Breakdown of triple-helical interstitial collagens is essential in embryonic development, organ morphogenesis and tissue remodelling and repair. Aberrant collagenolysis may result in diseases such as arthritis, cancer, atherosclerosis, aneurysm and fibrosis. In vertebrates, it is initiated by collagenases belonging to the matrix metalloproteinase (MMP) family. The three-dimensional structure of a prototypic collagenase, MMP-1, indicates that the substrate-binding site of the enzyme is too narrow to accommodate triple-helical collagen. Here we report that collagenases bind and locally unwind the triple-helical structure before hydrolyzing the peptide bonds. Mutation of the catalytically essential residue Glu200 of MMP-1 to Ala resulted in a catalytically inactive enzyme, but in its presence noncollagenolytic proteinases digested collagen into typical 3/4 and 1/4 fragments, indicating that the MMP-1(E200A) mutant unwinds the triple-helical collagen. The study also shows that MMP-1 preferentially interacts with the alpha2(I) chain of type I collagen and cleaves the three alpha chains in succession. Our results throw light on the basic mechanisms that control a wide range of biological and pathological processes associated with tissue remodelling.

Matrix metalloproteinase 1 (MMP-1) cleaves types I, II, and III collagen triple helices into 3 ⁄4 and 1 ⁄4 fragments. To understand the structural elements responsible for this activity, various lengths of MMP-1 segments have been introduced into MMP-3 (stromelysin 1) starting from the C-terminal end. MMP-3/MMP-1 chimeras and variants were overexpressed in Escherichia coli, folded from inclusion bodies, and isolated as zymogens. After activation, recombinant chimeras were tested for their ability to digest triple helical type I collagen at 25°C. The results indicate that the nine residues 183 RWTNNFREY191 located between the fifth ␤-strand and the second ␣-helix in the catalytic domain of MMP-1 are critical for the expression of collagenolytic activity. Mutation of Tyr191 of MMP-1 to Thr, the corresponding residue in MMP-3, reduced collagenolytic activity about 5-fold. Replacement of the nine residues with those of the MMP-3 sequence further decreased the activity 2-fold. Those variants exhibited significant changes in substrate specificity and activity against gelatin and synthetic substrates, further supporting the notion that this region plays a critical role in the expression of collagenolytic activity. However, introduction of this sequence into MMP-3 or a chimera consisting of the catalytic domain of MMP-3 with the hinge region and the C-terminal hemopexin domain of MMP-1 did not express any collagenolytic activity. It is therefore concluded that RWTNNFREY, together with the C-terminal hemopexin domain, is essential for collagenolytic activity but that additional structural elements in the catalytic domain are also required. These elements probably act in a concerted manner to cleave the collagen triple helix.Interstitial collagen types I, II, and III are the major structural proteins in connective tissues such as tendon, skin, bone, cartilage, and blood vessels. They consist of three ␣ chains with repeating Gly-X-Y triplets where X and Y are frequently Pro and Hyp, respectively. Each chain of the repeating tripeptide adopts a left-handed poly-Pro II helix conformation, and three left-handed chains then intertwine to form a right-handed superhelix (1-3). This triple helical conformation makes interstitial collagens resistant to most proteinases in vertebrates except for collagenases, cathepsin K (4), and neutrophil elastase (5). The action of cathepsin K is probably important in collagen breakdown in specialized environments such as during bone resorption in an acidic pH environment. Neutrophil elastase may degrade telopeptides of interstitial collagen (6) and the triple-helical region of type I collagen under inflammatory conditions, but the latter activity is much weaker than that of collagenase (5). Vertebrate collagenases, on the other hand, are synthesized by many cell types such as stromal fibroblasts, chondrocytes, keratinocytes, osteoblasts, endothelial cells, and macrophages in response to inflammatory cytokines, growth factors, cellular transformation, and other chemical and physical stimuli ...

