Catalytic Domain Of Matrix Metalloproteinases Research Articles

Bioinformatics and Molecular Insights to Anti-Metastasis Activity of Triethylene Glycol Derivatives.

The anti-metastatic and anti-angiogenic activities of triethylene glycol derivatives have been reported. In this study, we investigated their molecular mechanism(s) using bioinformatics and experimental tools. By molecular dynamics analysis, we found that (i) triethylene glycol dimethacrylate (TD-10) and tetraethylene glycol dimethacrylate (TD-11) can act as inhibitors of the catalytic domain of matrix metalloproteinases (MMP-2, MMP-7 and MMP-9) by binding to the S1’ pocket of MMP-2 and MMP-9 and the catalytic Zn ion binding site of MMP-7, and that (ii) TD-11 can cause local disruption of the secondary structure of vascular endothelial growth factor A (VEGFA) dimer and exhibit stable interaction at the binding interface of VEGFA receptor R1 complex. Cell-culture-based in vitro experiments showed anti-metastatic phenotypes as seen in migration and invasion assays in cancer cells by both TD-10 and TD-11. Underlying biochemical evidence revealed downregulation of VEGF and MMPs at the protein level; MMP-9 was also downregulated at the transcriptional level. By molecular analyses, we demonstrate that TD-10 and TD-11 target stress chaperone mortalin at the transcription and translational level, yielding decreased expression of vimentin, fibronectin and hnRNP-K, and increase in extracellular matrix (ECM) proteins (collagen IV and E-cadherin) endorsing reversal of epithelial–mesenchymal transition (EMT) signaling.

Directed evolution of the metalloproteinase inhibitor TIMP-1 reveals that its N- and C-terminal domains cooperate in matrix metalloproteinase recognition

Tissue inhibitors of metalloproteinases (TIMPs) are natural inhibitors of matrix metalloproteinases (MMPs), enzymes that contribute to cancer and many inflammatory and degenerative diseases. The TIMP N-terminal domain binds and inhibits an MMP catalytic domain, but the role of the TIMP C-terminal domain in MMP inhibition is poorly understood. Here, we employed yeast surface display for directed evolution of full-length human TIMP-1 to develop MMP-3-targeting ultrabinders. By simultaneously incorporating diversity into both domains, we identified TIMP-1 variants that were up to 10-fold improved in binding MMP-3 compared with WT TIMP-1, with inhibition constants (Ki ) in the low picomolar range. Analysis of individual and paired mutations from the selected TIMP-1 variants revealed cooperative effects between distant residues located on the N- and C-terminal TIMP domains, positioned on opposite sides of the interaction interface with MMP-3. Crystal structures of MMP-3 complexes with TIMP-1 variants revealed conformational changes in TIMP-1 near the cooperative mutation sites. Affinity was strengthened by cinching of a reciprocal "tyrosine clasp" formed between the N-terminal domain of TIMP-1 and proximal MMP-3 interface and by changes in secondary structure within the TIMP-1 C-terminal domain that stabilize interdomain interactions and improve complementarity to MMP-3. Our protein engineering and structural studies provide critical insight into the cooperative function of TIMP domains and the significance of peripheral TIMP epitopes in MMP recognition. Our findings suggest new strategies to engineer TIMP proteins for therapeutic applications, and our directed evolution approach may also enable exploration of functional domain interactions in other protein systems.

Atomic-Level Quality Assessment of Enzymes Encapsulated in Bioinspired Silica.

Among protein immobilization strategies, encapsulation in bioinspired silica is increasingly popular. Encapsulation offers high yields and the solid support is created through a protein-catalyzed polycondensation reaction that occurs under mild conditions. An integrated strategy is reported for the characterization of both the protein and bioinspired silica scaffold generated by the encapsulation of enzymes with an external silica-forming promoter or with the promoter expressed as a fusion to the enzyme. This strategy is applied to the catalytic domain of matrix metalloproteinase 12. Analysis reveals that the structure of the protein encapsulated by either method is not significantly altered with respect to the native form. The structural features of silica obtained by either strategy are also similar, but differ from those obtained by other approaches. In case of the covalently linked R5-enzyme construct, immobilization yields are higher. Encapsulation through a fusion protein, therefore, appears to be the method of choice.

