Crystal structure of the catalytic domain of human tumor necrosis factor-α-converting enzyme

Abstract

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...

Different ADAMs have distinct influences on Kit ligand processing: phorbol-ester-stimulated ectodomain shedding of Kitl1 by ADAM17 is reduced by ADAM19

“…One possible explanation for the lack of processing of KL2SSTL/KAAK is that ADAM17 has an extended recognition sequence in Kitl2 for processing that includes these two sequences, but requires only one to be present. This notion is consistent with the crystal structure of ADAM17, which shows an extended peptidebinding groove in the catalytic site (Maskos et al, 1998). Finally, it is possible that ADAM17 uses binding sites in the ectodomain of Kitl as a determinant for its recognition, and then proceeds to cleave the substrate in a membrane proximal position without a strong cleavage site preference, but nevertheless requires either SSTL141-144 or KAAK150-153 to be present in Kitl2.…”

CD30 Shedding from Karpas 299 Lymphoma Cells Is Mediated by TNF-α-Converting Enzyme

“…Total RNA from Karpas 299 cells was extracted with Trizol reagent (Life Technologies, Karlsruhe, Germany), digested with RNase-free DNase I (Roche Diagnostics, Mannheim, Germany), and cDNA synthesis was performed using a SuperScript Preamplification System and oligo(dT) [12][13][14][15][16][17][18] primers (Life Technologies). For an amplification of the cDNA fragment encoding the catalytic, disintegrin, and cysteine-rich domain of TACE, total cDNA was subjected to 30 cycles of PCR using proofreading polymerase (Elongase Enzyme mix; Life Technologies).…”

“…Metalloproteinase-disintegrins belong to the metzincin superfamily of metalloproteinases encompassing the physiologically important families of MMPs, reprolysins and astacins. Due to their x-ray crystal structure, metalloproteinase-disintegrins are closely related to snake venom metalloproteinases (reprolysins) and are therefore regarded as members of the reprolysin family of metalloproteinases (13). Both snake venom proteinases and metalloproteinase-disintegrins exhibit membrane protein sheddase activity through their catalytic domain (11,12,14) and binding to cellular disintegrin receptors mediated by the disintegrin domain (15)(16)(17).…”

A Comparison of the Binding Sites of Matrix Metalloproteinases and Tumor Necrosis Factor-α Converting Enzyme: Implications for Selectivity

“…Another interesting structural difference of TACE is that S1' and S3' subsites, separated on the surface by the side chains of residues Ala439 and Leu348, are merged below the surface. 25 This feature will be discussed in more detail in section Specificity Determinants and Comparison with Inhibitor Data.…”