Membrane type 1-matrix metalloproteinase (MT1-MMP) is a zinc-dependent, membrane-associated endoproteinase of the metzincin family. The enzyme regulates extracellular matrix remodeling and is capable of cleaving a wide variety of transmembrane proteins. The enzymatic activity of MT1-MMP is regulated by endogenous inhibitors, the tissue inhibitor of metalloproteinases (TIMP). To date, four variants of mammalian TIMP have been identified. Whereas TIMP-2-4 are potent inhibitors against MT1-MMP, TIMP-1 displays negligible inhibitory activity against the enzyme. The rationale for such selectivity is hitherto unknown. Here we identify the surface epitopes that render TIMP-1 inactive against MT1-MMP. We show that TIMP-1 can be transformed into an active inhibitor against MT1-MMP by the mutation of a single residue, namely threonine 98 to leucine (T98L). The resultant mutant displayed inhibitory characteristics of a typical slow, tight binding inhibitor. The potency of the mutant could be further enhanced by the introduction of valine 4 to alanine (V4A) and proline 6 to valine (P6V) mutations. Indeed, the inhibitory profile of the triple mutant (V4A/P6V/T98L) is indistinguishable from those of other TIMPs. Our findings suggest that threonine 98 is critical in initiating MMP binding and complex stabilization. Our findings also provide a potential mechanistic explanation for MMP-TIMP selectivity.Membrane type 1-matrix metalloproteinase (MT1-MMP, 1 MMP-14) is a member of the zinc-dependent endopeptidases of the metzincin family. The enzyme is involved in the degradation of extracellular matrix components and tissue remodeling (1-3). Among the six membrane-type MMPs identified, MT1-MMP is the most studied not only because of its role in activating other MMPs such as pro-MMP-2 (pro-gelatinase-A) and pro-MMP-13 (4, 5), but also because of its compelling link with cell invasion and tumor malignancy (6). As with other members of the matrixin family, MT1-MMP is a multidomain enzyme with clear structural compartmentation. Preceding the catalytic domain is a propeptide that contains a cysteine switch that regulates the catalytic activity of the enzyme. The catalytic domain displays a tertiary fold typical of an enzyme of the metzincin family (7). Succeeding the catalytic domain are a short hinge, a hemopexin domain, a transmembrane domain, and a cytotail of 2.5 kDa. A crystal structure of the catalytic domain of MT1-MMP was first reported by Fernandez-Catalan et al. in 1998 (8). To date, this is the only known variant of membrane-associated MMP to be crystallized and published.The activity of MMPs is regulated by their endogenous inhibitors, tissue inhibitor of metalloproteinases (TIMP). There are four mammalian TIMPs, namely TIMP-1-4. TIMP inhibit MT1-MMP by forming a tight, non-covalent 1:1 stoichiometric complex with the catalytic domain of the enzyme. The binding constants (K i app ) of wild type TIMP-2-4 with MT1-MMP are in the low picomolar range (9). Although TIMP-2-4 are superb inhibitors against MT1-MMP, TIMP-1, on the c...

Tissue inhibitors of metalloproteinases (TIMPs) are the endogenous modulators of the zinc-dependent mammalian matrix metalloproteinases (MMPs) and their close associates, proteinases of the ADAM (a disintegrin and metalloproteinase) and ADAM with thrombospondin repeats families. There are four variants of TIMPs, and each has its defined set of metalloproteinase (MP) targets. TIMP-1, in particular, is inactive against several of the membrane-type MMPs (MT-MMPs), MMP-19, and the ADAM proteinase TACE (tumor necrosis factor-␣-converting enzyme, ADAM-17). The molecular basis for such inactivity is unknown. Previously, we showed that TIMP-1 could be transformed into an active inhibitor against MT1-MMP by the replacement of threonine 98 residue with leucine (T98L). Here, we reveal that the T98L mutation has in fact transformed TIMP-1 into a versatile inhibitor against an array of MPs otherwise insensitive to wild-type TIMP-1; examples include TACE, MMP-19, and MT5-MMP. Using T98L as the scaffold, we created a TIMP-1 variant that is fully active against TACE. The binding affinity of the mutant (V4S/ TIMP-3-AB-loop/V69L/T98L) (K i app 0.14 nM) surpassed that of TIMP-3 (K i app 0.22 nM), the only natural TIMP inhibitor of the enzyme. The requirement for leucine is absolute for the transformation in inhibitory pattern. On the other hand, the mutation has minimal impact on the MPs already well inhibited by wild-type TIMP-1, such as gelatinase-A and stromelysin-1. Not only have we unlocked the molecular basis for the