Structural Basis for Potent Slow Binding Inhibition of Human Matrix Metalloproteinase-2 (MMP-2)
2003; Elsevier BV; Volume: 278; Issue: 29 Linguagem: Inglês
10.1074/jbc.m301139200
ISSN1083-351X
AutoresGabriel Rosenblum, Samy O. Meroueh, Oded Kleifeld, Stephen H.M. Brown, Steven P. Singson, Rafael Fridman, Shahriar Mobashery, Irit Sagi,
Tópico(s)Peptidase Inhibition and Analysis
ResumoThe zinc-dependent gelatinases belong to the family of matrix metalloproteinases (MMPs), enzymes that have been shown to play a key role in angiogenesis and tumor metastasis. These enzymes are capable of hydrolyzing extracellular matrix (ECM) components under physiological conditions. Specific and selective inhibitors aimed at blocking their activity are highly sought for use as potential therapeutic agents. We report herein on a novel mode of inhibition of gelatinase A (MMP-2) by the recently characterized inhibitors 4-(4-phenoxphenylsulfonyl)butane-1,2-dithiol (inhibitor 1) and 5-(4-phenoxphenylsulfonyl) pentane-1,2-dithiol (inhibitor 2). These synthetic inhibitors are selective for MMP-2 and MMP-9. We show that the dithiolate moiety of these inhibitors chelates the catalytic zinc ion of MMP-2 via two sulfur atoms. This mode of binding results in alternation of the coordination number of the metal ion and the induction of conformational changes at the microenvironment of the catalytic zinc ion; a set of events that is likely to be at the root of the potent slow binding inhibition behavior exhibited by these inhibitors. This study demonstrates a distinct approach for the understanding of the structural mechanism governing the molecular interactions between potent inhibitors and catalytic sites of MMPs, which may aid in the design of effective inhibitors. The zinc-dependent gelatinases belong to the family of matrix metalloproteinases (MMPs), enzymes that have been shown to play a key role in angiogenesis and tumor metastasis. These enzymes are capable of hydrolyzing extracellular matrix (ECM) components under physiological conditions. Specific and selective inhibitors aimed at blocking their activity are highly sought for use as potential therapeutic agents. We report herein on a novel mode of inhibition of gelatinase A (MMP-2) by the recently characterized inhibitors 4-(4-phenoxphenylsulfonyl)butane-1,2-dithiol (inhibitor 1) and 5-(4-phenoxphenylsulfonyl) pentane-1,2-dithiol (inhibitor 2). These synthetic inhibitors are selective for MMP-2 and MMP-9. We show that the dithiolate moiety of these inhibitors chelates the catalytic zinc ion of MMP-2 via two sulfur atoms. This mode of binding results in alternation of the coordination number of the metal ion and the induction of conformational changes at the microenvironment of the catalytic zinc ion; a set of events that is likely to be at the root of the potent slow binding inhibition behavior exhibited by these inhibitors. This study demonstrates a distinct approach for the understanding of the structural mechanism governing the molecular interactions between potent inhibitors and catalytic sites of MMPs, which may aid in the design of effective inhibitors. Matrix metalloproteinases (MMPs) 1The 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.1The 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. are major players in degradation of the extracellular matrix (ECM) components, and therefore they play key roles in normal and pathological conditions involving remodeling and turnover of ECM (1Kleiner D.E. Stetler-Stevenson W.G. Cancer Chemother. Pharmacol. 