Simultaneous use of solution NMR and X-ray data in REFMAC5 for joint refinement/detection of structural differences.

The program REFMAC5 from CCP4 was modified to allow the simultaneous use of X-ray crystallographic data and paramagnetic NMR data (pseudocontact shifts and self-orientation residual dipolar couplings) and/or diamagnetic residual dipolar couplings. Incorporation of these long-range NMR restraints in REFMAC5 can reveal differences between solid-state and solution conformations of molecules or, in their absence, can be used together with X-ray crystallographic data for structural refinement. Since NMR and X-ray data are complementary, when a single structure is consistent with both sets of data and still maintains reasonably `ideal' geometries, the reliability of the derived atomic model is expected to increase. The program was tested on five different proteins: the catalytic domain of matrix metalloproteinase 1, GB3, ubiquitin, free calmodulin and calmodulin complexed with a peptide. In some cases the joint refinement produced a single model consistent with both sets of observations, while in other cases it indicated, outside the experimental uncertainty, the presence of different protein conformations in solution and in the solid state.

Ultrafast MAS Solid-State NMR Permits Extensive 13C and 1H Detection in Paramagnetic Metalloproteins

We show here that by combining tailored approaches based on ultrafast (60 kHz) MAS on the Co(II)-replaced catalytic domain of matrix metalloproteinase 12 (CoMMP-12) we can observe and assign, in a highly paramagnetic protein in the solid state, (13)C and even (1)H resonances from the residues coordinating the metal center. In addition, by exploiting the enhanced relaxation caused by the paramagnetic center, and the low power irradiation enabled by the fast MAS, this can be achieved in remarkably short times and at very high field (21.2 T), with only less than 1 mg of sample. Furthermore, using the known crystal structure of the compound, we are able to distinguish and measure pseudocontact (PCS) contributions to the shifts up to the coordinating ligands and to unveil structural information.

High-Resolution Solid-State NMR Structure of a 17.6 kDa Protein

The use of pseudocontact shifts arising from paramagnetic metal ions in a microcrystalline protein sample is proposed as a strategy to obtain unambiguous signal assignments in solid-state NMR spectra enabling distance extraction for protein structure calculation. With this strategy, 777 unambiguous (281 sequential, 217 medium-range, and 279 long-range) distance restraints could be obtained from PDSD, DARR, CHHC, and the recently introduced PAR and PAIN-CP solid-state experiments for the cobalt(II)-substituted catalytic domain of matrix metalloproteinase 12 (159 amino acids, 17.6 kDa). The obtained structure is a high resolution one, with backbone rmsd of 1.0 +/- 0.2 A, and is in good agreement with the X-ray structure (rmsd to X-ray 1.3 A). The proposed strategy, which may be generalized for nonmetalloproteins with the use of paramagnetic tags, represents a significant step ahead in protein structure determination using solid-state NMR.

Solid‐State NMR of Matrix Metalloproteinase 12: An Approach Complementary to Solution NMR

Data transfer. The solid-state proton-driven spin diffusion (PDSD) and J-decoupled PDSD NMR spectra of the microcrystalline catalytic domain of matrix metalloproteinase 12 (MMP-12, 17 kDa) have been recorded. It is shown that such spectra can be largely assigned in a few days by using the available liquid-state assignment and validated with an independent sequential assignment based on 3D NCACX and NCOCX PDSD experiments. This demonstrates how quickly the liquid-state assignment of comparably large protein can be transferred to the solid state. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2268/2007/z600408_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Filter sterilization of highly infectious samples to prevent false negative analysis of matrix metalloproteinase activity

Matrix metalloproteinases (MMPs) are implicated in the immunopathology of numerous infectious diseases. High risk samples such as those generated after infection with Mycobacterium tuberculosis require filter sterilization for safe analysis of MMP concentrations. Here, we report that commercial filter membranes may cause artefacts by binding MMPs. Anopore 0.2 μM membrane filtration reduced MMP-1 concentrations to undetectable levels by zymography and Western blotting. Polypropylene 0.45 μM filtration removed some MMP-1, while Polysulphone, Durapore and Bio-inert 0.2 μM membranes did not remove MMP-1. Anopore filtration also removed all MMP-7 and -9 activity, suggesting that the conserved MMP catalytic domain binds the membrane. This study demonstrates the importance of selecting the appropriate filter in MMP analysis to avoid incorrectly excluding MMP involvement in infection-related immunopathology.