Matrix metalloproteinase (MMP) family members are involved in the physiological remodeling of tissues and embryonic development as well as pathological destruction of extracellular matrix components. To study the mechanisms of MMP action on collagenous substrates, we have constructed homotrimeric, fluorogenic triple-helical peptide (THP) models of the MMP-1 cleavage site in type II collagen. The substrates were designed to incorporate the fluorophore/quencher pair of (7-methoxycoumarin-4-yl)acetyl (Mca) and N-2,4-dinitrophenyl (Dnp) in the P(5) and P(5)' positions, respectively. In addition, Arg was incorporated in the P(2)' and P(8)' positions to enhance enzyme activity and improve substrate solubility. The desired sequences were Gly-Pro-Lys(Mca)-Gly-Pro-Gln-Gly approximately Leu-Arg-Gly-Gln-Lys(Dnp)-Gly-Ile/Val-Arg. Two fluorogenic substrates were prepared, one using a covalent branching protocol (fTHP-1) and one using a peptide self-assembly approach (fTHP-3). An analogous single-stranded substrate (fSSP-3) was also synthesized. Both THPs were hydrolyzed by MMP-1 at the Gly approximately Leu bond, analogous to the bond cleaved in the native collagen. The individual kinetic parameters for MMP-1 hydrolysis of fTHP-3 were k(cat) = 0.080 s(-1) and K(M) = 61.2 microM. Subsequent investigations showed fTHP-3 hydrolysis by MMP-2, MMP-3, MMP-13, a C-terminal domain-deleted MMP-1 [MMP-1(Delta(243-450))], and a C-terminal domain-deleted MMP-3 [MMP-3(Delta(248-460))]. The order of k(cat)/K(M) values was MMP-13 > MMP-1 approximately MMP-1(Delta(243-450)) approximately MMP-2 >> MMP-3 approximately MMP-3(Delta(248-460)). Studies on the effect of temperature on fTHP-3 and fSSP-3 hydrolysis by MMP-1 showed that the activation energies between these two substrates differed by 3.4-fold, similar to the difference in activation energies for MMP-1 hydrolysis of type I collagen and gelatin. This indicates that fluorogenic triple-helical substrates mimic the behavior of the native collagen substrate and may be useful for the investigation of collagenase triple-helical activity.

The unregulated activities of matrix metalloproteinases (MMPs) are implicated in disease processes including arthritis and tumor cell invasion and metastasis. MMP activities are controlled by four homologous endogenous protein inhibitors, tissue inhibitors of metalloproteinases (TIMPs), yet different TIMPs show little specificity for individual MMPs. The large interaction interface in the TIMP-1⅐MMP-3 complex includes a contiguous region of TIMP-1 around the disulfide bond between Cys 1 and Cys 70 that inserts into the active site of MMP-3. The effects of fifteen different substitutions for threonine 2 of this region reveal that this residue makes a large contribution to the stability of complexes with MMPs and has a dominant influence on the specificity for different MMPs. The size, charge, and hydrophobicity of residue 2 are key factors in the specificity of TIMP. Threonine 2 of TIMP-1 interacts with the S1 specificity pocket of MMP-3, which is a key to substrate specificity, but the structural requirements in TIMP-1 residue 2 for MMP binding differ greatly from those for the corresponding residue of a peptide substrate. These results demonstrate that TIMP variants with substitutions for Thr 2 represent suitable starting points for generating more targeted TIMPs for investigation and for intervention in MMP-related diseases.The matrix metalloproteinases (MMPs) 1 are a family of about twenty Zn 2ϩ -dependent endopeptidases that have important roles in connective tissue turnover during physiological processes including development, morphogenesis, and wound healing (1, 2). Their activities in the extracellular matrix are stringently regulated through transcriptional control, zymogen activation, and the actions of four endogenous inhibitory proteins, tissue inhibitors of metalloproteinases (TIMPs) 1 to 4 (3-7). Normal matrix homeostasis is associated with an appropriate balance between the levels of TIMPs and active MMPs, whereas an imbalance involving excess MMP activity is linked with disease processes including arthritis, tumor cell metastasis, and tissue invasion and atherosclerosis (1, 2).Mammalian TIMPs have an N-terminal domain of about 125 amino acids and a smaller C-terminal domain of about 65 amino acids; each domain is stabilized by three disulfide bonds (8). The N-terminal domains of different TIMPs fold into a correct native structure which carries the inhibitory activity against MMPs (9 -11). Although correctly folded and functional C-terminal domains have not been described, truncation experiments indicate that this region is responsible for the interactions of TIMPs with pro-MMPs (12, 13). There is little specificity in the inhibitory actions of TIMPs on metalloproteinases, with the exception of the ability of TIMP-2 and TIMP-3 to inhibit membrane-type metalloproteinases-1 and -2, whereas TIMP-1 is a poor inhibitor of these enzymes (12-14). However, the interactions of TIMPs with pro-MMPs are more specific. For example, TIMP-2 and TIMP-4 form specific complexes with pro-MMP-2 (progelatinase A), whe...

