The proteolytic activity of matrix metalloproteinases (MMPs) towards extracellular matrix components is held in check by the tissue inhibitors of metalloproteinases (TIMPs). The binary complex of TIMP-2 and membrane-type-1 MMP (MT1-MMP) forms a cell surface located 'receptor' involved in pro-MMP-2 activation. We have solved the 2.75 Å crystal structure of the complex between the catalytic domain of human MT1-MMP (cdMT1-MMP) and bovine TIMP-2. In comparison with our previously determined MMP-3-TIMP-1 complex, both proteins are considerably tilted to one another and show new features. CdMT1-MMP, apart from exhibiting the classical MMP fold, displays two large insertions remote from the active-site cleft that might be important for interaction with macromolecular substrates. The TIMP-2 polypeptide chain, as in TIMP-1, folds into a continuous wedge; the A-B edge loop is much more elongated and tilted, however, wrapping around the S-loop and the β-sheet rim of the MT1-MMP. In addition, both C-terminal edge loops make more interactions with the target enzyme. The C-terminal acidic tail of TIMP-2 is disordered but might adopt a defined structure upon binding to pro-MMP-2; the Ser2 side-chain of TIMP-2 extends into the voluminous S1Ј specificity pocket of cdMT1-MMP, with its Oγ pointing towards the carboxylate of the catalytic Glu240. The lower affinity of TIMP-1 for MT1-MMP compared with TIMP-2 might be explained by a reduced number of favourable interactions. Keywords: crystal structure/matrix metalloproteinase/ progelatinase A activator/proteinase complex/tissue inhibitor of metalloproteinases

Calpains (calcium-dependent cytoplasmic cysteine proteinases) are implicated in processes such as cytoskeleton remodeling and signal transduction. The 2.3-Å crystal structure of full-length heterodimeric [80-kDa (dI-dIV) ؉ 30-kDa (dV؉dVI)] human m-calpain crystallized in the absence of calcium reveals an oval disc-like shape, with the papain-like catalytic domain dII and the two calmodulin-like domains dIV؉dVI occupying opposite poles, and the tumor necrosis factor ␣-like ␤-sandwich domain dIII and the N-terminal segments dI؉dV located between. Compared with papain, the two subdomains dIIa؉dIIb of the catalytic unit are rotated against one another by 50°, disrupting the active site and the substrate binding site, explaining the inactivity of calpains in the absence of calcium. Calcium binding to an extremely negatively charged loop of domain dIII (an electrostatic switch) could release the adjacent barrel-like subdomain dIIb to move toward the helical subdomain dIIa, allowing formation of a functional catalytic center. This switch loop could also mediate membrane binding, thereby explaining calpains' strongly reduced calcium requirements in vivo. The activity status at the catalytic center might be further modulated by calcium binding to the calmodulin domains via the Nterminal linkers.T he calpains (EC 3.4.22.17; Clan CA, family C02) are a family of calcium-dependent cytosolic cysteine proteinases. They seem to catalyze limited proteolysis of proteins involved in cytoskeletal remodeling and signal transduction but are also implicated in other physiological and pathophysiological processes, such as cell cycle regulation, apoptosis, muscular dystrophies, cataractogenesis, and Alzheimer's or Parkinson's diseases (1-5). In mammals, the calpain family comprises several ''tissuespecific'' isoforms (n-calpains) besides two ''ubiquitous'' isoenzymes (-and m-calpains). In lower organisms such as insects, nematodes, fungi, and yeast, a number of ''atypical'' calpain homologues have been found.The ubiquitous -and m-calpains (calpains I and II), by far the best characterized calpains, are heterodimers comprising distinct but quite homologous 80-kDa ''large'' L-subunits and a common 30-kDa ''small'' S-subunit. On the basis of amino acid homologies, the L-and S-subunits have been described as consisting of four domains, dI to dIV, and of two domains, dV and dVI, respectively, with domain dII somewhat resembling papain and the calmodulin-like domains dIV and dVI containing EF-hands (6, 7). On exposure to calcium at concentrations of 5-50 M (-calpain) and 200-1,000 M (m-calpain), both calpains are activated and partially autolyzed. In vivo, both calpains seem to be active at physiological calcium concentrations of 100-300 nM, however, suggesting that other factors such as phospholipids might play a role in activation in addition.The crystal structures of rat and porcine domain dVI in the absence and presence of calcium have been determined (8, 9). For a full understanding of the activation mechanism and the functioning of calp...