1999; 43: S42-S51Crossref PubMed Scopus (624) Google Scholar). MMPs constitute a multigene family of at least 26 secreted and membrane-tethered zinc-dependent endopeptidases, which can be classified according to their structures and substrate specificities (2Massova I. Kotra L P. Fridman R. Mobashery S. FASEB J. 1998; 12: 1075-1095Crossref PubMed Scopus (695) Google Scholar, 3Nagase H. Woessner J.F. J. Biol. Chem. 1999; 274: 21491-21494Abstract Full Text Full Text PDF PubMed Scopus (3827) Google Scholar). Members of the family include: collagenases (MMP-1, -8, -13), gelatinases (MMP-2, -9), stromelysins (MMP-3, -7, -10, -11, -12), and membrane-type (MT)-MMPs (MT1-MMP to MT6-MMP). The catalytic domains of the MMPs have an ellipsoid shape with a small active site cleft. This cleft contains the catalytic zinc ion, which is essential for catalysis (4Morgunova E. Tuuttila A. Bergmann U. Isupov M. Lindqvist Y. Schneider G. Tryggvason K. Science. 1999; 284: 1667-1670Crossref PubMed Scopus (476) Google Scholar). 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 (5Moses M.A. Stem Cells. 1997; 15: 180-189Crossref PubMed Scopus (258) Google Scholar, 6Nguyen M. Arkell J. Jackson C.J. Int. J. Biochem. Cell Biol. 2001; 33: 960-970Crossref PubMed Scopus (259) Google Scholar). 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 (7Van Wart H.E. Birkedal-Hansen H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5578-5582Crossref PubMed Scopus (1182) Google Scholar) 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 (4Morgunova E. Tuuttila A. Bergmann U. Isupov M. Lindqvist Y. Schneider G. Tryggvason K. Science. 1999; 284: 1667-1670Crossref PubMed Scopus (476) Google Scholar). 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) (8Polette M. Birembaut P. Int. J. Biochem. Cell Biol. 1998; 30: 1195-1202Crossref PubMed Scopus (126) Google Scholar, 9Ellerbroek S.M. Stack M.S. Bioessays. 1999; 21: 940-949Crossref PubMed Scopus (133) Google Scholar, 10Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2361) Google Scholar, 11Strongin A.Y. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1993; 268: 14033-14039Abstract Full Text PDF PubMed Google Scholar, 12Hernandez-Barrantes S. Toth M. Bernardo M.M. Yurkova M. Gervasi D.C. Raz Y. Sang Q.A. Fridman R. J. Biol. Chem. 2000; 275: 12080-12089Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). 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 (13Strongin A.Y. Collier I. Bannikov G. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1995; 270: 5331-5338Abstract Full Text Full Text PDF PubMed Scopus (1432) Google Scholar). 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 (14Overall C. Lopez-Otin C. Nat. Rev. Cancer. 2002; 9: 657-672Crossref Scopus (1118) Google Scholar). 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 (15Rasmussen H.S. McCann P.P. Pharmacol. Ther. 1997; 75: 69-75Crossref PubMed Scopus (348) Google Scholar, 16Brown P.D. Breast Cancer Res. Treat. 1998; 52: 125-136Crossref PubMed Scopus (91) Google Scholar). 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 (17Coussens L.M. Fingleton B. Matrisian L.M. Science. 2002; 295: 2387-2392Crossref PubMed Scopus (2331) Google Scholar, 18Egeblad M. Werb Z. Nat. Rev. Cancer. 2002; 2: 161-174Crossref PubMed Scopus (5031) Google Scholar). 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 (19Toth M. Bernardo M.M. Gervasi D.C. Soloway P.D. Wang Z. Bigg H.F. Overall C.M. DeClerck Y.A. Tschesche H. Cher M. Brown S. Mobashery S. Fridman R. J. Biol. Chem. 2000; 275: 41415-41423Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). 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 (20Kleifeld O. Kotra L.P. Gervasi D.C. Brown S. Bernardo M.M. Fridman R. Mobashery S. Sagi I. J. Biol. Chem. 2001; 276: 17125-17131Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). 