Studies of the structural basis of the interactions of tissue inhibitors of metalloproteinases (TIMPs) and matrix metalloproteinases (MMPs) may provide clues for designing MMP-specific inhibitors. In this paper we report combinations of mutations in the major MMP-binding region that enhance the specificity of N-TIMP-1. Mutants with substitutions for residues 4 and 68 were characterized and combined with previously studied Thr 2 mutations to generate mutants with improved selectivity or binding affinity to specific MMPs. Some combinations of mutations had non-additive effects on ⌬G of binding to MMPs, suggesting interactions between subsites in the reactive site. The T2L/V4S mutation generates an inhibitor that binds to MMP-2 20-fold more tightly than to MMP-3(⌬C) and over 400-fold more tightly than to MMP-1. The T2S/V4A/S68Y mutant is the strongest inhibitor for stromelysin-1 among all mutants characterized to date, with an apparent K i for MMP-3(⌬C) in the picomolar range. A third mutant, T2R/V4I, has no detectable inhibitory activity for MMP-1 but is an effective inhibitor of MMP-2 and -3. These selective TIMP variants may provide useful tools for investigation of biological roles of specific MMPs and for possible therapy of MMP-related diseases.Degradation of the extracellular matrix is essential for normal biological processes including embryonic development and morphogenesis (1, 2), reproduction (3) and wound healing (4), and enhanced turnover is associated with diseases including arthritis (5, 6), tumor angiogenesis and metastasis (7), multiple sclerosis (8), and cardiovascular diseases (9). The matrix metalloproteinases (MMPs) 1 are a family of more than 20 zinc-dependent proteases that catalyze extracellular matrix turnover (10). Activity and zymogen activation in MMPs are regulated by a group of endogenous proteins named the tissue inhibitors of metalloproteinases (TIMPs) (11).TIMPs are distributed in both invertebrates and vertebrates (11-13). The mammalian TIMPs are a family of four members (TIMP-1-4) that have about 40% sequence identity and fold into two domains, each containing three disulfide bonds (11). The isolated N-terminal domains (N-TIMPs) are able to form the correct native structure that carries the inhibitory activity against the MMPs (14). Although there are four TIMPs, their inhibitory activities toward different MMPs are not particularly specific. A notable exception is that TIMP-1 is a weak inhibitor of MT-MMPs, whereas TIMP-2 and TIMP-3 are much more effective (15-17).Reported structures of TIMPs include crystal structures of TIMP-1 in a complex with the MMP-3 catalytic domain (18), TIMP-2 in a complex with the catalytic domain of membrane type MT1-MMP (19) and in a free form (20), and the solution NMR structures of N-TIMP-1 (21) and N-TIMP-2 (22). These structures show that the N-terminal inhibitory domain consists of a 5-stranded ␤-barrel with three associated ␣-helices resembling the folds of members of the oligonucleotide/oligosaccharide binding (OB) protein family (23). The st...

