on zinc chelation. We reported the first mechanism-based inhibitor (inhibitor 3) for MMPs, and initially we had targeted gelatinases due to their well-established role in cancer metastasis and angiogenesis. The dithiol inhibitors were designed based on the knowledge gained on the binding mode of the thiirane mechanism-based inhibitor 3 (40). The biphenyl moieties of 1, 2, and 3 are identical and this entity is designed to fit the S1' subsite of MMP-2. However, the functionalities of the inhibitors that interact with the catalytic zinc ion are different in the number of sulfur atoms or the length of the linker that connect the biphenyl moiety. Inhibitor 3 exhibits a higher degree of selectivity for gelatinases, and it is capable of discriminating between MMP-2 (Ki = 14 ± 4 nM) and MMP-9 (Ki = 600 ± 200 nM). Inhibitors 1 and 2 are slow-binding inhibitors that bind the active sites of gelatinases, facilitate a subtle conformational change that the end result of which enjoys excellent stability, such that it does not reverse itself readily (21). The slow-binding behavior is at the root of selectivity toward gelatinases. Yet, the principles that facilitate the manifestation of slow binding inhibition are not clear. In this respect, XAS, CD spectroscopy, and computational analyses of MMP-inhibitor complexes may serve as quantitative and analytical tools to closely examine the induced electronic and structural changes occurring upon enzyme inhibition at the nearest catalytic zinc environment.
Using XAS, we have shown that the binding of 3 to the active site of MMP-2 induces a structural state that resembles that of the latent enzyme (20). Here we show that inhibitors 1 and 2 bind the zinc ion via their dithiolate moieties. However, unlike inhibitor 3 the conformational changes detected by the raw edge spectra, as well as the effect of the inhibitor binding on the total effective charge of the zinc ion, resembles the active form conformation of the enzyme (Fig. 1). These results show that inhibitors 1 and 2, in contrast to 3, induce different structural conformations. This may suggest that each inhibitor stabilizes distinct conformational state of the active site, which may evolve at the catalytic zinc ion environment during the inhibition process.
Factors that control biological regulation and catalysis are often expected to be manifested by structural determinants that are associated with alternation in the protein architecture or conformational states. It was shown by Koshland and co-workers (38) that small structural perturbations in the active site of the enzyme isocitrate dehydrogenase contribute to the precise substrate alignment and to the catalytic power of the enzyme. The investigators demonstrated that the orbital overlap produced by optimal orientation of reacting orbitals play a major quantitative role in the catalytic power of the enzyme. These experiments as well as the results reported here suggest that small structural changes may results in a large catalytic or inhibition consequences. Consideration of these factors may aid in their being understood, and such level of fundamental understanding is critical in elucidation of properties of biomolecules and in de novo attempts at design of potential pharmaceuticals.
In summary, here we report the structural basis for inhibitory properties of two potent slow-binding inhibitors for gelatinases. These inhibitors chelate the catalytic zinc ion by inducing novel mode of binding as well as local conformational changes at the extended active site of the enzyme. In addition, we propose the use of XAS and CD spectroscopy as structural tools to quantify and analyze the binding modes, and the mechanisms of inhibition of new inhibitors for MMPs. The structural insight that emerges from these studies provides opportunities in the design of the future generations of molecules that would modulate the activities of a wide range of MMPs.
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FOOTNOTES |
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|| Incumbent of the Robert Edward Roselyn Rich Manson Career Development Chair. 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{at}weizmann.ac.il.
1 The abbreviations used are: MMPs, matrix metalloproteinases; XAS, x-ray
absorption spectroscopy; EXAFS, extended x-ray absorption fine structure;
XANES, x-ray absorption near-edge structure; PME, particle mesh Ewald; MT,
membrane-type; ECM, extracellular matrix.
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REFERENCES |
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