Engineering of Selective TIMPs

Nagase, Hideaki; Meng, Qing H.; Malinovskii, V. I.; Huang, Wen; Chung, Linda; Bode, Wolfram; Maskos, K.; Brew, Keith

doi:10.1111/j.1749-6632.1999.tb07670.x

Cited by 50 publications

(40 citation statements)

References 28 publications

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“…The effects of mutating six of these contact residues (Thr-2, Val-4, Met-66, Ser-68, Val-69, Cys-70) on the affinity for various MMPs has been previously investigated. Although multiple substitutions are required to produce N-TIMP-1 variants with high selectivity for MMPs (9,14,15,33), substitutions for Thr-2 of N-TIMP-1 show that this residue has a dominant influence on the free energy of binding selectivity for MMPs. Consequently, this residue, which is located centrally in the MMP binding ridge and interacts with the S1Ј specificity pocket of MMPs, has been designated as a hot spot in the interaction interface (14,15).…”

Section: Correlation Of Structural Data With Results From Mutagenesismentioning

confidence: 99%

Crystal Structure of the Catalytic Domain of Matrix Metalloproteinase-1 in Complex with the Inhibitory Domain of Tissue Inhibitor of Metalloproteinase-1

Iyer

Wei

Brew

et al. 2007

Journal of Biological Chemistry

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The mammalian collagenases are a subgroup of the matrix metalloproteinases (MMPs) that are uniquely able to cleave triple helical fibrillar collagens. Collagen breakdown is an essential part of extracellular matrix turnover in key physiological processes including morphogenesis and wound healing; however, unregulated collagenolysis is linked to important diseases such as arthritis and cancer. The tissue inhibitors of metalloproteinases (TIMPs) function in controlling the activity of MMPs, including collagenases. We report here the structure of a complex of the catalytic domain of fibroblast collagenase (MMP-1) with the N-terminal inhibitory domain of human TIMP-1 (N-TIMP-1) at 2.54 Å resolution. Comparison with the previously reported structure of the TIMP-1/stromelysin-1 (MMP-3) complex shows that the mechanisms of inhibition of both MMPs are generally similar, yet there are significant differences in the protein-protein interfaces in the two complexes. Specifically, the loop between ␤-strands A and B of TIMP-1 makes contact with MMP-3 but not with MMP-1, and there are marked differences in the roles of individual residues in the C-D connector of TIMP-1 in binding to the two MMPs. Structural rearrangements in the bound MMPs are also strikingly different. This is the first crystallographic structure that contains the truncated N-terminal domain of a TIMP, which shows only minor differences from the corresponding region of the full-length protein. Differences in the interactions in the two TIMP-1 complexes provide a structural explanation for the results of previous mutational studies and a basis for designing new N-TIMP-1 variants with restricted specificity.The matrix metalloproteinases (MMPs) 4 are a family of zinc endopeptidases that share homologous catalytic domains. These enzymes catalyze the cleavage the protein components of the extracellular matrix and play a pivotal role in connective tissue remodeling during development, wound healing, and other physiological processes (1). There are a total of 24 MMPs in humans that have broad and overlapping specificities. The collagenases, a subgroup of MMPs that cleave collagens type I, II, and III, include MMP-1 (fibroblast collagenase), MMP-8 (neutrophil collagenase), and MMP-13 (collagenase-3) (2-4). MMP-1 is considered a prototype for all interstitial collagenases and plays an important role in the turnover of collagen fibrils in the matrix. Much evidence indicates that collagenolysis is an important event in diverse physiologic processes such as development, tissue morphogenesis, and wound repair (5), whereas unregulated collagen breakdown has been implicated in a variety of human diseases including cancer, rheumatoid arthritis, pulmonary emphysema, and fibrotic disorders (6). Therefore, knowledge of the structural basis of collagenase action and its regulation is crucial for understanding the molecular basis of these diseases and opening new therapeutic avenues.MMPs are initially synthesized as inactive precursors (proMMPs), and the levels of MMP activity ...