Previous studies have established that ligation of keratinocyte ␣ 2 ␤ 1 integrin by type I collagen induces expression of matrix metalloproteinase-1 (MMP-1) and that MMP-1 activity is required for the ␣ 2 ␤ 1 integrin-dependent migration of primary keratinocytes across collagenous matrices. We now present evidence that MMP-1 binds the ␣ 2 ␤ 1 integrin via the I domain of the ␣ 2 integrin subunit. Using an enzyme-linked immunosorbent assay with purified human MMP-1 and recombinant ␣ 2 integrin I domain, we showed that the ␣ 2 integrin I domain specifically bound in a divalent cation-dependent manner to both the pro and active forms of MMP-1, but not to MMP-3 or MMP-13. Although both the I domain and MMP-1 bind divalent cations, MMP-1 bound, in a divalent cation-dependent manner, to ␣ 2 integrin I domains containing metal ion-dependent adhesion sites motif mutations that prevent divalent cation binding to the I domain, demonstrating that the metal ion dependence is a function of MMP-1. Using a series of MMP-1-MMP-3 and MMP-1-MMP-13 chimeras, we determined that both the linker domain and the hemopexin-like domain of MMP-1 were required for optimal binding to the I domain. The ␣ 2 integrin/MMP-1 interaction described here extends an emerging paradigm in matrix biology involving anchoring of proteinases to the cell surface to regulate their biological activities.The extracellular matrix is not a static environment. Remodeling and degradation of the extracellular matrix is a vital component of physiological and pathophysiological processes, such as development and differentiation, cell migration, angiogenesis, wound healing, and metastasis. Matrix metalloproteinases (MMPs) 1 play a central role in the turnover of extracellular matrix components (1).MMPs constitute a large family of metal-dependent endoproteases with varying substrate specificities for many extracellular proteins. The structure of native triple helical type I collagen makes it resistant to proteolysis, and only six MMPs, MMP-1, MMP-8, MMP-13, MMP-14 (MT1-MMP), MMP-18, and MMP-2, exhibit an ability to cleave native fibrillar collagen within its triple helical domain (2-8). Similar to most MMPs, the collagenases (MMP-1, MMP-8, and MMP-13) have several structural features in common, including an N-terminal prodomain, a catalytic domain, and a short proline-rich linker connected to a hemopexin-like domain at the C terminus (9). The catalytic domain contains a Zn 2ϩ -binding site that is conserved in all MMPs and is required for catalytic activity (10, 11). The catalytic domain of the collagenases contains an additional structural Zn 2ϩ , as well as three structural Ca 2ϩ ions (12). The hemopexin-like domain contains a Ca 2ϩ and a Ca 2ϩ -Cl Ϫ ion pair (12).The three collagenases differ in patterns of tissue expression. In humans, MMP-1, which is expressed by epithelium, endothelium, fibroblasts, chondrocytes, and macrophages, seems to be the enzyme principally responsible for collagen turnover in most tissues (13-18). During cutaneous wound healing, human k...

Differences in proteinase susceptibility between free TIMP-1 and the TIMP-1-MMP-3 complex and mutagenesis studies suggested that the residues around the disulfide bond between Cys1 and Cys70 in TIMP-1 may interact with MMPs. The crystal structure of the complex between TIMP-1 and the catalytic domain of MMP-3 has revealed that the alpha-amino group of Cys1 bidentately chelates the catalytic zinc of MMP-3 and the Thr2 side chain occupies the S1' pocket. Generation of the N-terminal domain of TIMP-1 (N-TIMP-1) variants with 15 different amino acid substitutions for Thr2 has indicated that the nature of the side chain of residue 2 has a major effect on the affinity of N-TIMP-1 for three different MMPs (MMPs-1, -2 and -3). The results also demonstrate that the mode of binding of N-TIMP-1 residue 2 differs from the binding of the P1' residue of a peptide substrate.

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