Abstract
Tumor necrosis factor-alpha-converting enzyme (TACE) is a disintegrin metalloproteinase that processes tumor necrosis factor and a host of other ectodomains. TACE is biosynthesized as a zymogen, and activation requires the removal of an inhibitory pro domain. Little is known about how the pro domain exerts inhibition for this class of enzymes. To study the inhibitory properties of the pro domain of TACE, we have expressed it in isolation from the rest of the protease. Here we show that the TACE pro domain (TACE Pro) is a stably folded protein that is able to inhibit this enzyme. TACE Pro inhibited the catalytic domain of TACE with an IC(50) of 70 nm. In contrast, this inhibitory potency decreased over 30-fold against a TACE form containing the catalytic plus disintegrin/cysteine-rich domains (IC(50) greater that 2 microm). The disintegrin/cysteine-rich region in isolation also decreases the interaction of TACE Pro with the catalytic domain. Surprisingly, we found that the cysteine switch motif located in TACE Pro was not essential for inhibition of the enzymatic activity of TACE; the pro domain variant C184A showed the same inhibitory potency against both TACE forms as wild type TACE Pro. X-ray absorption spectroscopy experiments indicate that binding of TACE Pro to the catalytic domain does include ligation of the catalytic zinc ion via the sulfur atom of its conserved Cys(184) residue. Moreover, the binding of TACE Pro to the catalytic zinc ion partially oxidizes the catalytic zinc ion of the enzyme. Despite this, the nature of the interaction between the pro and catalytic domains of TACE is not consistent with a simple competitive model of inhibition based on cysteine switch ligation of the zinc ion within the active site of TACE.
Highlights
The tumor necrosis factor-␣-converting enzyme (TACE1 or ADAM 17) is a zinc metalloproteinase that cleaves the precur
This is similar to the secreted bacterial serine proteases subtilisin and ␣-lytic protease, in which the pro domain accelerates the folding of the catalytic domain by several orders of magnitude, apparently by lowering the conformational energy barrier between the unfolded and native states (14 –17)
To characterize the role of Tumor necrosis factor-␣-converting enzyme (TACE) Pro as an enzyme inhibitor, we attempted its expression in E. coli
Summary
The tumor necrosis factor-␣-converting enzyme (TACE1 or ADAM 17) is a zinc metalloproteinase that cleaves the precur-. TACE Pro includes a cysteine switch box (PKVCGY186), a feature present in most metzincins, including matrix metalloproteinases and ADAMs. It has been proposed that the pro domains of metzincins act as inhibitors of their catalytic domains through a mechanism that involves ligation of the cysteinyl thiol within the cysteine switch box to the zinc ion in the active site (9 –11). Similar results have been reported for other members of the ADAM family [12, 13] At first approximation, this is similar to the secreted bacterial serine proteases subtilisin and ␣-lytic protease, in which the pro domain accelerates the folding of the catalytic domain by several orders of magnitude, apparently by lowering the conformational energy barrier between the unfolded and native states (14 –17). We show that the cysteine switch of TACE, present in the zymogen form, is not required for the interaction between the pro and catalytic domains of TACE
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