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Section: Correlation Of Structural Data With Results From Mutagenesismentioning

confidence: 99%

Crystal Structure of the Catalytic Domain of Matrix Metalloproteinase-1 in Complex with the Inhibitory Domain of Tissue Inhibitor of Metalloproteinase-1

Iyer

Wei

Brew

et al. 2007

Journal of Biological Chemistry

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“…Hence, the fundamental questions confronting the TIMP engineer are: how does a TIMP recognize and select its MP targets, and is it possible to create one or more tailor-made TIMP(s) for specific MP targeting? There have been many reviews written on the subject of TIMP engineering, but few have given much consideration to the most fundamental issue, the exact molecular elements that govern the recognition and selectivity profile of a TIMP species (25)(26)(27). It is against this background that our current series of TIMP-engineering projects is configured and initiated.…”

Section: Discussionmentioning

confidence: 99%

Delineating the Molecular Basis of the Inactivity of Tissue Inhibitor of Metalloproteinase-2 against Tumor Necrosis Factor-α-converting Enzyme

Lee

Rapti

Murphy

2004

Journal of Biological Chemistry

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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)...

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“…MMPs and TIMPs are known to be involved in various physiological processes both in normal bone remodeling and under pathological conditions such as osteoporosis and rheumatoid arthritis. [12][13][14] Direct regulation of MMPs or TIMPs by mechanical loading would provide an interesting means by which mechanical loading can regulate the spatial control of bone metabolism. Yet only very limited data are available on the interaction between mechanical loading and MMPs or TIMPs in osteoblasts.…”

Section: Introductionmentioning

confidence: 99%

Stretch-induced modulation of matrix metalloproteinases in mineralizing osteoblasts via extracellular signal-regulated kinase-1/2

et al. 2006

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Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) produced by osteoblasts play an essential role in bone remodeling. Hence, these proteins could provide an interesting means by which mechanical loading leads to adaptation of bone. Here, we examined the effect of stretch on MMP-1, -2, -3, -8, -9, -13, and -14, as well as TIMP-1 and -2 gene expression in differentiating, mineralizing, and nonmineralizing human SV-40 immortalized preosteoblast cells. In the mineralizing osteoblast culture, but not in the nonmineralizing cultures, cyclic stretch for only 15 min resulted in an increase of MMP-1 (fourfold) and -3 (depending on differentiation stage up to 25-fold) transcript abundance. No clear effect was observed for other MMPs, TIMP-1 or -2. The increase of MMP-1 and -3 was confirmed on the protein level. Stretching experiments performed in the presence of a specific inhibitor of extracellular signal-regulated kinase (ERK) showed a strong suppression of the stretch-induced increase in MMP-1 and -3. In conclusion, we show that MMP-1 and MMP-3 are mechanosensitive genes in mineralizing the human osteoblast, and that the mechano-induction of these genes is mediated via the ERK pathway. Our findings implicate that these MMPs are important factors in the mechanoregulation of bone turnover. With the ability to generate MMPs at highly stretched sites, osteoblasts can potantially direct osteoclasts to specific bone surface areas prepared for resorption. ß

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Engineering of Selective TIMPs

Cited by 50 publications

References 28 publications

Crystal Structure of the Catalytic Domain of Matrix Metalloproteinase-1 in Complex with the Inhibitory Domain of Tissue Inhibitor of Metalloproteinase-1

Crystal Structure of the Catalytic Domain of Matrix Metalloproteinase-1 in Complex with the Inhibitory Domain of Tissue Inhibitor of Metalloproteinase-1

Delineating the Molecular Basis of the Inactivity of Tissue Inhibitor of Metalloproteinase-2 against Tumor Necrosis Factor-α-converting Enzyme

Stretch-induced modulation of matrix metalloproteinases in mineralizing osteoblasts via extracellular signal-regulated kinase-1/2

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