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...