Noninvasive Targeted Imaging of Matrix Metalloproteinase Activation in a Murine Model of Postinfarction Remodeling

Time-dependent activation of matrix metalloproteinases (MMPs) after myocardial infarction (MI) contributes to adverse left ventricular (LV) remodeling; however, noninvasive methods to monitor this process serially are needed. MMP-targeted radiotracers were developed that displayed selective binding kinetics to the active MMP catalytic domain. Initial nonimaging studies were performed with a (111)In-labeled MMP-targeted radiotracer ((111)In-RP782) and negative control compound ((111)In-RP788) in control mice (Ctrl) and in mice 1 week after surgically induced MI. Localization of (111)In-RP782 was demonstrated within the MI by microautoradiography. A 334+/-44% increase (P<0.001 versus Ctrl) in relative retention of (111)In-RP782 was confirmed by gamma well counting of myocardium. Subsequent high-resolution dual-isotope planar and hybrid micro-single-photon emission computed tomography/CT imaging studies with an analogous 99mTc-labeled MMP-targeted radiotracer (99mTc-RP805) and 201Tl demonstrated favorable biodistribution and clearance kinetics of 99mTc-RP805 for in vivo cardiac imaging, with robust retention 1 to 3 weeks after MI in regions of decreased 201Tl perfusion. Gamma well counting yielded a similar approximately 300% increase in relative myocardial retention of 99mTc-RP805 in MI regions (Ctrl, 102+/-9%; 1 week, 351+/-77%; 2 weeks, 291+/-45%; 3 weeks, 292+/-41%; P<0.05 versus Ctrl). Myocardial uptake in the MI region was also significantly increased approximately 5-fold when expressed as percentage injected dose per gram tissue. There was also a significant 2-fold increase in myocardial activity in remote regions relative to control mice, suggesting activation of MMPs in regions remote from the MI. This novel noninvasive targeted MMP radiotracer imaging approach holds significant diagnostic potential for in vivo localization of MMP activation and tracking of MMP-mediated post-MI remodeling.

Conformational variability of matrix metalloproteinases: Beyond a single 3D structure

The structures of the catalytic domain of matrix metalloproteinase 12 in the presence of acetohydroxamic acid and N-isobutyl-N-[4-methoxyphenylsulfonyl]glycyl hydroxamic acid have been solved by x-ray diffraction in the crystalline state at 1.0 and 1.3-A resolution, respectively, and compared with the previously published x-ray structure at 1.2-A resolution of the adduct with batimastat. The structure of the N-isobutyl-N-[4-methoxyphenylsulfonyl]glycyl hydroxamic acid adduct has been solved by NMR in solution. The three x-ray structures and the solution structure are similar but not identical to one another, the differences being sizably higher in the loops. We propose that many of the loops show a dynamical behavior in solution on a variety of time scales. Different conformations of some flexible regions of the protein can be observed as "frozen" in different crystalline environments. The mobility in solution studied by NMR reveals conformational equilibria in accessible time scales, i.e., from 10(-5) s to ms and more. Averaging of some residual dipolar couplings is consistent with further motions down to 10(-9) s. Finally, local thermal motions of each frozen conformation in the crystalline state at 100 K correlate well with local motions on the picosecond time scale. Flexibility/conformational heterogeneity in crucial parts of the catalytic domain is a rule rather than an exception in matrix metalloproteinases, and its extent may be underestimated by inspection of one x-ray structure. Backbone flexibility may play a role in the difficulties encountered in the design of selective inhibitors, whereas it may be a requisite for substrate binding and broad substrate specificity.

Matrix metalloproteinaes and bone metastasis.