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. (21Bernardo M.M. Brown S. Li Z. Fridman R. Mobashery S. J. Biol. Chem. 2002; 277: 11201-11207Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) 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 K i values in the range of 46–260 nm (21Bernardo M.M. Brown S. Li Z. Fridman R. Mobashery S. J. Biol. Chem. 2002; 277: 11201-11207Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). 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 (21Bernardo M.M. Brown S. Li Z. Fridman R. Mobashery S. J. Biol. Chem. 2002; 277: 11201-11207Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), as we reported for the interaction of the first mechanism-based inhibitor selective for the gelatinases (20Kleifeld O. Kotra L.P. Gervasi D.C. Brown S. Bernardo M.M. Fridman R. Mobashery S. Sagi I. J. Biol. Chem. 2001; 276: 17125-17131Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). 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. Because of the need to use relatively large amounts of enzyme for the various biophysical experiments, human pro-MMP-2 was obtained from two sources. Specifically, TIMP-2-free pro-MMP-α was purified from the media of HeLa S3 cells infected with a recombinant vaccinia virus encoding the full-length cDNA of human pro-MMP-2 as described previously (22Olson M.W. Bernardo M.M. Pietila M. Gervasi D.C. Toth M. Kotra L.P. Massova I. Mobashery S. Fridman R. J. Biol. Chem. 2000; 275: 2661-2668Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), and pro-MMP-2 purified from HT-1080 cells was generously provided by Prof. Alex Strongin of the Burngham Institute. The protein concentration of the enzyme was determined using the molar extinction coefficient of 122,800 m–1cm–1 (23Murphy G. Crabbe. T. Methods Enzymol. 1995; 248: 470-484Crossref PubMed Scopus (174) Google Scholar). Pro-MMP-2 was activated with 1 mm p-aminophenylmercuric acetate (APMA), dissolved in 200 mm Tris, for 30 min at 37 °C. Inhibitors 1 and 2 were synthesized as described earlier (21Bernardo M.M. Brown S. Li Z. Fridman R. Mobashery S. J. Biol. Chem. 2002; 277: 11201-11207Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Both 1 and 2 were dissolved in 10% Me2SO. The inhibitors were complexed with MMP-2 using a 1:100 enzyme to inhibitor ratio. 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. All 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 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 × 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 Zn k-edge absorption coefficient μ(E), which was obtained after 10–12 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 (24Stern E.A. Newville M. Ravel B. Haskel D. Yacoby Y. Physica B. 1995; 208 & 209: 117-122Crossref Scopus (920) Google Scholar). The same energy, E 0 = 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 χ(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 (PDB code 1CK7). Using the computer code FEFF7 (26Rehr J.J. Mustre de Leon J. Zabinsky S.I. Albers R.C. J. Am. Chem. Soc. 1991; 113: 5135-5140Crossref Scopus (1808) Google Scholar, 27Zabinsky S.I. Rehr J.J. Ankudinov A. Albers R.C. Eller M.J. Phys. Rev. B. 1995; 52: 2995-3009Crossref PubMed Scopus (2645) Google Scholar), we calculated the theoretical photoelectron scattering amplitudes and phase shifts. Total theoretical χ(k) was constructed by adding the most important partial χ(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 (24Stern E.A. Newville M. Ravel B. Haskel D. Yacoby Y. Physica B. 1995; 208 & 209: 117-122Crossref Scopus (920) Google Scholar) 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. 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 (180–240 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. A recently published x-ray structure of MMP-2 (4Morgunova E. Tuuttila A. Bergmann U. Isupov M. Lindqvist Y. Schneider G. Tryggvason K. Science. 