inactivity of TIMP-1 against several of the MPs, but also our findings fundamentally modify the current beliefs on the molecular mechanism of TIMP-MP recognition and selectivity.The mammalian matrix metalloproteinases (MMPs, 1 matrixins) and their close associates, ADAM (a disintegrin and metalloproteinase, adamalysins) proteinases, are important modulators of cell-cell and cell-matrix interactions. The enzymes degrade various pericellular components and regulate cell proliferation, matrix remodeling, and the release of a vast array of bioactive molecules (1-3). Uncontrolled activities of the enzymes often lead to severe pathological conditions such as rheumatoid arthritis, cardiovascular disorders, and tumor metastasis (4 -8). Under normal physiological conditions, the activities of the metalloproteinases (MPs) are tightly controlled by their natural inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). There are four variants of TIMPs (TIMP-1 to -4), and each has its own profile of MP targets (9 -11). Although the majority of the MMPs are inhibited by all TIMPs, some are notably less sensitive to TIMP-1 inhibition. To name a few, MMP-19, the membrane-type 1 (MT1)-MMP (MMP-14), MT3-MMP (MMP-16), MT5-MMP (MMP-24), and the best known ADAM proteinase, tumor necrosis factor-␣-converting enzyme (TACE), have all been shown to be intractable to TIMP-1 inhibition (12-14). Despite the similarity in sequence identity (40 -50%) and tertiary conformation, the molecular basis of TIMP selectivity remains hitherto ...

Tumor necrosis factor-␣ (TNF-␣)-converting enzyme (TACE, ADAM-17) is a zinc-dependent ADAM (a disintegrin and metalloproteinase) metalloproteinase (MP) of the metzincin superfamily. The enzyme regulates the shedding of a variety of cell surface-anchored molecules such as cytokines, growth factors, and receptors. The activities of the MPs are modulated by the endogenous inhibitors, the tissue inhibitor of metalloproteinases (TIMPs). Among the four mammalian TIMPs (TIMP-1 to -4), TACE is selectively inhibited by TIMP-3. The rationale for such selectivity is not fully understood. Here, we examine the molecular basis of TIMP-TACE selectivity using TIMP-2 as the scaffold. By systematically replacing the surface epitopes of TIMP-2 with those of TIMP-3 and a TIMP-1 variant V4S/TIMP-3 AB-loop/V69L/T98L, we created a novel TIMP-2 mutant that exhibits inhibitory potency almost equal to that of the TIMP-3. The affinity of the mutant with TACE is 1.49 nM, a marked improvement in comparison to that of the wild-type protein (K i 893 nM). The inhibitory pattern of the mutant is typical of that of a slow, tight binding inhibitor. We identify phenylalanine 34, a residue unique to the TIMP-3 AB-loop, as a vital element in TACE association. Mutagenesis carried out on leucine 100 also upholds our previous findings that a leucine on the EF-loop is critical for TACE recognition. Replacement of the residue by other amino acids resulted in a dramatic decrease in binding affinity, although isoleucine (L100I) and methionine (L100M) are still capable of producing the slow, tight binding effect. Our findings here represent a significant advance toward designing tailor-made TIMPs for specific MP targeting.The matrix metalloproteinases (MMPs), 1 the ADAM (a disintegrin and metalloproteinase) and the ADAM-TS (ADAM with thrombospondin repeats) proteinases are members of the mammalian metalloproteinases (MPs) of the metzincin superfamily. The enzymes are multidomain, zinc-dependent endopeptidases with a common structural feature, they all share the same tertiary configuration, termed "metzincin" fold, at the catalytic domain. Despite the similarity in structure, the functions of MPs are enormously diverse. To name a few, matrix degradation, transmembrane protein shedding, and regulation of growth factors are just some of the many physiologic processes that are known to involve the MPs (reviewed in Refs. 1-3).The enzymatic activities of the MPs are regulated by the endogenous inhibitors, the tissue inhibitor of metalloproteinases (TIMPs). There are four variants of mammalian TIMPs (TIMP-1 to -4), and each TIMP has its own profile of MP selectivity. For instance, whereas the majority of the soluble MMPs are well inhibited by all TIMP-1 to -4, membrane type MMPs and MMP-19 are less sensitive to TIMP-1 (4, 5). The ADAMs, on the other hand, are generally more sensitive to TIMP-3 than TIMP-1, -2, or -4 (6 -10). TIMPs are small molecules of ϳ22 kDa in molecular mass and variably glycosylated. The molecules are composed of two recognizable domains: (i)...