The degradation of stromal and epithelial extracellular matrices (ECM) is partly mediated by a family of zinc-dependent endopeptidases, the matrix metalloproteinases (MMPs), which have the potential to cleave virtually all structural ECM components and thus are major mediators of extracellular proteolysis in normal and pathological conditions. In humans, the MMP family comprises at least 25 closely homologous multidomain enzymes that share common structural and functional features. Overall, the MMP family has evolved to include both secreted and plasma membrane-tethered members, thus conferring the MMPs with the ability to mediate proteolytic events at both the cell surface and in the immediate peri-cellular milieu. With few exceptions, MMPs comprise four basic domains: a signal peptide, a propeptide domain, a catalytic domain and a C-terminal domain known as the hemopexin-like domain. The propeptide domain is a short stretch of residues containing the conserved PRCG(V/N)PD, which maintains the enzyme in its inactive, zymogen state (referred here as pro-MMP) until its proteolytic removal, via the so-called “cysteine switch” (Nagase, 1999). A cysteine residue within the propeptide domain occupies a coordination site of the catalytic zinc to maintain enzyme latency (Becker et al., 1995; Van Wart and Birkedal-Hansen, 1990). The catalytic domain of MMPs contains two zinc ions, which are important for either structural or catalytic competence, and at least one calcium ion. The catalytic zinc ion is coordinated to three histidine residues, and it is necessary for MMP proteolytic activity (Bode et al, 1994; Salowe et al., 1992). All MMPs, except MMP-7 and MMP-26, contain a hemopexin-like domain, which is connected to the catalytic domain via a hinge region. Depending on the type of MMP, the hemopexin-like domain plays a role in substrate binding and interactions with tissue inhibitors of metalloproteinases (TIMPs), which affect MMP substrate recognition, zymogen activation and inhibition of activity. Structurally, the hemopexin-like domain has an ellipsoid shape, composed of a four-bladed ß -propeller structure (). In the case of the zymogenic form of the gelatinases, the hemopexin-like domain can bind TIMPs, a family of endogenous MMP inhibitors, with proMMP-2 binding TIMP-2, TIMP-3, and TIMP-4 and pro-MMP-9 binding TIMP-1 and TIMP-3 (Baker et al., 2002). For a long time, the significance of the binding of TIMPs to the latent form of the gelatinases was not understood because the zymogens are devoid of enzymatic activity. It turned out that in the case of pro-MMP-2, binding of TIMP-2 plays a role in zymogen activation by MT-MMPs (described below).KeywordsVascular Endothelial Growth FactorBone MetastasisBone Marrow Stromal CellSkeletal MetastasisNational Academy ofSciencesThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Identification of a Negatively Charged Peptide Motif within the Catalytic Domain of Progelatinases That Mediates Binding to Leukocyte β2 Integrins

The alpha M beta 2 integrin of leukocytes can bind a variety of ligands. We screened phage display libraries to isolate peptides that bind to the alpha M I domain, the principal ligand binding site of the integrin. Only one peptide motif, (D/E)(D/E)(G/L)W, was obtained with this approach despite the known ligand binding promiscuity of the I domain. Interestingly, such negatively charged sequences are present in many known beta 2 integrin ligands and also in the catalytic domain of matrix metalloproteinases (MMPs). We show that purified beta 2 integrins bind to pro-MMP-2 and pro-MMP-9 gelatinases and that that the negatively charged sequence of the MMP catalytic domain is an active beta 2 integrin-binding site. Furthermore, a synthetic DDGW-containing phage display peptide inhibited the ability of beta 2 integrin to bind progelatinases but did not inhibit the binding of cell adhesion-mediating substrates such as intercellular adhesion molecule-1, fibrinogen, or an LLG-containing peptide. Immunoprecipitation and cell surface labeling demonstrated complexes of pro-MMP-9 with both the alpha M beta 2 and alpha L beta 2 integrins in leukocytes, and pro-MMP-9 colocalized with alpha M beta 2 in cell surface protrusions. The DDGW peptide and the gelatinase-specific inhibitor peptide CTTHWGFTLC blocked beta 2 integrin-dependent leukocyte migration in a transwell assay. These results suggest that leukocytes may move in a progelatinase-beta 2 integrin complex-dependent manner.