1999; 284: 1667-1670Crossref PubMed Scopus (476) Google Scholar) (PDB code 1CK7) 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 (28Case D.A. Pearlman D.A. Caldwell J.W. Cheatham III, T.E. Ross W.S. Simmerling C.L. Darden T.A. Merz K.M. Seibel G.L. Cheng A.L. Vincent J.J. Crowley M. Tsui V. Radmer R.J. Duan Y. Pitera J. Massova I. Seibel G.L. Singh U.C. Weiner P.K. Kollman P.A. AMBER. 6th Ed. University of California, San Francisco1999Google Scholar) 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 (29Bayly C.I. Cieplak P. Cornell W.D. Kollman P.A. J. Chem. Phys. 1993; 97: 10269Crossref Scopus (5357) Google Scholar). This consisted of first optimizing the molecules at the HF/3–21G* level of theory and basis set, followed by a HF/6–31G* 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 (30Frisch M.J. Truck G.W. Schlegel H.B. Scuseria G.E. Robb M.A. Cheeseman J.R. Zakrzewski V.G. Montgomery Jr., J.A. Stratmann R.E. Burant J.C. Dapprich S. Millam J.M. Daniels A.D. Kudin K.N. Strain M.C. Farkas O. Tomasi J. Barone V. Cossi M. Cammi R. Mennucci B. Pomelli C. Adamo C. Clifford S. Ochterski J. Petersson G.A. Ayala P.Y. Cui Q. Morokuma K. Malick D.K. Rabuck A.D. Raghavachari K. Foresman J.B. Cioslowski J. Ortiz J.V. Stefanov B.B. Liu G. Liashenko A. Piskorz P. Komaromi I. Gomperts R. Marti R.L. Fox D.J. Keith T. Al-Laham M.A. Peng C.Y. Nanayakkara A. Gonzalez C. Challacombe M. Gill P.M.W. Johnson B. Chen W. Wong M.W. Andres J.L. Gonzalez C. Head-Gordon M. Replogle E.S. Pople J.A. GAUSSIAN 98. Gaussian Inc., Pisttsburgh, PA1998Google Scholar). 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 (31Darden T.A.M. York D. Pedersen L.G. J. Chem. Phys. 1993; 98: 10089-10092Crossref Scopus (19731) Google Scholar). The AMBER 6 software package using the Cornell et al. force field (32Cornell W.D.C.P. Bayley C.I. Gould I.R. Merz K.M. Ferguson D.M. Spellmeyer D.C. Fox T. Caldwell J.W. Kollman P.A. J. Am. Chem. Soc. 1995; 117: 5179-5197Crossref Scopus (11375) Google Scholar) 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. The local structures around the catalytic zinc ion in latent and active MMP-2, and in the complexes of active MMP-2 with inhibitors 1 and 2 were studied by XAS. XAS refers to modulations in x-ray absorption coefficient around an x-ray absorption edge of a given atom. The technique measures the transition core electronic states of the metal to excited electronic states or continuum states. Spectral analysis near the electronic transition, x-ray absorption near-edge structure (XANES), provides information on the metal charge state and geometry (33Scott R.A. Methods Enzymol. 1985; 117: 414-459Crossref Scopus (189) Google Scholar). Spectral analysis above the absorption edge, in the extended x-ray absorption fine structure (EXAFS) region, provides complementary structural information such as coordination numbers, types, thermal disorder, Debye-Waller factors, and distances from neighboring atoms to the central (absorbing) atom. XAS is a valuable technique for elucidating the structures of a variety of metal-binding sites in metalloproteins (33Scott R.A. Methods Enzymol. 1985; 117: 414-459Crossref Scopus (189) Google Scholar). XANES Studies of Pro-MMP-2, Active MMP-2, and of the Complexes of MMP-2:1 and MMP-2:2— Fig. 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 (20Kleifeld O. Kotra L.P. Gervasi D.C. Brown S. Bernardo M.M. Fridman R. Mobashery S. Sagi I. J. Biol. Chem. 2001; 276: 17125-17131Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). 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 (20Kleifeld O. Kotra L.P. Gervasi D.C. Brown S. Bernardo M.M. Fridman R. Mobashery S. Sagi I. J. Biol. Chem. 2001; 276: 17125-17131Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 34Kleifeld O. Frenkel A. Martin J.M.L. Sagi I. Nat. Struct. Biol. 2000; 10: 98-103Crossref Scopus (90) Google Scholar). 