Tumor necrosis factor-␣ (TNF␣) is a cytokine that induces protective inf lammatory reactions and kills tumor cells but also causes severe damage when produced in excess, as in rheumatoid arthritis and septic shock. Soluble TNF␣ is released from its membrane-bound precursor by a membrane-anchored proteinase, recently identified as a multidomain metalloproteinase called TNF␣-converting enzyme or TACE. We have cocrystallized the catalytic domain of TACE with a hydroxamic acid inhibitor and have solved its 2.0 Å crystal structure. This structure reveals a polypeptide fold and a catalytic zinc environment resembling that of the snake venom metalloproteinases, identifying TACE as a member of the adamalysin͞ADAM family. However, a number of large insertion loops generate unique surface features. The pro-TNF␣ cleavage site fits to the active site of TACE but seems also to be determined by its position relative to the base of the compact trimeric TNF␣ cone. The active-site cleft of TACE shares properties with the matrix metalloproteinases but exhibits unique features such as a deep S3 pocket merging with the S1 specificity pocket below the surface. The structure thus opens a different approach toward the design of specific synthetic TACE inhibitors, which could act as effective therapeutic agents in vivo to modulate TNF␣-induced pathophysiological effects, and might also help to control related shedding processes.Tumor necrosis factor-␣ (TNF␣) (1), a major immunomodulatory and proinflammatory cytokine, is synthesized as a 223-aa membrane-anchored precursor. The soluble form of TNF␣, comprising the C-terminal two-thirds of this precursor, is released into extracellular space by limited proteolysis at the Ala-76 3 Val-77 bond. The proteinase responsible for this cleavage, called TACE or ADAM 17, has recently been identified (2, 3) as a zinc-endopeptidase consisting of a multidomain extracellular part, an apparent transmembrane helix and an intracellular C-terminal tail. The extracellular part comprises an N-terminal pro domain, a 259-residue catalytic domain, and a Cys-rich moiety that has been hypothesized to be composed of a disintegrin-like, an epidermal growth factorlike, and a crambin-like domain (2). Its polypeptide sequence, in particular, that accounting for the catalytic domain, indicates some similarity with other metzincins (4, 5), especially with the adamalysins͞ADAMs (6-8) (a protein family comprising snake venom metalloproteinases and membraneanchored surface proteins containing an adamalysin-like catalytic domain) and the matrix metalloproteinases (MMPs). In comparison to enzymes in these families, however, the polypeptide chain of the TACE catalytic domain is clearly longer and is stable in the absence of calcium. Further, in contrast to the MMPs, TACE is relatively insensitive to the tissue inhibitor of metalloproteinases-1 (TIMP-1) (9) and exhibits a different inhibition pattern toward synthetic inhibitors (9-12). In contrast to the MMPs, TACE cleaves a 12-mer peptide spanning the cleavage site in...

Matrix metalloproteinases (MMPs) are involved in extracellular matrix degradation. Their proteolytic activity must be precisely regulated by their endogenous protein inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance results in serious diseases such as arthritis, tumour growth and metastasis. Knowledge of the tertiary structures of the proteins involved is crucial for understanding their functional properties and interference with associated dysfunctions. Within the last few years, several three-dimensional MMP and MMP-TIMP structures became available, showing the domain organization, polypeptide fold and main specificity determinants. Complexes of the catalytic MMP domains with various synthetic inhibitors enabled the structure-based design and improvement of high-affinity ligands, which might be elaborated into drugs. A multitude of reviews surveying work done on all aspects of MMPs have appeared in recent years, but none of them has focused on the three-dimensional structures. This review was written to close the gap.