Expression and high yield production of the catalytic domain of matrix metalloproteinase 12 and of an active mutant with increased solubility

Abstract In the general frame of a project aiming at screening candidate drugs against several different matrix metalloproteinases (MMP) to find a rationale for selectivity, the catalytic domain of MMP-12 (metalloelastase) has been expressed in E. coli strain BL21D3 and its production optimized to about 30 mg/dm 3 . The chosen construct spans residues 106–267 of the whole MMP-12 and contains two additional methionines at positions 104–105. This is at variance with the previously published constructs which span residues 99–279 [J. Mol. Biol. 312 (2001) 743] and 100–262 [J. Mol. Biol. 312 (2001) 731], respectively. The protein, expressed in inclusion bodies, is solubilized in high urea concentration and properly refolded, as judged from 1 H to 15 N HSQC, by stepwise urea dilution in the presence of the mild inhibitor acetohydroxamic acid. The latter can be easily dialysed out when needed for activity or inhibition studies. The solubility of this catalytic domain construct of MMP-12 is around 250–300 μM. To increase solubility, a mutant (F171D) has been designed that should not alter the activity and should not interfere with the contacts between the catalytic domain and either the pro-domain or the C-terminal domains that precede and follow it in the full-length protein. The F171D mutant was produced and indeed resulted fully active and three times more soluble than the WT, greatly facilitating its use in NMR screening experiments. Comparison of X-ray data for the present [Angew. Chem. Int. Ed. 42 (2003) 2673] and previous [J. Mol. Biol. 312 (2001) 731; J. Mol. Biol. 312 (2001) 743] constructs of the catalytic domain of MMP-12 suggested that elimination of Met 105 and of the stretch 264–267 should lead to an even more soluble and stable construct. The latter was produced and showed to have the desired properties.

Catalytic Activities and Substrate Specificity of the Human Membrane Type 4 Matrix Metalloproteinase Catalytic Domain

Membrane type (MT) matrix metalloproteinases (MMPs) are recently recognized members of the family of Zn(2+)- and Ca(2+)-dependent MMPs. To investigate the proteolytic capabilities of human MT4-MMP (i.e. MMP-17), we have cloned DNA encoding its catalytic domain (CD) from a breast carcinoma cDNA library. Human membrane type 4 MMP CD (MT4-MMPCD) protein, expressed as inclusion bodies in Escherichia coli, was purified to homogeneity and refolded in the presence of Zn(2+) and Ca(2+). While MT4-MMPCD cleaved synthetic MMP substrates Ac-PLG-[2-mercapto-4-methylpentanoyl]-LG-OEt and Mca-PLGL-Dpa-AR-NH(2) with modest efficiency, it catalyzed with much higher efficiency the hydrolysis of a pro-tumor necrosis factor-alpha converting enzyme synthetic substrate, Mca-PLAQAV-Dpa-RSSSR-NH(2). Catalytic efficiency with the pro-tumor necrosis factor-alpha converting enzyme substrate was maximal at pH 7.4 and was modulated by three ionizable enzyme groups (pK(a3) = 6.2, pK(a2) = 8.3, and pK(a1) = 10.6). MT4-MMPCD cleaved gelatin but was inactive toward type I collagen, type IV collagen, fibronectin, and laminin. Like all known MT-MMPs, MT4-MMPCD was also able to activate 72-kDa progelatinase A to its 68-kDa form. EDTA, 1,10-phenanthroline, reference hydroxamic acid MMP inhibitors, tissue inhibitor of metalloproteinases-1, and tissue inhibitor of metalloproteinases-2 all potently blocked MT4-MMPCD enzymatic activity. MT4-MMP is, therefore, a competent Zn(2+)-dependent MMP with unique specificity among synthetic substrates and the capability to both degrade gelatin and activate progelatinase A.

Retrospective analysis of a secondary structure prediction: the catalytic domain of matrix metalloproteinases.

Secondary structure prediction of the catalytic domain of matrix metalloproteinases is evaluated in the light of recently published experimentally determined structures. The prediction was made by combining conformational propensity, surface probability, and residue conservation calculated for an alignment of 19 sequences. The position of each observed secondary structure element was correctly predicted with a high degree of accuracy, with a single beta-strand falsely predicted. The domain fold was also anticipated from the prediction by analogy with the structural elements found in the distantly related metalloproteinases thermolysin, astacin, and adamalysin.