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. EXAFS Data Analysis of Pro-MMP-2, Active MMP-2, and MMP-2:1 and MMP-2:2 Complexes—The 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) into the FEFF7 program (26Rehr J.J. Mustre de Leon J. Zabinsky S.I. Albers R.C. J. Am. Chem. Soc. 1991; 113: 5135-5140Crossref Scopus (1808) Google Scholar). 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 χ(k). The k 2 weighting factor and the Hanning window function, defined between 2 and 9 Å–1 were used in the Fourier transforms of all data sets. During the fitting procedure, the corrections to the energy origin (ΔE 0), bond distances (ΔR), and mean square disorders of the distances (σ2) were varied until the best fit was obtained. The number of relevant independent data points N idp in the data was calculated using Equation 1 (35Stern E.A. Phys. Rev. 1993; B48: 9825Crossref Scopus (501) Google Scholar), Nidp=2ΔkΔRπ+2(Eq. 1) where Δk and ΔR represent the data ranges in k and R spaces, respectively. Equation 1 implies that the number of fit variables should be smaller than N idp. To reduce the number of fit variables, we fixed the many-body factor S02 at 0.9. 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 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 ΔE 0 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 (20Kleifeld O. Kotra L.P. Gervasi D.C. Brown S. Bernardo M.M. Fridman R. Mobashery S. Sagi I. J. Biol. Chem. 2001; 276: 17125-17131Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). 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.Table IEXAFS curve fitting parameters of pro-MMP-2, active MMP-2, MMP-2:1 and MMP-2:2 inhibitor complexesFitPathΔE0R [Å]σ [Å2]Pro-MMP-2Fit 1.Zn-N × 2-3.17[F]1.88 [F]1.0E-03 [V]Zn-N × 1-3.17[F]1.91 (1)1.0E-03[VA]Zn-S × 1-3.17[F]2.24 (1)1.0E-03 [V]Zn-C × 26.9 [F]2.63 [F]6.0E-03 [V]Zn-C × 56.9 [F]3.01 (4)6.0E-03[VA]Active MMP-2Fit 1.Zn-N/O × 45.65[F]1.99 (2)3.0E-03 [V]Zn-C × 47.1 [F]3.00 (1)1.0E-03 [V]MMP-2:1 complexFit 1.Zn-N × 3-7.1 [F]1.91 (1)6.0E-03 [V]Zn-S × 2-7.1 [F]2.24 (1)5.0E-03 [V]Zn-C × 46.8 [F]2.76 (4)1.9E-02 [V]Zn-C × 46.8 [F]3.05 (5)1.9E-02[VA]Fit 2.Zn-N × 3-7.3 [F]1.01 [F]6.0E-03 [V]Zn-S × 2-7.3 [F]2.24 (1)5.0E-03 [V]Zn-C × 46.8 [F]2.76 (3)5.0E-03 [V]Zn-C × 46.8 [F]3.05 (4)1.8E-02[VA]Fit 3.Zn-N × 3-3.6 [F]1.94 (1)3.0E-03 [V]Zn-S × 1-3.6 [F]2.27 (1)1.0E-06 [V]Zn-C × 46.8 [F]2.78 (5)1.9E-02 [V]Zn-C × 46.8 [F]3.07 (7)1.9E-02[VA]Fit 4.Zn-N × 3-8 [F]1.91 (2)1.0E-02 [V]Zn-S × 3-8 [F]2.23 (1)1.1E-02 [V]Zn-C × 46.8 [F]2.75 (4)3.0E-02 [V]Zn-C × 46.8 [F]3.04 (6)3.0E-02[VA]MMP-2:2 complexFit 1.Zn-N × 3-0.5 [F]1.95 (1)2.0E-03 [V]Zn-S × 2-0.5 [F]2.29 (1)4.0E-03 [V]Zn-C × 21.95[V]3.34 (9)1.4E-02 [V]Zn-C × 51.95[V]2.96 (9)1.4E-02[VA]Fit 2.Zn-N × 1-1.6 [F]1.87 [F]1.0E-03[VA]Zn-N × 2-1.6 [F]1.99 (1)1.0E-03 [V]Zn-S × 1-1.8 [F]2.22 [F]2.0E-03[VA]Zn-S × 1-1.8 [F]2.36 (2)2.0E-03 [V]Zn-C × 24.68[V]2.61 (7)1.2E-02 [V]Zn-C × 54.68[V]2.97 [F]1.2E-02[VA]Fit 3.Zn-N × 3-0.5 [F]1.97 (1)2.0E-03 [V]Zn-S × 1-0.5 [F]2.31 (2)1.0E-08 [V]Zn-C × 25.5 [V]2.64 (9)1.0E-02 [V]Zn-C × 55.5 [V]2.96 (9)1.0E-02[VA]Fit 4.Zn-N × 3-7 [F]1.98 (1)5.0E-03 [V]Zn-S × 3-7 [F]2.3 (1)1.1E-02 [V]Zn-C × 21.1 [V]3.3(1)1.4E-02 [V]Zn-C × 51.1 [V]2.96(1)1.4E-02[VA] Open table in a new tab Stable and reproducible fits of pro-MMP-2 were consistent with a tetrahedral coordination of the zinc ion with three Zn–N(His) at 1.90 ± 0.01 Å, one Zn–S(Cys) at 2.24 ± 0.01 Å contributions (in the first coordination shell), and seven Zn–C contributions were two Zn–C 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 (4Morgunova E. Tuuttila A. Bergmann U. Isupov M. Lindqvist Y. Schneider G. Tryggvason K. Science. 1999; 284: 1667-1670Crossref PubMed Scopus (
Referência(s)